effects of korean red ginseng and haart on vif gene in 10 long

13
Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 871648, 12 pages http://dx.doi.org/10.1155/2013/871648 Research Article Effects of Korean Red Ginseng and HAART on vif Gene in 10 Long-Term Slow Progressors over 20 Years: High Frequency of Deletions and G-to-A Hypermutation Young Keol Cho, 1 Ba Reum Kim, 1 Mee Soo Chang, 2 and Jung Eun Kim 1 1 Department of Microbiology, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 138-736, Republic of Korea 2 Department of Pathology, Seoul National University Boramae Hospital, Seoul 156-707, Republic of Korea Correspondence should be addressed to Young Keol Cho; [email protected] Received 12 June 2013; Accepted 3 September 2013 Academic Editor: K. B. Harikumar Copyright © 2013 Young Keol Cho et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To investigate if Korean red ginseng (KRG) affects vif gene, we determined vif gene over 20 years in 10 long-term slowly progressing patients (LTSP) who were treated with KRG alone and then KRG plus HAART. We also compared these data with those of 21 control patients who did not receive KRG. Control patient group harbored only one premature stop codon (PSC) (0.9%), whereas the 10 LTSP revealed 78 defective genes (18.1%) ( < 0.001). e frequency of small in-frame deletions was found to be significantly higher in patients who received KRG alone (10.5%) than 0% in the pre-KRG or control patients ( < 0.01). Regarding HAART, vif genes containing PSCs were more frequently detected in patients receiving KRG plus HAART than patients receiving KRG alone or control patients ( < 0.01). In conclusion, our current data suggest that the high frequency of deletions and PSC in the vif gene is associated with KRG intake and HAART, respectively. 1. Introduction Panax ginseng has a long history of medicinal use in Asia. At present, ginseng is the best-selling herbal medicine in the world [1]. About 200 constituents of Korean ginseng have been isolated and characterized. Its major compo- nents include ginseng saponins and polysaccharides. e major pharmacological effects of ginseng include adapto- genic effects [2]; that is, ginseng nonspecifically increases the resistance to physical, chemical, and biological stress by immunomodulation of the hypothalamic-pituitary-adrenal axis [3]. Recent studies have also demonstrated ginseng’s potential use in adjuvant and immunotherapies [48]. Persistent immune activation and inflammation despite sustained antiretroviral therapy (ART)-mediated viral sup- pression have emerged as a major challenge in the modern era of HIV treatment [9]. In particular, the saponin fraction of ginseng downregulates proinflammatory mediators in LPS-stimulated cells and protects mice against endotoxic shock [1012]. e absence of microbial translocation and immune activation in well-adapted, natural hosts with simian immunodeficiency virus [13] is a very important mechanism in our understanding of the slow progression of HIV-1- infected patients who have been treated with Korean red ginseng (KRG) [14]. We previously reported that KRG induces gross deletions in the nef gene [14] and frequent genetic defects in the 5 LTR/gag gene [15]. Interestingly, the detection of genetic defects was inhibited during the administration of highly active antiretroviral therapy (HAART) [16]. However, there are only a few studies that have reported the gross deletion of the vif gene because it is the second most highly conserved gene aſter pol [1719]. Hence, to determine if KRG affects the vif gene, as shown for the nef and gag genes [1416], we amplified vif gene in peripheral blood mononuclear cells (PBMCs) obtained over 20 years from 10 long-term slowly progressing (LTSP) patients. It appears from our analyses that KRG intake might induce gross and small in-frame deletions.

Upload: duongcong

Post on 10-Feb-2017

214 views

Category:

Documents


1 download

TRANSCRIPT

Page 1: Effects of Korean Red Ginseng and HAART on vif Gene in 10 Long

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 871648 12 pageshttpdxdoiorg1011552013871648

Research ArticleEffects of Korean Red Ginseng and HAART on vif Gene in10 Long-Term Slow Progressors over 20 Years High Frequencyof Deletions and G-to-A Hypermutation

Young Keol Cho1 Ba Reum Kim1 Mee Soo Chang2 and Jung Eun Kim1

1 Department of Microbiology University of Ulsan College of Medicine 88 Olympic-ro 43-gil Songpa-guSeoul 138-736 Republic of Korea

2Department of Pathology Seoul National University Boramae Hospital Seoul 156-707 Republic of Korea

Correspondence should be addressed to Young Keol Cho ykcho2amcseoulkr

Received 12 June 2013 Accepted 3 September 2013

Academic Editor K B Harikumar

Copyright copy 2013 Young Keol Cho et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

To investigate if Korean red ginseng (KRG) affects vif gene we determined vif gene over 20 years in 10 long-term slowly progressingpatients (LTSP) whowere treated with KRG alone and thenKRGplusHAARTWe also compared these data with those of 21 controlpatients who did not receive KRG Control patient group harbored only one premature stop codon (PSC) (09) whereas the 10LTSP revealed 78 defective genes (181) (119875 lt 0001) The frequency of small in-frame deletions was found to be significantlyhigher in patients who received KRG alone (105) than 0 in the pre-KRG or control patients (119875 lt 001) Regarding HAART vifgenes containing PSCs were more frequently detected in patients receiving KRG plus HAART than patients receiving KRG aloneor control patients (119875 lt 001) In conclusion our current data suggest that the high frequency of deletions and PSC in the vif geneis associated with KRG intake and HAART respectively

1 Introduction

Panax ginseng has a long history of medicinal use in AsiaAt present ginseng is the best-selling herbal medicine inthe world [1] About 200 constituents of Korean ginsenghave been isolated and characterized Its major compo-nents include ginseng saponins and polysaccharides Themajor pharmacological effects of ginseng include adapto-genic effects [2] that is ginseng nonspecifically increasesthe resistance to physical chemical and biological stress byimmunomodulation of the hypothalamic-pituitary-adrenalaxis [3] Recent studies have also demonstrated ginsengrsquospotential use in adjuvant and immunotherapies [4ndash8]

Persistent immune activation and inflammation despitesustained antiretroviral therapy (ART)-mediated viral sup-pression have emerged as a major challenge in the modernera of HIV treatment [9] In particular the saponin fractionof ginseng downregulates proinflammatory mediators inLPS-stimulated cells and protects mice against endotoxic

shock [10ndash12] The absence of microbial translocation andimmune activation inwell-adapted natural hosts with simianimmunodeficiency virus [13] is a very important mechanismin our understanding of the slow progression of HIV-1-infected patients who have been treated with Korean redginseng (KRG) [14]

We previously reported that KRG induces gross deletionsin the nef gene [14] and frequent genetic defects in the51015840 LTRgag gene [15] Interestingly the detection of geneticdefects was inhibited during the administration of highlyactive antiretroviral therapy (HAART) [16] However thereare only a few studies that have reported the gross deletion ofthe vif gene because it is the second most highly conservedgene after pol [17ndash19] Hence to determine if KRG affectsthe vif gene as shown for the nef and gag genes [14ndash16]we amplified vif gene in peripheral blood mononuclear cells(PBMCs) obtained over 20 years from 10 long-term slowlyprogressing (LTSP) patients It appears fromour analyses thatKRG intakemight induce gross and small in-frame deletions

2 Evidence-Based Complementary and Alternative Medicine

Table 1 Characteristics of 10 long-term slow progressors

Patient code Year ofdiagnosis

Date of AIDSdiagnosis

CD4+ Tcells120583La

Viral load(copymL)a

Follow-up from Dxbto HAART (Mo) HAARTc Duration of

HAART87-05 1987 NA 256 115000 314 None None89-17 1989 Jul 02 106 162000 188 Mar 05 6290-05 1990 May 08 103 94376 221 July 08 5890-18 1990 Mar 04 112 124000 167 Apr 04 10490-50 1990 Mar 07 116 244000 197 May 07 6891-20 1991 Aug 07 55 3886 214 Aug 07 6592-13 1992 June 07 47 5800 181 Jun 07 4593-04 1993 NA 297 656000 166 Nov 06 993-60 1993 NA 109 121000 182 May 08 4796-51 1996 NA 169 386543 164 Dec 09 36aCD4+ T cell count and viral load measured just before the initiation of HAARTbDx diagnosiscHAART highly active antiretroviral therapy

In addition we found that HAART increases the frequency ofpremature stop codons (PSCs) in the vif gene

2 Materials and Methods

21 Patients Ten patients whose annual decrease in CD4+T cells was lt20 cells120583L were diagnosed as LTSP patients[14]Their clinical characteristics including changes inCD4+T-cell count and RNA copy number KRG therapy andfrequent genetic defects in the nef and 51015840 LTRgag geneshave been previously described [14 15] However the follow-up period of the present study was extended to include theKRG plus HAART periodThe control patients (119899 = 21) wereselected from 169 patients [20] who had not been exposedto KRG or any antiretroviral therapies (eg AZT) at thetime of sampling and whose PBMCs were available for geneamplification This study was approved by the InstitutionalReview Board of the Asan Medical Center

22 KRG Intake KRG powder was manufactured by theKorea Ginseng Corporation (Daejeon Korea) from theroots of a 6-year-old Panax ginseng Meyer red ginseng andharvested in the Republic of Korea KRG was made bysteaming fresh ginseng at 90ndash100∘C for three hours anddrying at 50ndash80∘C KRG powder was prepared from groundred ginseng (500mg KRGcapsule) and analyzed using high-performance liquid chromatography

23 Amplification of the vif Gene Nested PCR was used toamplify the proviral vif gene from the patientsrsquo PBMCs aspreviously described [14ndash16 19] Total RNA was extractedfrom 300 120583L serum using theQIAampUltra Sense Viral RNAkit (Qiagen Hilden Germany) as previously described [20]

24 Determination of G-to-A Hypermutation Sequenceswere analyzed using the Hypermut 20 program(httpwwwhivlanlgovcontentsequenceHYPERMUThypermuthtml) which compares each patientrsquos sequence

to a patient-specific consensus in order to determine thefrequency and context of the G-to-A mutations [21]

25 Statistical Analysis Data are expressed as the means plusmn2 standard deviations (for continuous variables) or as countsand percentages (categorical variables) Comparing the pro-portions between groups was analyzed by using the Chi-squared test or Fisher exact test In this study 119875 lt 005 wasconsidered statistically significant

26 Accession Numbers of the Nucleotide Sequence Theinvestigated sequences were previously assigned the fol-lowing GeneBank accession numbers DQ072735 JF957893JQ067069-76 AY581367 JF957902 JQ248188-201 AY581377AY581378 JQ248228-50 JF957922 AY581382-5 DQ072750-51 JF957924 JQ248256-92 DQ072759-60 AY581386-89JF957933 JQ248313-29 AY581394-95 JF957940 JQ248339-55 AY581398-AY581399 JQ327719 JQ327794-97 JF958044-45 JQ066879-927 AY581341-44 JF957976 JQ268920-39AY581412-15 JQ268940-61 AF462782 JF957977 AY581416-18 JQ269006-32 JF957983 AY581419-20 KC247158-315 andKF270357-459

3 Results

31 Effects of KRG during the Follow-Up Period prior toHAART Administration Ten LTSP patients received follow-up without HAART for 199 plusmn 45 months (166 years range164ndash314months) followingHIV-1 diagnosis (Table 1) Duringthis period KRG therapy was administered for 173 plusmn 34months and thereafter KRG plus HAART was administeredfor 55 plusmn 26 months The amounts of KRG administeredduring these periods were 14398 plusmn 5775 and 5648 plusmn 3677 grespectively Patient 89-17rsquos compliance with KRG was poorand the amount supplied prior to HAART therapy was theleast (5076 g) of the 10 enrolled LTSP patients Patient 87-05began to take KRG in December 1991 though his compliancewas also poor He has taken KRG since June 1994 but his

Evidence-Based Complementary and Alternative Medicine 3

CD4+ T-cell count remained low (Figure 1) The remainingnine patients progressed to AIDS demonstrating CD4+ T-cell counts of lt200 cells120583L (Table 1)

32 Effects of HAART on PSC The 10 LTSP patients weincluded in our present study were untreated before Decem-ber 1991 were treated with only KRG between 2004 and2009 and have been treated with KRG plus HAART since2009 (Figure 1) We analyzed 432 vif genes over 20 years inthese 10 LTSP patients Among these 15 vif genes (35)demonstrated PSC In total 275 and 157 vif genes wereobtained during KRG and KRG plus HAART respectivelyEach group demonstrated 3 (11) and 12 (76) vif geneswith PSCs respectively When receiving only KRG threepatients (90-50 93-04 and 93-60) demonstrated PSC at 14years 1 year and 9months and 7 years after starting to receiveKRG respectively The frequency was significantly higherwhen receivingKRGplusHAART thanKRGalone (119875 lt 001Table 2) This suggests that HAART itself might induce G-to-A hypermutation thereby resulting in PSC and possiblylethal hypermutations This is consistent with the findings ofother studies [22] However we found no difference in thefrequency of PSCs between our KRG (3 of 275 genes) andcontrol groups (1 of 106 genes) Interestingly of the 15 vifgenes that demonstrated PSCs in our present analysis elevendid not satisfy the criteria of Hypermut 20 in comparisonwith the earliest sequences obtained from each patient (119875 lt005 Figure 3)

33 Effects of KRG and HAART on Genetic Defects Wedetected 11 gross deletions in 432 amplicons obtained fromour 10 LTSP patients (Table 2 Figure 2) No deleted genesincluding gross deletions were detected in the controlpatients In total five patients demonstrated gross deletionsafter gt19 months of KRG intake Four patients demonstratedgross deletions during KRG intake prior toHAART althoughthe frequency was very low (15) Specifically a sequencecontaining a gross deletion and duplicationrecombination(KC247159) was identified in patient 87-05 A 362-base pair(bp) deletion and 1 bp insertion (KC247195) 309 bp deletion(JQ327719) and 870 bp deletion (JQ327722) were detectedas short bands together with the wild-type genotype after139 months 115 months and 125 months of KRG intakein patients 90-05 91-20 and 93-60 respectively (patient90-05 demonstrated 9 bp insertions in 16 amplicons after104 months of KRG intake including KC247191 JQ248236DQ295192 and JQ248237-50) In addition two patientsdemonstrated gross deletions during KRG plus HAARTSpecifically patient 93-04 demonstrated 386 bp deletionsin one-third of amplicons after 14 years of KRG intakePatient 93-60 demonstrated six deletions (including 374 bp[JQ327723] and 401 bp deletions [KC247314] Figure 2 andTable 2) Each deletion was one (JQ327723) of 3 ampliconsand one (KC247314) of 2 amplicons obtained between August2008 and October 2010 and all 4 amplicons respectively(Figure 1) This frequency is the highest among the relatedstudies to date although we found no significant differencesin terms of the frequencies of gross deletions between patients

who received KRG (15) or KRG plus HAART (45Table 2) in our present study If both PSCs and gross deletionsare only defined as nonfunctional vif genes the proportion ofnonfunctional genes is 25 (7 of 275 genes) during KRG and121 (19 of 157 genes) during KRG plus HAART (119875 lt 001)

34 Position and Frequency of PSCs in the vif Gene Thereare 8 tryptophan residues in the Vif protein In this study15 vif genes from 5 patients (90-05 90-18 90-50 93-04and 93-60 Figure 3) demonstrated PSCs The frequencies ofthe PSCs in 5 tryptophan residues (21 38 70 89 and 174)were 3 7 13 2 and 7 respectively In the control group onepatient demonstrated stop codons at residues 21 70 and 174(JQ066980)

35 Small in-FrameDeletions Are Associated with KRG IntakeWe did not find any specific changes in the nucleotide oramino acid sequences due to KRG intake However inter-estingly two of our patients demonstrated small deletionsduring KRG intake one patient (90-18) demonstrated a 9 bpin-frame deletion (detected in 15 of 26 amplicons) at aminoacid positions 182ndash184 in April 1998 and another patient(92-13) demonstrated a 12 bp deletion (detected in 14 of 16amplicons) at positions 186ndash189 in May 2005 (Figure 2) Thefrequencies of these deletions were similar between patientswho received KRG (105 29 of 275 genes) and KRG plusHAART (159 25 of 157 genes) However the frequencyof these deletions was zero (0 of 52 genes as determinedusing RT-PCR) during pre-KRG and 105 (29 of 275) duringKRG only without HAART (119875 lt 001) These deletionsmanifested after 63 and 120 years of KRG intake in patients90-18 and 92-13 respectively Interestingly patient 90-18 alsodemonstrated a 6 bp deletion in the nef gene after 11 yearsof KRG intake with HAART although we did not categorizethis as a gross deletion [14] Deletions were conservedduring KRG plus HAART therapy (Figure 2) and compriseda higher proportion thanwild-type alleles In particular threeamplicons containing PSC also demonstrated small deletionsin patient 90-18 The presence of both wild-type and mutantalleles in the same samples differed from the findings forthe samples obtained from patients who were not treatedwith KRG in which all amplicons contained deletions in fivepatients (data not shown) In addition there were no suchdeletions in the control group (0 of 106 genes)

36 Overall Rate of Defective Genes Whenwe included smallin-frame deletions of defective genes the overall proportionsof defective genes were 131 (36 of 275 genes) when receivingonly KRG and 268 (42 of 157 genes) when receiving KRGplus HAART (119875 lt 001) In our current study the frequencyof defective genes identified in PBMCs obtained from HIV-1-infected patients prior to receiving HAART was similar tothe results of previous reports byWieland et al (10) [23] andSova et al (13) [24] although exceptional cases have beenreported byYamada and Iwamoto (205) [25] andTominagaet al (31) [26] Overall the proportions of defective genesidentified in our present study were significantly lower thanthe proportions of defective nef (94 of 479 genes including

4 Evidence-Based Complementary and Alternative Medicine

0

1

2

3

4

5

6

7

0100200300400500600700

87-05Korean Red Ginseng (KRG) KRG KRG KRG

Jun

87Se

p 89

Apr

91

Nov

91

Nov

92

Dec

93

Jun

94D

ec 9

4O

ct 9

5O

ct 9

6A

pr 9

7A

pr 9

8O

ct 9

8A

pr 9

9O

ct 9

9M

ar 0

0Fe

b 01

Jul 0

1Se

p 02

Sep

03O

ct 0

4M

ar 0

5M

ay 0

5O

ct 0

5M

ay 0

6O

ct 0

6Ja

n 07

May

07

Aug

07

Oct

07

Feb

08A

ug 0

8D

ec 0

8Ju

n 09

Dec

09

Jul 1

0M

ar 1

1Se

p 11

Feb

12A

ug 1

2D

ec 1

2

CD4

+ T

cells

(120583

L)

CD4+ T cells (120583L)

minus100 0

1

2

3

4

5

6

7

0200400600800

1000120014001600 89-17

KRG KRG HAARTKRGKRG KRG

Aug

89

Jan

90Se

p 90

Apr

91

Dec

91

Jan

92M

ar 9

2Ju

n 92

Nov

92

Feb

93A

pr 9

3Ju

l 93

Oct

93

Apr

94

Oct

94

Sep

95Ju

l 96

Oct

96

Mar

97

Aug

97

Apr

98

Jun

99N

ov 9

9M

ar 0

0Se

p 00

Feb

01Ju

l 01

Jan

02M

ar 0

2Se

p 02

Feb

03M

ay 0

3Ja

n 04

Aug

04

Mar

05

Dec

05

Oct

06

Nov

06

Feb

07A

pr 0

7A

ug 0

7D

ec 0

7Ju

l 08

May

09

May

10

Mar

12

RNA

copy

(log

mL)

minus200

0

1

2

3

4

5

6

7

050

100150200250300350400450 90-05

KRG HAARTKRG

lowast lowast lowast

Jul 9

1N

ov 9

1N

ov 9

2A

pr 9

3O

ct 9

3Ju

n 94

Jan

95M

ay 9

5A

ug 9

6A

ug 9

7A

pr 9

8M

ar 9

9A

ug 0

0Fe

b 01

May

01

Mar

02

Aug

02

Jan

03M

ar 0

3Ju

n 03

Oct

03

Mar

04

Aug

04

Feb

05Ju

l 05

Oct

05

Jan

06Ju

l 06

Oct

06

Feb

07M

ay 0

7A

ug 0

7O

ct 0

7Ja

n 08

Apr

08

May

08

Aug

08

Nov

08

Apr

09

Oct

09

Apr

10

Oct

10

Apr

11

Oct

11

Apr

12

Nov

12

May

13

CD4

+ T

cells

(120583

L)

0

1

2

3

4

5

6

7

0100200300400500600700800900

1000 90-18

HAARTKRGKRG KRG KRGKRGKRGKRG KRG

Aug

90

Feb

91A

ug 9

1D

ec 9

1M

ar 9

2Ju

n 92

Nov

92

Mar

93

May

93

Jun

93O

ct 9

3Fe

b 94

May

94

Aug

94

Nov

94

Feb

95M

ay 9

5O

ct 9

5Fe

b 96

May

96

Dec

96

Mar

97

Mar

97

Jul 9

7M

ar 9

8A

pr 9

8Se

p 98

Feb

99A

pr 9

9Ju

n 99

Nov

99

Mar

00

Jul 0

0A

ug 0

0O

ct 0

0M

ar 0

1Ju

l 01

Dec

01

Apr

02

Oct

02

Jan

03M

ay 0

3O

ct 0

3Ju

l 04

Dec

04

Mar

05

Feb

06Ju

n 06

Jul 0

6A

ug 0

6N

ov 0

6Ja

n 07

Apr

07

Jun

07O

ct 0

7N

ov 0

7D

ec 0

7M

ay 0

8N

ov 0

8Fe

b 09

Apr

09

Oct

09

Apr

10

Oct

10

Oct

11

Jun

12D

ec 1

2

minus100

lowastlowast lowast

RNA

copy

(log

mL)

CD4

+ T

cells

(120583

L)

0

1

2

3

4

5

6

7

0

200

400

600

800

1000

1200 90-50HAARTKRGKRGKRGKRG

Dec

90

Jul 9

1Ja

n 92

Jul 9

2Fe

b 93

Apr

93

Sep

93M

ar 9

4Se

p 94

Apr

95

Oct

95

Apr

96

Nov

96

May

97

Nov

97

Jun

98N

ov 9

8M

ay 9

9N

ov 9

9Se

p 00

Jul 0

1N

ov 0

1Ju

n 02

Dec

02

Mar

03

Jun

03Se

p 03

Mar

04

Aug

04

Jan

05A

ug 0

5Ja

n 06

Jul 0

6Se

p 06

Mar

07

May

07

Jun

07Ju

l 07

Oct

07

Nov

07

Jul 0

9D

ec 0

9A

pr 1

0Ju

l 10

Jan

11A

pr 1

1D

ec 1

1Ju

l 12

Jan

13

lowast

CD4+

T ce

lls (120583

L)

minus200 0

1

2

3

4

5

6

7

0

200

400

600

800

1000

120091-20

KRG HAARTKRGKRG

Jun

91D

ec 9

1M

ar 9

2M

ay 9

2Ja

n 93

May

93

Dec

93

Jul 9

4N

ov 9

4D

ec 9

4Fe

b 95

Mar

95

May

95

Oct

95

Apr

96

Aug

96

Mar

97

Jul 9

7A

pr 9

8N

ov 9

8M

ay 9

9N

ov 9

9M

ar 0

0A

pr 0

0Ju

l 00

Dec

00

Mar

01

Jul 0

1N

ov 0

1M

ar 0

2A

ug 0

2Ja

n 03

Mar

03

Aug

03

Jan

04Ju

l 04

Jan

05Ju

l 05

Jan

06Ju

l 06

Dec

06

May

07

Jul 0

7A

ug 0

7Se

p 07

Nov

07

Jan

08A

pr 0

8Ju

l 08

Dec

08

Oct

10

Apr

11

Jan

12Ju

l 12

Jan

13

minus200

RNA

copy

(log

mL)

CD4+

T ce

lls (120583

L)

0

1

2

3

4

5

6

7

050

100150200250300350400450

92-13KRG HAART HAART

KRGKRGKRGKRGKRG

Expired in 2011

May

92

Nov

92

May

93

Jul 9

3Ja

n 94

Oct

94

Dec

94

Feb

95A

pr 9

5O

ct 9

5A

pr 9

6N

ov 9

6Fe

b 97

Jun

97A

pr 9

8Ja

n 99

Oct

99

Feb

01D

ec 0

1Ju

l 02

Dec

02

Mar

03

Jun

03O

ct 0

3M

ay 0

4N

ov 0

4M

ay 0

5Ju

l 05

Feb

06A

ug 0

6N

ov 0

6A

pr 0

7Ju

n 07

Jul 0

7A

ug 0

7Se

p 07

Dec

07

May

08

Aug

08

Dec

08

May

09

Dec

09

Apr

10

Jun

10Ja

n 11

CD4

+ T

cells

(120583

L)

minus500

1

2

3

4

5

67

0100200300400500600700800900 93-04

KRG HAARTKRG KRG KRGKRG

Feb

93M

ar 9

3Ju

l 93

Dec

93

Feb

94Ju

n 94

Dec

94

May

95

Nov

95

Feb

96Se

p 96

Jan

97Fe

b 97

Jul 9

7A

ug 9

7A

pr 9

8Ju

l 98

Sep

00D

ec 0

0M

ar 0

1Ju

n 01

Sep

01Ju

n 02

Jul 0

2Ja

n 03

May

03

Oct

03

Mar

04

Aug

04

Jan

05M

ay 0

5Ju

n 05

Aug

05

Oct

05

Mar

06

Jul 0

6N

ov 0

6M

ar 0

7A

ug 0

7

lowastlowast

RNA

copy

(log

mL)

CD4

+ T

cells

(120583

L)

RNA

copy

(log

mL)

RNA copy (logmL)

RNA

copy

(log

mL)

RNA

copy

(log

mL)

RNA

copy

(log

mL)

if if

if

if

ifif

ifif

Sampling date Sampling date

Sampling date Sampling date

Sampling date Sampling date

Sampling date Sampling dateCD4+ T cells (120583L)RNA copy (logmL)

CD4

+ T

cells

(120583

L)

Figure 1 Continued

Evidence-Based Complementary and Alternative Medicine 5

0

1

2

3

4

5

6

7

0100200300400500600700800

93-60

HAARTKRGKRG

Apr

94

Nov

95

May

96

Nov

96

Apr

97

Jun

97Ju

n 99

Nov

99

Nov

00

Jul 0

1N

ov 0

1Ja

n 02

Sep

02Fe

b 03

May

03

Nov

03

May

04

Mar

05

Sep

05A

pr 0

6O

ct 0

6N

ov 0

6A

pr 0

7Ju

l 07

Aug

07

Oct

07

Jan

08M

ay 0

8M

ay 0

8Ju

l 08

Aug

08

Dec

08

May

09

Dec

09

Oct

10

Apr

11

Apr

12

lowast

0

1

2

3

4

5

6

7

0

200

400

600

800

1000

120096-51

KRG HAARTKRGKRG

Jul 9

6Ja

n 97

Jul 9

7A

pr 9

8N

ov 9

8M

ay 9

9Ju

n 00

Oct

00

Sep

01A

pr 0

2D

ec 0

2O

ct 0

3Fe

b 04

Aug

04

Feb

05A

ug 0

5N

ov 0

6Ju

l 06

Oct

06

Feb

07A

ug 0

7M

ar 0

8A

ug 0

8Fe

b 09

Jul 0

9D

ec 0

9M

ay 1

0Ju

n 10

Apr

11

Oct

11

Jul 1

2Ja

n 13

RNA

copy

(log

mL)

CD4

+ T

cells

(120583

L)

Sampling dateCD4+ T cells (120583L)RNA copy (logmL)

RNA

copy

(log

mL)

CD4

+ T

cells

(120583

L)

Sampling dateCD4+ T cells (120583L)RNA copy (logmL)

if if

Figure 1 Changes in the CD4+ T-cell count plasma viral load and genetic defects in terms of Korean red ginseng (KRG) intake and highlyactive antiretroviral therapy (HAART) The durations of KRG intake and HAART are indicated by the bars Solid and dotted lines denotegood (gt90) and poor (lt90) compliance according to self-administered responses respectively The upward arrow (uarr) downward arrow(darr) plus sign (+) and asterisk (lowast) denote the sequences of the vif gene gross deletions 3- and- 6-base pair (bp) in-frame insertions and stopcodons respectively

Table 2 Distribution of defective vif gene

Patient On-KRG On-HAARTKRGNo of genes Stop codon gΔ sΔ No of genes Stop codon gΔ sΔ

87-05 20 1 ND ND ND ND89-17 9 9 0 0 090-05 37 1 18 4 0 090-18 35 15 20 3a 0 12a

90-50 14 1 15 0 0 091-20 35 1 20 0 0 092-13 53 14 22 0 0 1393-04 18 1 12 5 1 093-60 28 1 1 20 0 6 096-51 26 21 0 0 0Total 275 3b 4 29 157 12b 7 25gΔ and sΔ denote gross deletion and in-frame small deletion respectivelyHAART highly active antiretroviral therapy ND not determinedaTwo out of three genes contained both stop codon and sΔb119875 lt 001119875 lt 001 for the sum of 3 kinds of defective genes (36275 versus 42157)Fifty-two vif genes at baseline obtained from serum using RT-PCR were all wild types lowastControl patients (119899 = 21) revealed premature stop codon in one outof 106 vif genes

stop codons 119875 lt 005) and 51015840 LTRgag genes (71 of 189 genes119875 lt 0001) identified in the same patients [14 15]

4 Discussion

Todate we have identified the vif gene in 194Korean patientsOf these 145 patients were infected with the Korean subcladeB ofHIV-1 (KSB) and the remaining 49 patients were infectedwith non-KSB Of these 194 patients 10 demonstrated smallin-frame deletions (3ndash15 bp) and all were diagnosed withKSB whereas no such deletions were detected in non-KSBpatients (119875 = 0068) Of the 10 patients with small deletionsthree patients were included in a group of 20 hemophiliacs

who were infected with KSB from plasma donors O and P[16 20] Interestingly plasma donors O and P revealed wild-type vif only between 1991 and 2002 (AY581320-21 JQ248127-33 JF957909 JF957921 JQ248134 and JF957935-37) In otherwords original vif gene was wild type without in-framesmall deletion In our current study only two patients (90-18 and 92-13) demonstrated 9 (119899 = 15) and 12 (119899 = 14)bp in-frame deletions in about two-thirds of the ampliconsobtained after 63 and 120 years of KRG intake respectivelywhereas five patients without KRG intake demonstrated in-frame deletions in all amplicons (data not shown) To ourknowledge these in-frame deletions are very rare and haveonly been reported in two patients in other countries [26 27]

6 Evidence-Based Complementary and Alternative Medicine

87-05

89-17

90-05

90-18

90-50

91-20

1 20 40 60 80 96Kor consensus MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKEWVYRHHYESTHPRISSEVHIPLGDAKLVITTYWGLHTGEREWHLGQGVSIEWRKKRYNT

91CSR4-10026 RGNVVD91CSR11-6922 RGNVVD92CSR5-9885 RGNVVD93CSR6-10159 RGNVVD93CSR6-10162s1 ------------------------------------------------------------------------------------------------03CSR9-805 SGGNVVD05CSR3 RGNIVVDG06CSR10-5280 RGNIVVDRG08CSR3-5334 RGNNVDG08CSR8-5279 RGNIVVD10CSR7-8126 VRGNLVVDG12CSR2-10141 VRGNLVVDG

91KJS12-8538 I92KJS3-6819 GI93KJS7-10155 IAI97KJS3-3814 IGKEI01KJS2-5667 IGKI03KJS2-745 IGAI03KJS7-775 IGAEI05KJS12-3264 YMIVGAEI08KJS5-5157 IVNGAAI10KJS5-10133 IGKAES

92LSK11-9417 K94LSK6-759 KGNPRS96LSK6-11491 AKNPIRS98LSK4-11495 KHN00LSK8-9999 KGNR03LSK6-10001s1 ------------------------------------------------------------------------------04LSK7 KKGNPRST06LSK1-3236 KKGNPVRST07LSK2-3232 KKGNPVRST08LSK1-4762 KKGIPVRST09LSK4-6143 IKNPIGRST10LSK4-9165 KGNPRS10LSK4-9168 KKGNPVRTRST12LSK4-10131 KAVKALKNPEKST 12LSK4-10132 KAKNPGRST13LSK5-11470 KKGNPGRST13LSK5-11471 KKVGNPVRST13LSK5-11472 IKANNRI

92LSH3-6951 K92LSH11-8548 TKK93LSH10-1252 94LSH2-740 95LSH5-10562 V96LSH5-10609 V98LSH4-10567 VG99LSH1-5776 VG99LSH1-5777 VG99LSH1-5778 VG99LSH6-5781 VGE99LSH6-5782 VG00LSH3-5704 KVGR00LSH3-5705 VG00LSH3-5706 VGK02LSH10-5707 VGR02LSH10-5708 VG02LSH10-5710 VG03LSH5-5650 VGG03LSH5-5651 VG03LSH5-5652 VG03LSH10-5653 VG04LSH7-4848 VGKES04LSH8-3011 V06LSH8-5647 VGES06LSH8-5649 VGES06LSH11-3238 VGIS08LSH5-5644 VG08LSH5-5645 VGR08LSH5-5646 KVGE08LSH5-5647 KVGE08LSH11-5694 KVG10LSH4-9190 KVGES10LSH4-9192 VG10LSH4-9193 VGG12LSH6-10100 VGES12LSH6-10101 VG

91KJin7-6723 N93KJin2-10024 N93KJin9-8530 NHVKINVDT01KJin7-5383 KVN03KJin6-683 KVINT04KJin8-1955 KVINDT06KJin1-3030 KVKINDT06KJin9-3012 KVINT07KJin3-5148 VNKINID07KJin3-5961 VNKKINEN10KJin4-9162 NHVKKINVT10KJin9-8176 NHVKKINVDT11KJin12-10119 KVKKINVDT13KJin1-11373 VNNVNDT

K94JWK12-5524 K97JWK K99JWK5-11441 KNE00JWK3-11453 KN00JWK12-11445 KN01JWK7-5603 KVA01JWK7-5603s1 KKNN02JWK8-11435 KVA03JWK8-677 KN05JWK1-5600 KVA06JWK7-3042 KVNAN

KKNVNVVN08JWK1-4772 KVNAN

KVA11JWK4-8993 KVA12JWK1-10125 KVA13JWK1-11366 KVA

minus minusminus minusminus minusminus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ +

Patient KRG HAART

10JWK4 9159

06JWK12 5624

91JWK6 6949

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 7

92-13

93-04

93-60

96-51

92CWS5-9398 G93CWS3-9925 GK93CWS5-6669 GK93CWS7-802 G96CWS11-5341 G99CWS10-5338 KVGE01CWS2-10532 VG03CWS6-684 VGTG03CWS10-5773 VG04CWS5-3239 VGKSTG05CWS5-5783 GTG05CWS5-5785 VGKITG06CWS2-3071 VGTG06CWS2-3203 VGKITG06CWS2-3221 VGTG06CWS2-3222 NVGTG06CWS2-3223 NVGTG06CWS8-3038 NVGKTG06CWS8-3039 NVGKTG07CWS4-3820 VGKIATG07CWS4-3821 VGKIATG07CWS4-10174 HWVGKIATG07CWS4-10175 VGKIATG07CWS4-10176 VGKITG07CWS4-10177 VGKKTG07CWS9-6252 VGTG07CWS9-6253 VGKITG07CWS9-6254 VGKIATG07CWS12-4777 VGQITG08CWS5-6214 NKVGQITG08CWS8-5160 NVGKTG08CWS8-5161 NVGKNQG08CWS12-5720 VGQITG08CWS12-5721 VGQITG08CWS12-5722 VGKQITG09CWS5-6256 VGKQTG09CWS5-6257 VGKQTG09CWS5-6258 VGKDQTG09CWS12-9108 LVGTG09CWS12-9109 VGKQITG

93KYR2-9472 VNN94KYR6-10137 VNN94KYR12-9441 VNN95KYR5-815 VGNN00KYR9-9913 VGNN02KYR7-9911 VGNN03KYR1-817 VNN06KYR7-5236 VGNN06KYR11-5249 VGNNS07KYR3-5265 VGNN07KYR3-11387s1 -------------------------------------------------------------------------------------------------07KYR8-4845 VGNN07KYR8-4846 VGNN07KYR8-5166 IVRNKN07KYR8-5167 VGNRN07KYR8-5168 VGNN

94KMH5 EI96KMH11-8534 IAEI97KMH4-9452 ISEI01KMH11-5526 ISIEI03KMH5-687 EI03KMH11-5725 ISKIEI04KMH5 EI05KMH3-5727 ISEI06KMH11-3026 ISKEI07KMH4-3281s1 --------------------------------------------------------------------EI07KMH10-5732 VNEI08KMH5-5165 VGNEI08KMH8-5427s1 ------------------------------------------------------------------------------------------------09KMH5-11475 ISEI09KMH5-11475s2 HQS----------------------10KMH10-9907s1 ------------------------------------------------------------------------------------------------11KMH4-9917 VDI12KMH4-10096 VGNEI

00YJN10-9385 TRGNMS01YJN9-4850 RGNMS03YJN10-840 VVRGNMS05YJN2-1948 VVRGNMS06YJN10-3044 VRGNMS07YJN4-3248 VVRGNMQI07YJN8-5627 VRGNMQS08YJN3-5606 VRGNMQS08YJN8-5408 VRGNPVMQS09YJN2-6041 VRGNPVMQS09YJN12-9156 VRGNVMQS10YJN6-9918 VRGNVMQS11YJN2-9921 VRGNVMS12YJN7-11382 VRGNVMQS13YJN1-11381 VRGNVMSKor consensus MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKEWVYRHHYESTHPRISSEVHIPLGDAKLVITTYWGLHTGEREWHLGQGVSIEWRKKRYNT

1

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ +minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

87-05

89-17

97 120 140 160 180 192QVDPDLADKLIHLHYFDCFSDSAIRHAILGRLVRPKCEYQAGHNKVGSLQYLALTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH

91CSR4-10026 TQRYKHISSTA91CSR11-6922 TQRYKHISSTA92CSR5-9885 TQRYKHISSTA93CSR6-10159 TQRYKHISSTA93CSR6-10162s1 ------------------------------------------------------------------------------------------------03CSR9-805 AQRYEKHISSTA05CSR3 TQRYKHISSTAK06CSR10-5280 TQRYKHISRTA08CSR3-5334 TQRYKHISSTA08CSR8-5279 TQRYKHISSTA10CSR7-8126 TQRYKHISSTIA12CSR2-10141 TQRYKHISSTIA

91KJS12-8538 92KJS3-6819 93KJS7-10155 97KJS3-3814 01KJS2-5667 03KJS3-745 03KJS7-775 05KJS12-3264 08KJS5-5157 10KJS5-10133

minus minusminus minusminus minusminus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ +

Patient Kor consensus KRG HAART

20 60 80 9640

RR

VS

VRE

VR

VRS

VRRES

VRRES

PKVPVIFRHIES

EVRRHES

VRRHES

Figure 2 Continued

8 Evidence-Based Complementary and Alternative Medicine

90-05

90-18

90-50

91-20

92LSK11-9417 YETRQE94LSK6-759 YETRPQES96LSK6-11491 GRYETRQ98LSK4-11495 YENTRPQES00LSK8-9999 YPQE03LSK6-10001s1 -------------------------------------------QRES04LSK7 YETRPQES06LSK1-3236 YEVTRPQRES07LSK2-3232 YEVTRPQES08LSK1-4762 YEVTRPQAESS09LSK4-6143 YEVTRPQES10LSK4-9165 YEVTRPQES10LSK4-9168 YEVTRPQES12LSK4-10131 NYKVTRPQRAES12LSK4-10132 YEVTRPQAESQ13LSK5-11470 PYVRRPTQETESS13LSK5-11471 YVTRPQRES13LSK5-11472 YENRTRQRES

92LSH3-6951 YRDH92LSH11-8548 GYRD93LSH10-1252 YRDR94LSH2-740 YRDR95LSH5-10562 YTRDR96LSH5-10609 YTR98LSH4-10567 YQTRRG---H99LSH1-5776 YQTRRG---RH99LSH1-5777 YQTRRHG---H99LSH1-5778 YQTRG---H99LSH6-5781 YQTRRQG---H99LSH6-5782 YQTRG---H00LSH3-5704 YQTRRG---H00LSH3-5705 YQTRRQSG---H00LSH3-5706 YQTRAG---H02LSH10-5707 YETRQG---H02LSH10-5708 YQTRG---H02LSH10-5710 YQTRRQR---H03LSH5-5650 YQTRRR---03LSH5-5651 YQTRRR---H03LSH5-5652 YQTRRER---03LSH10-5653 YQTRRQRS04LSH7-4848 YQTRRKQG---H04LSH8-3011 YR06LSH8-5647 YQTRRG---H06LSH8-5649 YQTRRG---H06LSH11-3238 YQHTRRQES08LSH5-5644 YQTRRSG---H08LSH5-5645 YQTRQAKS08LSH5-5646 YQTRRR---H08LSH5-5647 YQTRRR---H08LSH11-5694 YQTRRSRKG---H10LSH4-9190 YEQCTRRRG---H10LSH4-9192 YQRTRRRRG---H10LSH4-9193 YQTRRG---H12LSH6-10100 YQTRRG---H12LSH6-10101 YQTRRSRG---H

91KJin7-6723 TT93KJin2-10024 TT93KJin9-8530 VTPAKH01KJin7-5383 ISTT03KJin6-683 ISTPQH04KJin8-1955 ITPTH06KJin1-3030 VTPTH06KJin9-3012 VAPT07KJin3-5148 QEPTN07KJin3-5961 NLEPTAN10KJin4-9162 VTTAH10KJin9-8176 QVTAAH11KJin12-10119 VTPTADH13KJin1-11373 VTPTH

PE94JWK12-5524 VQ97JWK HR99JWK5-11441 HR00JWK3-11453 HR00JWK12-11445 HR01JWK7-5603 NHRQ01JWK7-5603s1 -------------------------------------------------------------------------------02JWK8-11436 NRQQ03JWK8-677 HR05JWK1-5600 NRQQ06JWK7-3042 HRQ

HRQ08JWK1-4772 NRQQ

NRQQ11JWK4-8993 NRQQ12JWK1-10125 NRQQ13JWK1-11366 NRQQ

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ +

10JWK4 9159

06 JWK12 5624

91JWK6 6949

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 9

92-13

93-04

93-60

92CWS5-9398 HSE93CWS3-9925 HE93CWS5-6669 EHTE93CWS7-802 KHTE96CWS11-5341 KHTE99CWS10-5338 HPES01CWS2-10532 HPDE03CWS6-684 HPE03CWS10-5773 HPDE04CWS5-3239 HPTE05CWS5-5783 HPTD----05CWS5-5785 HPTD----06CWS2-3071 HPTDE06CWS2-3203 HPTD----06CWS2-3221 HPTD----06CWS2-3222 VHPTD----06CWS2-3223 HPTD----06CWS8-3038 HPTD----06CWS8-3039 HPTD----07CWS4-3820 HPTATD----07CWS4-3821 HPTATD----07CWS4-10174 HPTATD----07CWS4-10175 HPTATD----07CWS4-10176 HPTATD----07CWS4-10177 VHRD----07CWS9-6252 HPE07CWS9-6253 HPTATD----07CWS9-6254 HPTATD----07CWS12-4777 HPTATD----08CWS5-6214 HPTATD----08CWS8-5160 HPTATD----08CWS8-5161 HPTATD----08CWS12-5720 HPTATD----08CWS12-5721 HPTATD----08CWS12-5722 HPTATD----09CWS5-6256 HPTATD----09CWS5-6257 HPTTATD----09CWS5-6258 HPTAD----09CWS12-9108 HPDE09CWS12-9109 HPTATTD----

93KYR2-9472 NSRIRKE94KYR6-10137 NSRRKE94KYR12-9441 NSRIRKER95KYR5-815 NSRIRKE00KYR9-9913 NSRRRKE02KYR7-9911 NSRRRKE03KYR1-817 NSRIRKE06KYR7-5236 QKPHRIRKE06KYR11-5249 QKPHRIRKE07KYR3-5265 NSHRIRKE07KYR3-11387s1 ---------------QNSHRIRRKER07KYR8-4845 QNSRRIRKER07KYR8-4846 NSRRIRKE07KYR8-5166 KNSHRRRIRKER07KYR8-5167 NKHRRIRRKE07KYR8-5168 IQRKPHRRIRKE

94KMH5 HVPR96KMH11-8534 VRT97KMH4-9452 HVQR01KMH11-5526 HVPR03KMH5-687 HVPR03KMH11-5725 HVQRS04KMH5 VQRS05KMH3-5727 HVPR06KMH11-3026 HVPR07KMH4-3281s1 VHVPRV----------------------------07KMH10-5732 HVQPRVRS08KMH5-5165 HIQPRVRS08KMH8-5427s1 ----------------------------HVQPTRVRS09KMH5-11475 HVQPRVRS09KMH5-11475s2 ------------------------------------------------------------------------------------------------10KMH10-9907s1 -----------------------------------------RVRS11KMH4-9917 HVPRVRS12KMH4-10096 HVPRVRRS

00YJN10-9385 ETVSRE01YJN9-4850 ETVSRE03YJN10-840 ETVSRE05YJN2-1948 ETQVSRE06YJN10-3044 ETQVSRE07YJN4-3248 ETQVSREY07YJN8-5627 ETQVSREY08YJN3-5606 ETQVSREY08YJN8-5408 ETQVSREY09YJN2-6041 ETQVSREY09YJN12-9156 ETQVSREY10YJN6-9918 ETQVSRIEY11YJN2-9921 ETQVSREY12YJN7-11382 ETQVSREY13YJN1-11381 ETQVSREYKor consensus QVDPDLADKLIHLHYFDCFSDSAIRHAILGRLVRPKCEYQAGHNKVGSLQYLALTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH

97 120 140 160 180 192

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ +

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

96-51

Figure 2 Sequential alignment of the amino acids of the Vif protein over 20 years All sequences that demonstrated small in-frame deletionsgross deletions and stop codons are depicted together with the baseline and last sequences of each patient Patients 90-18 demonstrated a9 bp deletion (positions 182ndash184) in April 1998 and patient 92-13 demonstrated a 12 bp deletion (positions 185ndash188) in May 2005 Patient90-05 consistently demonstrated a 9 bp insertion (RQTRAR-RAR-NGASRP) at the same position at the terminus of vpr beginning in August2000 (data not shown) The code 92LSH3-6951 (sampling year initials of the patient sampling month and sequence number) denotes thatthe sampling date is March 1992 and the sequence number is 6951 in patient LSH (90-18) The dot (sdot) hyphen (-) and asterisk (lowast) denotesequence identity deletions and premature stop codons respectively compared with the Korean consensus [20] In the right column theplus (+) and minus (minus) signs denote the presence or absence of the corresponding therapy respectively

10 Evidence-Based Complementary and Alternative Medicine

61 69 112 120 208 216 264 272 520 528Patient WT nucleotide TGGAAAAGT TGGGTCTAT TGGGGTCTG TGGAGGAAA TGGAACAAG Hypermut 20

sequence no P value90-05 10LSK4-9168 G A lt005

12LSK4-10131 A A AA A A lt000113LSK5-11470 A A A 015 13LSK5-11472 A AA lt001

90-18 08LSH5-5645 AA 019 08LSH11-5694 A 05710LSH4-9192 AA 013

90-54 07KJin3-5961 A 00693-04 94KYR12-9441 AA AG 009

07KYR8-5166 A A AA A AG lt000107KYR8-4846 A AA G 00507KYR8-5167 A A A AG 00507KYR8-5168 A A AG 02907KYR8-4845 AA G 005

93-60 03KMH11-5725 AA A 015

WT amino acid W K S W V Y W G L W R K W N K21 22 23 38 39 40 70 71 72 89 90 91 174 175 176

Figure 3 Positions of the premature stop codons in the 15 vif genes from five patients All stop codons resulted from GG rarr GA orGG rarr AG changes APOBEC3G exhibits an intrinsic preference for the second cytosine in a 51015840CC dinucleotide motif leading to 51015840GG-to-AG mutations [38]

In total five of our patients demonstrated in-frame deletionsof the vif gene of the 75 KSB-infected patients who weretreated with KRG Also in our present study cohort fivepatients including 90-18 and 92-13 demonstrated deletedvif genes (small in-frame and gross deletions) during KRGintake whereas all of our 10 LTSP patients demonstrateddeletions in the nef gene (119875 lt 005) [14] In additioncompared with the reported deletions of the nef and 51015840LTRgag genes the locations of the deleted vif gene werepositioned within a narrow range at the terminus [15 16 23]

Regarding gross deletions to date only two patientshave demonstrated gross deletions in the vif gene [17 18]and only one patient has demonstrated the insertion of twoamino acids and these patients were part of a nonprogress-ing mother-child pair [28] However in our present studyof the 10 LTSP patients who were assessed four patientsdemonstrated gross deletion in the vif gene during KRGintake prior to receiving HAART (15) This frequency issignificantly lower than the previously reported incidence of10 of 10 patients demonstrating gross deletions in the nef(187) and 51015840 LTRgag genes (376 119875 lt 001) [27 28]The frequency of deletion in the nef gene was reported tosignificantly decrease during KRG plus HAART (119875 lt 001)[16] In contrast to previous studies on the nef and gag genes[16 29] the detection of in-frame deletions in the vif genewas not suppressed in the patients receiving HAART in ourpresent cohort The reasons for this include the following (1)the proportion and frequency (approximately two-thirds) ofdeleted vif genes after the first occurrence were much higherthan in the nef and 51015840 LTRgag genes (lt20 and 30 resp)(2) small deletions in the vif gene occurred as a single bandwhereas gross deletions in the nef and 51015840 LTRgag genes weremainly detected as two bands (wild type and short) per PCRreaction and (3) the number of amplicons indicating PSCdueto G-to-A hypermutation was significantly higher in patients

receiving KRG plus HAART than patients receiving onlyKRG This phenomenon is consistent with our previous datain 51015840 LTRgag gene [29] However the frequency of G-to-Ahypermutationwas significantly lower in vif gene than that in51015840 LTRgag gene [29] Thus for the vif gene the detection ofsmall deletionsmight be less affected by limit-diluting effectsas has been shown for the nef gene [29] Taken together theseadditional defects in the vif genes in the same patients mightbe related to the replicative impairment in vif defective HIV-1 which ultimately results in defects in the synthesis of viralDNA [30]

In our present study it appears that the frequency ofhypermutation was higher in patients who maintained goodcompliance with HAART during HAART plus KRG (90-0590-18 and 93-04) than in the patients who demonstratedpoor compliance (Figure 1) In our extended data analysis124 (60485) 31 (5161) 30 (19617) and 09 (1108)of vif genes during KRG plus HAART HAART aloneKRG alone and control revealed PSC respectively Thesedata indicate the presence of synergistic effect of KRG plusHAART on PSC (119875 lt 001) There was no case that smalldeletions were induced by HAART alone About 6ndash43 ofthe integrated proviral pol gene is hypermutated by HAART[22 31 32] although hypermutated viral genomes are notpresent in plasma [32]

ApoBec3G-induced hypermutation in LTNPs has beenreported previously [33] Indeed it has been reported alsothat LTNP patients harbor PSC-containing vif genes morefrequently than progressors [25] although the reportedfrequencies of defective genes vary among studies [34] Inour current analyses vif gene-containing PSC due to G-to-A hypermutation was found to be significantly higher duringKRG plus HAART than only KRG intake prior to HAARTThis finding is consistent with our previous data obtainedfrom a cohort of the Korean hemophiliacs (21 incidence

Evidence-Based Complementary and Alternative Medicine 11

of G-to-A hypermutation while receiving only KRG versus98 on KRG plus HAART unpublished data) and otherobservations regarding the pol gene [22]

Previous studies report that certain vif mutations such asL81M R132S S130I and the insertion of two amino acids areassociatedwith slow progression or low viral loads [17 18 28]In our present study L81M [35] and R132S were consistentlydetected in patient 96-51 and patients 87-05 and 96-51respectively although both mutations were found in only in2 and 3 of 169 Korean patients respectively Although thevif gene is highly conserved in HIV-1 genomes the recoveryof hypermutants might be severely underestimated in ourpresent analysis because of the primer mismatching andthe difficulties involved in detection following amplification[36 37] Overall our current data suggest that small in-frame deletion and PSCs are associated with KRG intake andHAART respectively although further studies are needed

5 Conclusions

Our data suggest that HAART and KRG intake might inducePSCs and small in-frame deletions respectively

Conflict of Interests

The authors have no conflict of interests to declare

Acknowledgments

This work was supported by a grant from the Korean Societyof Ginseng (2010ndash2012) The authors thank Professor Seung-Chul Yun the Division of Biostatistics University of UlsanCollege of Medicine Asan Medical Center for the statisticalconsultation

References

[1] E Ernst ldquoPanax ginseng an overview of the clinical evidencerdquoJournal of Ginseng Research vol 34 no 4 pp 259ndash263 2010

[2] K T Choi ldquoBotanical characteristics pharmacological effectsand medicinal components of Korean Panax ginseng C AMeyerrdquoActa Pharmacologica Sinica vol 29 no 9 pp 1109ndash11182008

[3] S J Fulder ldquoGinseng and the hypothalamic-pituitary control ofstressrdquo The American Journal of Chinese Medicine vol 9 no 2pp 112ndash118 1981

[4] V K Singh S S Agarwal and B M Gupta ldquoImmunomodula-tory activity of Panax ginseng extractrdquo Planta Medica vol 50no 6 pp 462ndash465 1984

[5] X Song and S Hu ldquoAdjuvant activities of saponins fromtraditional Chinese medicinal herbsrdquo Vaccine vol 27 no 36pp 4883ndash4890 2009

[6] D G Yoo M C Kim M K Park et al ldquoProtective effectof ginseng polysaccharides on influenza viral infectionrdquo PLoSONE vol 7 no 3 Article ID e33678 2012

[7] H X Sun Y P Ye H J Pan and Y J Pan ldquoAdjuvant effectof Panax notoginseng saponins on the immune responses toovalbumin in micerdquo Vaccine vol 22 no 29-30 pp 3882ndash38892004

[8] J Sun S Hu and X Song ldquoAdjuvant effects of protopanaxadioland protopanaxatriol saponins from ginseng roots on theimmune responses to ovalbumin in micerdquo Vaccine vol 25 no6 pp 1114ndash1120 2007

[9] P W Hunt ldquoHIV and inflammation mechanisms and conse-quencesrdquo Current HIVAIDS Reports vol 9 no 2 pp 139ndash1472012

[10] T Yayeh K H Jung H Y Jeong et al ldquoKorean red ginsengsaponin fraction downregulates proinflammatory mediators inLPS stimulated RAW264 7 cells and protects mice againstendotoxic shockrdquo Journal of Ginseng Research vol 36 no 3 pp263ndash269 2012

[11] A T Smolinski and J J Pestka ldquoModulation of lipopolysaccha-ride-induced proinflammatory cytokine production in vitroand in vivo by the herbal constituents apigenin (chamomile)ginsenoside Rb1 (ginseng) and parthenolide (feverfew)rdquo Foodand Chemical Toxicology vol 41 no 10 pp 1381ndash1390 2003

[12] A Rhule S Navarro J R Smith and D M ShepherdldquoPanax notoginseng attenuates LPS-induced pro-inflammatorymediators in RAW2647 cellsrdquo Journal of Ethnopharmacologyvol 106 no 1 pp 121ndash128 2006

[13] J M Brenchley D A Price T W Schacker et al ldquoMicrobialtranslocation is a cause of systemic immune activation inchronic HIV infectionrdquo Nature Medicine vol 12 no 12 pp1365ndash1371 2006

[14] Y K Cho J Y Lim Y S Jung S K Oh H J Lee and H SungldquoHigh frequency of grossly deleted nef genes in HIV-1 infectedlong-term slow progressors treated with Korean red ginsengrdquoCurrent HIV Research vol 4 no 4 pp 447ndash457 2006

[15] Y K Cho and Y S Jung ldquoHigh frequency of gross deletions inthe 51015840 LTR and gag regions in HIV type 1-infected long-termsurvivors treated with Korean red ginsengrdquo AIDS Research andHuman Retroviruses vol 24 no 2 pp 181ndash193 2008

[16] Y K Cho Y S Jung andH S Sung ldquoFrequent gross deletion intheHIV type 1 nef gene in hemophiliacs treatedwith korean redginseng inhibition of detection by highly active antiretroviraltherapyrdquo AIDS Research and Human Retroviruses vol 25 no 4pp 419ndash424 2009

[17] V Sandonıs C Casado T Alvaro et al ldquoA combination ofdefective DNA and protective host factors are found in a setof HIV-1 ancestral LTNPsrdquo Virology vol 391 no 1 pp 73ndash822009

[18] H R Rangel D Garzaro A K Rodrıguez et al ldquoDeletioninsertion and stop codon mutations in vif genes of HIV-1 infecting slow progressor patientsrdquo Journal of Infection inDeveloping Countries vol 3 no 7 pp 531ndash538 2009

[19] Y K Cho J E Kim and B T Foley ldquoPhylogenetic analysisof near full-length HIV-1 genomic sequences in 21 Koreanindividualsrdquo AIDS Research and Human Retroviruses vol 29no 4 pp 738ndash743 2013

[20] Y K Cho Y S Jung J S Lee and B T Foley ldquoMolecu-lar evidence of HIV-1 transmission in 20 Korean individu-als with haemophilia phylogenetic analysis of the vif generdquoHaemophilia vol 18 no 2 pp 291ndash299 2012

[21] P P Rose and B T Korber ldquoDetecting hypermutations inviral sequences with an emphasis on GrarrA hypermutationrdquoBioinformatics vol 16 no 4 pp 400ndash401 2000

[22] S Fourati S Lambert-Niclot C Soulie et al ldquoHIV-1 genomeis often defective in PBMCs and rectal tissues after long-termHAART as a result of APOBEC3 editing and correlates with thesize of reservoirsrdquo Journal of Antimicrobial Chemotherapy vol67 no 10 pp 2323ndash2326 2012

12 Evidence-Based Complementary and Alternative Medicine

[23] U Wieland A Seelhoff A Hofmann et al ldquoDiversity of thevif gene of human immunodeficiency virus type 1 in UgandardquoJournal of General Virology vol 78 no 2 pp 393ndash400 1997

[24] P Sova M van Ranst P Gupta et al ldquoConservation of an intacthuman immunodeficiency virus type 1 vif gene in vitro and invivordquo Journal of Virology vol 69 no 4 pp 2557ndash2564 1995

[25] T Yamada and A Iwamoto ldquoComparison of proviral accessorygenes between long-term nonprogressors and progressors ofhuman immunodeficiency virus type 1 infectionrdquo Archives ofVirology vol 145 no 5 pp 1021ndash1027 2000

[26] K Tominaga S Kato M Negishi and T Takano ldquoA high fre-quency of defective vif genes in peripheral blood mononuclearcells from HIV type 1-infected individualsrdquo AIDS Research andHuman Retroviruses vol 12 no 16 pp 1543ndash1549 1996

[27] Y K Cho H Sung I G Bae et al ldquoFull sequence of HIV type 1Korean subtype B in anAIDS case with atypical seroconversionTAAAA at TATA boxrdquoAIDS Research andHuman Retrovirusesvol 21 no 11 pp 961ndash964 2005

[28] L Alexander M J Aquino-DeJesus M Chan and W AAndiman ldquoInhibition of human immunodeficiency virus type 1(HIV-1) replication by a two-amino-acid insertion in HIV-1 Viffrom a nonprogressing mother and childrdquo Journal of Virologyvol 76 no 20 pp 10533ndash10539 2002

[29] YK Cho Y S JungH S Sung andCH Joo ldquoFrequent geneticdefects in the HIV-1 51015840LTRgag gene in hemophiliacs treatedwith Korean red ginseng decreased detection of genetic defectsby highly active antiretroviral therapyrdquo Journal of GinsengResearch vol 35 no 4 pp 413ndash420 2011

[30] M Nascimbeni M Bouyac F Rey B Spire and F Clavel ldquoThereplicative impairment of Vif-mutants of human immunodefi-ciency virus type 1 correlates with an overall defect in viral DNAsynthesisrdquo Journal of General Virology vol 79 no 8 pp 1945ndash1950 1998

[31] J P Vartanian M Henry and S Wain-Hobson ldquoSustainedGrarrA hypermutation during reverse transcription of an entirehuman immunodeficiency virus type 1 strain Vau group Ogenomerdquo Journal of General Virology vol 83 no 4 pp 801ndash8052002

[32] T L Kieffer P Kwon R E Nettles Y Han S C Ray andR F Siliciano ldquoGrarrA hypermutation in protease and reversetranscriptase regions of human immunodeficiency virus type 1residing in resting CD4+ T cells in vivordquo Journal of Virology vol79 no 3 pp 1975ndash1980 2005

[33] B Wang M Mikhail W B Dyer J J Zaunders A D Kelleherand N K Saksena ldquoFirst demonstration of a lack of viralsequence evolution in a nonprogressor defining replication-incompetent HIV-1 infectionrdquo Virology vol 312 no 1 pp 135ndash150 2003

[34] G Hassaine I Agostini D Candotti et al ldquoCharacterizationof human immunodeficiency virus type 1 vif gene in long-termasymptomatic individualsrdquoVirology vol 276 no 1 pp 169ndash1802000

[35] F A de Maio C A Rocco P C Aulicino R Bologna AMangano and L Sen ldquoUnusual substitutions in HIV-1 Vif fromchildren infected perinatally without progression to AIDS formore than 8 years without therapyrdquo Journal of Medical Virologyvol 84 no 12 pp 1844ndash1852 2012

[36] M Janini M Rogers D R Birx and F E McCutchan ldquoHumanimmunodeficiency virus type 1 DNA sequences geneticallydamaged by hypermutation are often abundant in patientperipheral blood mononuclear cells and may be generated

during near-simultaneous infection and activation of CD4+ Tcellsrdquo Journal of Virology vol 75 no 17 pp 7973ndash7986 2001

[37] G H Kijak L M Janini S Tovanabutra et al ldquoVariablecontexts and levels of hypermutation inHIV-1 proviral genomesrecovered from primary peripheral blood mononuclear cellsrdquoVirology vol 376 no 1 pp 101ndash111 2008

[38] E W Refsland J F Hultquist and R S Harris ldquoEndogenousorigins of HIV-1 G-to-A hypermutation and restriction in thenonpermissive T cell line CEM2nrdquo PLoS Pathogens vol 8 no7 Article ID e1002800 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 2: Effects of Korean Red Ginseng and HAART on vif Gene in 10 Long

2 Evidence-Based Complementary and Alternative Medicine

Table 1 Characteristics of 10 long-term slow progressors

Patient code Year ofdiagnosis

Date of AIDSdiagnosis

CD4+ Tcells120583La

Viral load(copymL)a

Follow-up from Dxbto HAART (Mo) HAARTc Duration of

HAART87-05 1987 NA 256 115000 314 None None89-17 1989 Jul 02 106 162000 188 Mar 05 6290-05 1990 May 08 103 94376 221 July 08 5890-18 1990 Mar 04 112 124000 167 Apr 04 10490-50 1990 Mar 07 116 244000 197 May 07 6891-20 1991 Aug 07 55 3886 214 Aug 07 6592-13 1992 June 07 47 5800 181 Jun 07 4593-04 1993 NA 297 656000 166 Nov 06 993-60 1993 NA 109 121000 182 May 08 4796-51 1996 NA 169 386543 164 Dec 09 36aCD4+ T cell count and viral load measured just before the initiation of HAARTbDx diagnosiscHAART highly active antiretroviral therapy

In addition we found that HAART increases the frequency ofpremature stop codons (PSCs) in the vif gene

2 Materials and Methods

21 Patients Ten patients whose annual decrease in CD4+T cells was lt20 cells120583L were diagnosed as LTSP patients[14]Their clinical characteristics including changes inCD4+T-cell count and RNA copy number KRG therapy andfrequent genetic defects in the nef and 51015840 LTRgag geneshave been previously described [14 15] However the follow-up period of the present study was extended to include theKRG plus HAART periodThe control patients (119899 = 21) wereselected from 169 patients [20] who had not been exposedto KRG or any antiretroviral therapies (eg AZT) at thetime of sampling and whose PBMCs were available for geneamplification This study was approved by the InstitutionalReview Board of the Asan Medical Center

22 KRG Intake KRG powder was manufactured by theKorea Ginseng Corporation (Daejeon Korea) from theroots of a 6-year-old Panax ginseng Meyer red ginseng andharvested in the Republic of Korea KRG was made bysteaming fresh ginseng at 90ndash100∘C for three hours anddrying at 50ndash80∘C KRG powder was prepared from groundred ginseng (500mg KRGcapsule) and analyzed using high-performance liquid chromatography

23 Amplification of the vif Gene Nested PCR was used toamplify the proviral vif gene from the patientsrsquo PBMCs aspreviously described [14ndash16 19] Total RNA was extractedfrom 300 120583L serum using theQIAampUltra Sense Viral RNAkit (Qiagen Hilden Germany) as previously described [20]

24 Determination of G-to-A Hypermutation Sequenceswere analyzed using the Hypermut 20 program(httpwwwhivlanlgovcontentsequenceHYPERMUThypermuthtml) which compares each patientrsquos sequence

to a patient-specific consensus in order to determine thefrequency and context of the G-to-A mutations [21]

25 Statistical Analysis Data are expressed as the means plusmn2 standard deviations (for continuous variables) or as countsand percentages (categorical variables) Comparing the pro-portions between groups was analyzed by using the Chi-squared test or Fisher exact test In this study 119875 lt 005 wasconsidered statistically significant

26 Accession Numbers of the Nucleotide Sequence Theinvestigated sequences were previously assigned the fol-lowing GeneBank accession numbers DQ072735 JF957893JQ067069-76 AY581367 JF957902 JQ248188-201 AY581377AY581378 JQ248228-50 JF957922 AY581382-5 DQ072750-51 JF957924 JQ248256-92 DQ072759-60 AY581386-89JF957933 JQ248313-29 AY581394-95 JF957940 JQ248339-55 AY581398-AY581399 JQ327719 JQ327794-97 JF958044-45 JQ066879-927 AY581341-44 JF957976 JQ268920-39AY581412-15 JQ268940-61 AF462782 JF957977 AY581416-18 JQ269006-32 JF957983 AY581419-20 KC247158-315 andKF270357-459

3 Results

31 Effects of KRG during the Follow-Up Period prior toHAART Administration Ten LTSP patients received follow-up without HAART for 199 plusmn 45 months (166 years range164ndash314months) followingHIV-1 diagnosis (Table 1) Duringthis period KRG therapy was administered for 173 plusmn 34months and thereafter KRG plus HAART was administeredfor 55 plusmn 26 months The amounts of KRG administeredduring these periods were 14398 plusmn 5775 and 5648 plusmn 3677 grespectively Patient 89-17rsquos compliance with KRG was poorand the amount supplied prior to HAART therapy was theleast (5076 g) of the 10 enrolled LTSP patients Patient 87-05began to take KRG in December 1991 though his compliancewas also poor He has taken KRG since June 1994 but his

Evidence-Based Complementary and Alternative Medicine 3

CD4+ T-cell count remained low (Figure 1) The remainingnine patients progressed to AIDS demonstrating CD4+ T-cell counts of lt200 cells120583L (Table 1)

32 Effects of HAART on PSC The 10 LTSP patients weincluded in our present study were untreated before Decem-ber 1991 were treated with only KRG between 2004 and2009 and have been treated with KRG plus HAART since2009 (Figure 1) We analyzed 432 vif genes over 20 years inthese 10 LTSP patients Among these 15 vif genes (35)demonstrated PSC In total 275 and 157 vif genes wereobtained during KRG and KRG plus HAART respectivelyEach group demonstrated 3 (11) and 12 (76) vif geneswith PSCs respectively When receiving only KRG threepatients (90-50 93-04 and 93-60) demonstrated PSC at 14years 1 year and 9months and 7 years after starting to receiveKRG respectively The frequency was significantly higherwhen receivingKRGplusHAART thanKRGalone (119875 lt 001Table 2) This suggests that HAART itself might induce G-to-A hypermutation thereby resulting in PSC and possiblylethal hypermutations This is consistent with the findings ofother studies [22] However we found no difference in thefrequency of PSCs between our KRG (3 of 275 genes) andcontrol groups (1 of 106 genes) Interestingly of the 15 vifgenes that demonstrated PSCs in our present analysis elevendid not satisfy the criteria of Hypermut 20 in comparisonwith the earliest sequences obtained from each patient (119875 lt005 Figure 3)

33 Effects of KRG and HAART on Genetic Defects Wedetected 11 gross deletions in 432 amplicons obtained fromour 10 LTSP patients (Table 2 Figure 2) No deleted genesincluding gross deletions were detected in the controlpatients In total five patients demonstrated gross deletionsafter gt19 months of KRG intake Four patients demonstratedgross deletions during KRG intake prior toHAART althoughthe frequency was very low (15) Specifically a sequencecontaining a gross deletion and duplicationrecombination(KC247159) was identified in patient 87-05 A 362-base pair(bp) deletion and 1 bp insertion (KC247195) 309 bp deletion(JQ327719) and 870 bp deletion (JQ327722) were detectedas short bands together with the wild-type genotype after139 months 115 months and 125 months of KRG intakein patients 90-05 91-20 and 93-60 respectively (patient90-05 demonstrated 9 bp insertions in 16 amplicons after104 months of KRG intake including KC247191 JQ248236DQ295192 and JQ248237-50) In addition two patientsdemonstrated gross deletions during KRG plus HAARTSpecifically patient 93-04 demonstrated 386 bp deletionsin one-third of amplicons after 14 years of KRG intakePatient 93-60 demonstrated six deletions (including 374 bp[JQ327723] and 401 bp deletions [KC247314] Figure 2 andTable 2) Each deletion was one (JQ327723) of 3 ampliconsand one (KC247314) of 2 amplicons obtained between August2008 and October 2010 and all 4 amplicons respectively(Figure 1) This frequency is the highest among the relatedstudies to date although we found no significant differencesin terms of the frequencies of gross deletions between patients

who received KRG (15) or KRG plus HAART (45Table 2) in our present study If both PSCs and gross deletionsare only defined as nonfunctional vif genes the proportion ofnonfunctional genes is 25 (7 of 275 genes) during KRG and121 (19 of 157 genes) during KRG plus HAART (119875 lt 001)

34 Position and Frequency of PSCs in the vif Gene Thereare 8 tryptophan residues in the Vif protein In this study15 vif genes from 5 patients (90-05 90-18 90-50 93-04and 93-60 Figure 3) demonstrated PSCs The frequencies ofthe PSCs in 5 tryptophan residues (21 38 70 89 and 174)were 3 7 13 2 and 7 respectively In the control group onepatient demonstrated stop codons at residues 21 70 and 174(JQ066980)

35 Small in-FrameDeletions Are Associated with KRG IntakeWe did not find any specific changes in the nucleotide oramino acid sequences due to KRG intake However inter-estingly two of our patients demonstrated small deletionsduring KRG intake one patient (90-18) demonstrated a 9 bpin-frame deletion (detected in 15 of 26 amplicons) at aminoacid positions 182ndash184 in April 1998 and another patient(92-13) demonstrated a 12 bp deletion (detected in 14 of 16amplicons) at positions 186ndash189 in May 2005 (Figure 2) Thefrequencies of these deletions were similar between patientswho received KRG (105 29 of 275 genes) and KRG plusHAART (159 25 of 157 genes) However the frequencyof these deletions was zero (0 of 52 genes as determinedusing RT-PCR) during pre-KRG and 105 (29 of 275) duringKRG only without HAART (119875 lt 001) These deletionsmanifested after 63 and 120 years of KRG intake in patients90-18 and 92-13 respectively Interestingly patient 90-18 alsodemonstrated a 6 bp deletion in the nef gene after 11 yearsof KRG intake with HAART although we did not categorizethis as a gross deletion [14] Deletions were conservedduring KRG plus HAART therapy (Figure 2) and compriseda higher proportion thanwild-type alleles In particular threeamplicons containing PSC also demonstrated small deletionsin patient 90-18 The presence of both wild-type and mutantalleles in the same samples differed from the findings forthe samples obtained from patients who were not treatedwith KRG in which all amplicons contained deletions in fivepatients (data not shown) In addition there were no suchdeletions in the control group (0 of 106 genes)

36 Overall Rate of Defective Genes Whenwe included smallin-frame deletions of defective genes the overall proportionsof defective genes were 131 (36 of 275 genes) when receivingonly KRG and 268 (42 of 157 genes) when receiving KRGplus HAART (119875 lt 001) In our current study the frequencyof defective genes identified in PBMCs obtained from HIV-1-infected patients prior to receiving HAART was similar tothe results of previous reports byWieland et al (10) [23] andSova et al (13) [24] although exceptional cases have beenreported byYamada and Iwamoto (205) [25] andTominagaet al (31) [26] Overall the proportions of defective genesidentified in our present study were significantly lower thanthe proportions of defective nef (94 of 479 genes including

4 Evidence-Based Complementary and Alternative Medicine

0

1

2

3

4

5

6

7

0100200300400500600700

87-05Korean Red Ginseng (KRG) KRG KRG KRG

Jun

87Se

p 89

Apr

91

Nov

91

Nov

92

Dec

93

Jun

94D

ec 9

4O

ct 9

5O

ct 9

6A

pr 9

7A

pr 9

8O

ct 9

8A

pr 9

9O

ct 9

9M

ar 0

0Fe

b 01

Jul 0

1Se

p 02

Sep

03O

ct 0

4M

ar 0

5M

ay 0

5O

ct 0

5M

ay 0

6O

ct 0

6Ja

n 07

May

07

Aug

07

Oct

07

Feb

08A

ug 0

8D

ec 0

8Ju

n 09

Dec

09

Jul 1

0M

ar 1

1Se

p 11

Feb

12A

ug 1

2D

ec 1

2

CD4

+ T

cells

(120583

L)

CD4+ T cells (120583L)

minus100 0

1

2

3

4

5

6

7

0200400600800

1000120014001600 89-17

KRG KRG HAARTKRGKRG KRG

Aug

89

Jan

90Se

p 90

Apr

91

Dec

91

Jan

92M

ar 9

2Ju

n 92

Nov

92

Feb

93A

pr 9

3Ju

l 93

Oct

93

Apr

94

Oct

94

Sep

95Ju

l 96

Oct

96

Mar

97

Aug

97

Apr

98

Jun

99N

ov 9

9M

ar 0

0Se

p 00

Feb

01Ju

l 01

Jan

02M

ar 0

2Se

p 02

Feb

03M

ay 0

3Ja

n 04

Aug

04

Mar

05

Dec

05

Oct

06

Nov

06

Feb

07A

pr 0

7A

ug 0

7D

ec 0

7Ju

l 08

May

09

May

10

Mar

12

RNA

copy

(log

mL)

minus200

0

1

2

3

4

5

6

7

050

100150200250300350400450 90-05

KRG HAARTKRG

lowast lowast lowast

Jul 9

1N

ov 9

1N

ov 9

2A

pr 9

3O

ct 9

3Ju

n 94

Jan

95M

ay 9

5A

ug 9

6A

ug 9

7A

pr 9

8M

ar 9

9A

ug 0

0Fe

b 01

May

01

Mar

02

Aug

02

Jan

03M

ar 0

3Ju

n 03

Oct

03

Mar

04

Aug

04

Feb

05Ju

l 05

Oct

05

Jan

06Ju

l 06

Oct

06

Feb

07M

ay 0

7A

ug 0

7O

ct 0

7Ja

n 08

Apr

08

May

08

Aug

08

Nov

08

Apr

09

Oct

09

Apr

10

Oct

10

Apr

11

Oct

11

Apr

12

Nov

12

May

13

CD4

+ T

cells

(120583

L)

0

1

2

3

4

5

6

7

0100200300400500600700800900

1000 90-18

HAARTKRGKRG KRG KRGKRGKRGKRG KRG

Aug

90

Feb

91A

ug 9

1D

ec 9

1M

ar 9

2Ju

n 92

Nov

92

Mar

93

May

93

Jun

93O

ct 9

3Fe

b 94

May

94

Aug

94

Nov

94

Feb

95M

ay 9

5O

ct 9

5Fe

b 96

May

96

Dec

96

Mar

97

Mar

97

Jul 9

7M

ar 9

8A

pr 9

8Se

p 98

Feb

99A

pr 9

9Ju

n 99

Nov

99

Mar

00

Jul 0

0A

ug 0

0O

ct 0

0M

ar 0

1Ju

l 01

Dec

01

Apr

02

Oct

02

Jan

03M

ay 0

3O

ct 0

3Ju

l 04

Dec

04

Mar

05

Feb

06Ju

n 06

Jul 0

6A

ug 0

6N

ov 0

6Ja

n 07

Apr

07

Jun

07O

ct 0

7N

ov 0

7D

ec 0

7M

ay 0

8N

ov 0

8Fe

b 09

Apr

09

Oct

09

Apr

10

Oct

10

Oct

11

Jun

12D

ec 1

2

minus100

lowastlowast lowast

RNA

copy

(log

mL)

CD4

+ T

cells

(120583

L)

0

1

2

3

4

5

6

7

0

200

400

600

800

1000

1200 90-50HAARTKRGKRGKRGKRG

Dec

90

Jul 9

1Ja

n 92

Jul 9

2Fe

b 93

Apr

93

Sep

93M

ar 9

4Se

p 94

Apr

95

Oct

95

Apr

96

Nov

96

May

97

Nov

97

Jun

98N

ov 9

8M

ay 9

9N

ov 9

9Se

p 00

Jul 0

1N

ov 0

1Ju

n 02

Dec

02

Mar

03

Jun

03Se

p 03

Mar

04

Aug

04

Jan

05A

ug 0

5Ja

n 06

Jul 0

6Se

p 06

Mar

07

May

07

Jun

07Ju

l 07

Oct

07

Nov

07

Jul 0

9D

ec 0

9A

pr 1

0Ju

l 10

Jan

11A

pr 1

1D

ec 1

1Ju

l 12

Jan

13

lowast

CD4+

T ce

lls (120583

L)

minus200 0

1

2

3

4

5

6

7

0

200

400

600

800

1000

120091-20

KRG HAARTKRGKRG

Jun

91D

ec 9

1M

ar 9

2M

ay 9

2Ja

n 93

May

93

Dec

93

Jul 9

4N

ov 9

4D

ec 9

4Fe

b 95

Mar

95

May

95

Oct

95

Apr

96

Aug

96

Mar

97

Jul 9

7A

pr 9

8N

ov 9

8M

ay 9

9N

ov 9

9M

ar 0

0A

pr 0

0Ju

l 00

Dec

00

Mar

01

Jul 0

1N

ov 0

1M

ar 0

2A

ug 0

2Ja

n 03

Mar

03

Aug

03

Jan

04Ju

l 04

Jan

05Ju

l 05

Jan

06Ju

l 06

Dec

06

May

07

Jul 0

7A

ug 0

7Se

p 07

Nov

07

Jan

08A

pr 0

8Ju

l 08

Dec

08

Oct

10

Apr

11

Jan

12Ju

l 12

Jan

13

minus200

RNA

copy

(log

mL)

CD4+

T ce

lls (120583

L)

0

1

2

3

4

5

6

7

050

100150200250300350400450

92-13KRG HAART HAART

KRGKRGKRGKRGKRG

Expired in 2011

May

92

Nov

92

May

93

Jul 9

3Ja

n 94

Oct

94

Dec

94

Feb

95A

pr 9

5O

ct 9

5A

pr 9

6N

ov 9

6Fe

b 97

Jun

97A

pr 9

8Ja

n 99

Oct

99

Feb

01D

ec 0

1Ju

l 02

Dec

02

Mar

03

Jun

03O

ct 0

3M

ay 0

4N

ov 0

4M

ay 0

5Ju

l 05

Feb

06A

ug 0

6N

ov 0

6A

pr 0

7Ju

n 07

Jul 0

7A

ug 0

7Se

p 07

Dec

07

May

08

Aug

08

Dec

08

May

09

Dec

09

Apr

10

Jun

10Ja

n 11

CD4

+ T

cells

(120583

L)

minus500

1

2

3

4

5

67

0100200300400500600700800900 93-04

KRG HAARTKRG KRG KRGKRG

Feb

93M

ar 9

3Ju

l 93

Dec

93

Feb

94Ju

n 94

Dec

94

May

95

Nov

95

Feb

96Se

p 96

Jan

97Fe

b 97

Jul 9

7A

ug 9

7A

pr 9

8Ju

l 98

Sep

00D

ec 0

0M

ar 0

1Ju

n 01

Sep

01Ju

n 02

Jul 0

2Ja

n 03

May

03

Oct

03

Mar

04

Aug

04

Jan

05M

ay 0

5Ju

n 05

Aug

05

Oct

05

Mar

06

Jul 0

6N

ov 0

6M

ar 0

7A

ug 0

7

lowastlowast

RNA

copy

(log

mL)

CD4

+ T

cells

(120583

L)

RNA

copy

(log

mL)

RNA copy (logmL)

RNA

copy

(log

mL)

RNA

copy

(log

mL)

RNA

copy

(log

mL)

if if

if

if

ifif

ifif

Sampling date Sampling date

Sampling date Sampling date

Sampling date Sampling date

Sampling date Sampling dateCD4+ T cells (120583L)RNA copy (logmL)

CD4

+ T

cells

(120583

L)

Figure 1 Continued

Evidence-Based Complementary and Alternative Medicine 5

0

1

2

3

4

5

6

7

0100200300400500600700800

93-60

HAARTKRGKRG

Apr

94

Nov

95

May

96

Nov

96

Apr

97

Jun

97Ju

n 99

Nov

99

Nov

00

Jul 0

1N

ov 0

1Ja

n 02

Sep

02Fe

b 03

May

03

Nov

03

May

04

Mar

05

Sep

05A

pr 0

6O

ct 0

6N

ov 0

6A

pr 0

7Ju

l 07

Aug

07

Oct

07

Jan

08M

ay 0

8M

ay 0

8Ju

l 08

Aug

08

Dec

08

May

09

Dec

09

Oct

10

Apr

11

Apr

12

lowast

0

1

2

3

4

5

6

7

0

200

400

600

800

1000

120096-51

KRG HAARTKRGKRG

Jul 9

6Ja

n 97

Jul 9

7A

pr 9

8N

ov 9

8M

ay 9

9Ju

n 00

Oct

00

Sep

01A

pr 0

2D

ec 0

2O

ct 0

3Fe

b 04

Aug

04

Feb

05A

ug 0

5N

ov 0

6Ju

l 06

Oct

06

Feb

07A

ug 0

7M

ar 0

8A

ug 0

8Fe

b 09

Jul 0

9D

ec 0

9M

ay 1

0Ju

n 10

Apr

11

Oct

11

Jul 1

2Ja

n 13

RNA

copy

(log

mL)

CD4

+ T

cells

(120583

L)

Sampling dateCD4+ T cells (120583L)RNA copy (logmL)

RNA

copy

(log

mL)

CD4

+ T

cells

(120583

L)

Sampling dateCD4+ T cells (120583L)RNA copy (logmL)

if if

Figure 1 Changes in the CD4+ T-cell count plasma viral load and genetic defects in terms of Korean red ginseng (KRG) intake and highlyactive antiretroviral therapy (HAART) The durations of KRG intake and HAART are indicated by the bars Solid and dotted lines denotegood (gt90) and poor (lt90) compliance according to self-administered responses respectively The upward arrow (uarr) downward arrow(darr) plus sign (+) and asterisk (lowast) denote the sequences of the vif gene gross deletions 3- and- 6-base pair (bp) in-frame insertions and stopcodons respectively

Table 2 Distribution of defective vif gene

Patient On-KRG On-HAARTKRGNo of genes Stop codon gΔ sΔ No of genes Stop codon gΔ sΔ

87-05 20 1 ND ND ND ND89-17 9 9 0 0 090-05 37 1 18 4 0 090-18 35 15 20 3a 0 12a

90-50 14 1 15 0 0 091-20 35 1 20 0 0 092-13 53 14 22 0 0 1393-04 18 1 12 5 1 093-60 28 1 1 20 0 6 096-51 26 21 0 0 0Total 275 3b 4 29 157 12b 7 25gΔ and sΔ denote gross deletion and in-frame small deletion respectivelyHAART highly active antiretroviral therapy ND not determinedaTwo out of three genes contained both stop codon and sΔb119875 lt 001119875 lt 001 for the sum of 3 kinds of defective genes (36275 versus 42157)Fifty-two vif genes at baseline obtained from serum using RT-PCR were all wild types lowastControl patients (119899 = 21) revealed premature stop codon in one outof 106 vif genes

stop codons 119875 lt 005) and 51015840 LTRgag genes (71 of 189 genes119875 lt 0001) identified in the same patients [14 15]

4 Discussion

Todate we have identified the vif gene in 194Korean patientsOf these 145 patients were infected with the Korean subcladeB ofHIV-1 (KSB) and the remaining 49 patients were infectedwith non-KSB Of these 194 patients 10 demonstrated smallin-frame deletions (3ndash15 bp) and all were diagnosed withKSB whereas no such deletions were detected in non-KSBpatients (119875 = 0068) Of the 10 patients with small deletionsthree patients were included in a group of 20 hemophiliacs

who were infected with KSB from plasma donors O and P[16 20] Interestingly plasma donors O and P revealed wild-type vif only between 1991 and 2002 (AY581320-21 JQ248127-33 JF957909 JF957921 JQ248134 and JF957935-37) In otherwords original vif gene was wild type without in-framesmall deletion In our current study only two patients (90-18 and 92-13) demonstrated 9 (119899 = 15) and 12 (119899 = 14)bp in-frame deletions in about two-thirds of the ampliconsobtained after 63 and 120 years of KRG intake respectivelywhereas five patients without KRG intake demonstrated in-frame deletions in all amplicons (data not shown) To ourknowledge these in-frame deletions are very rare and haveonly been reported in two patients in other countries [26 27]

6 Evidence-Based Complementary and Alternative Medicine

87-05

89-17

90-05

90-18

90-50

91-20

1 20 40 60 80 96Kor consensus MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKEWVYRHHYESTHPRISSEVHIPLGDAKLVITTYWGLHTGEREWHLGQGVSIEWRKKRYNT

91CSR4-10026 RGNVVD91CSR11-6922 RGNVVD92CSR5-9885 RGNVVD93CSR6-10159 RGNVVD93CSR6-10162s1 ------------------------------------------------------------------------------------------------03CSR9-805 SGGNVVD05CSR3 RGNIVVDG06CSR10-5280 RGNIVVDRG08CSR3-5334 RGNNVDG08CSR8-5279 RGNIVVD10CSR7-8126 VRGNLVVDG12CSR2-10141 VRGNLVVDG

91KJS12-8538 I92KJS3-6819 GI93KJS7-10155 IAI97KJS3-3814 IGKEI01KJS2-5667 IGKI03KJS2-745 IGAI03KJS7-775 IGAEI05KJS12-3264 YMIVGAEI08KJS5-5157 IVNGAAI10KJS5-10133 IGKAES

92LSK11-9417 K94LSK6-759 KGNPRS96LSK6-11491 AKNPIRS98LSK4-11495 KHN00LSK8-9999 KGNR03LSK6-10001s1 ------------------------------------------------------------------------------04LSK7 KKGNPRST06LSK1-3236 KKGNPVRST07LSK2-3232 KKGNPVRST08LSK1-4762 KKGIPVRST09LSK4-6143 IKNPIGRST10LSK4-9165 KGNPRS10LSK4-9168 KKGNPVRTRST12LSK4-10131 KAVKALKNPEKST 12LSK4-10132 KAKNPGRST13LSK5-11470 KKGNPGRST13LSK5-11471 KKVGNPVRST13LSK5-11472 IKANNRI

92LSH3-6951 K92LSH11-8548 TKK93LSH10-1252 94LSH2-740 95LSH5-10562 V96LSH5-10609 V98LSH4-10567 VG99LSH1-5776 VG99LSH1-5777 VG99LSH1-5778 VG99LSH6-5781 VGE99LSH6-5782 VG00LSH3-5704 KVGR00LSH3-5705 VG00LSH3-5706 VGK02LSH10-5707 VGR02LSH10-5708 VG02LSH10-5710 VG03LSH5-5650 VGG03LSH5-5651 VG03LSH5-5652 VG03LSH10-5653 VG04LSH7-4848 VGKES04LSH8-3011 V06LSH8-5647 VGES06LSH8-5649 VGES06LSH11-3238 VGIS08LSH5-5644 VG08LSH5-5645 VGR08LSH5-5646 KVGE08LSH5-5647 KVGE08LSH11-5694 KVG10LSH4-9190 KVGES10LSH4-9192 VG10LSH4-9193 VGG12LSH6-10100 VGES12LSH6-10101 VG

91KJin7-6723 N93KJin2-10024 N93KJin9-8530 NHVKINVDT01KJin7-5383 KVN03KJin6-683 KVINT04KJin8-1955 KVINDT06KJin1-3030 KVKINDT06KJin9-3012 KVINT07KJin3-5148 VNKINID07KJin3-5961 VNKKINEN10KJin4-9162 NHVKKINVT10KJin9-8176 NHVKKINVDT11KJin12-10119 KVKKINVDT13KJin1-11373 VNNVNDT

K94JWK12-5524 K97JWK K99JWK5-11441 KNE00JWK3-11453 KN00JWK12-11445 KN01JWK7-5603 KVA01JWK7-5603s1 KKNN02JWK8-11435 KVA03JWK8-677 KN05JWK1-5600 KVA06JWK7-3042 KVNAN

KKNVNVVN08JWK1-4772 KVNAN

KVA11JWK4-8993 KVA12JWK1-10125 KVA13JWK1-11366 KVA

minus minusminus minusminus minusminus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ +

Patient KRG HAART

10JWK4 9159

06JWK12 5624

91JWK6 6949

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 7

92-13

93-04

93-60

96-51

92CWS5-9398 G93CWS3-9925 GK93CWS5-6669 GK93CWS7-802 G96CWS11-5341 G99CWS10-5338 KVGE01CWS2-10532 VG03CWS6-684 VGTG03CWS10-5773 VG04CWS5-3239 VGKSTG05CWS5-5783 GTG05CWS5-5785 VGKITG06CWS2-3071 VGTG06CWS2-3203 VGKITG06CWS2-3221 VGTG06CWS2-3222 NVGTG06CWS2-3223 NVGTG06CWS8-3038 NVGKTG06CWS8-3039 NVGKTG07CWS4-3820 VGKIATG07CWS4-3821 VGKIATG07CWS4-10174 HWVGKIATG07CWS4-10175 VGKIATG07CWS4-10176 VGKITG07CWS4-10177 VGKKTG07CWS9-6252 VGTG07CWS9-6253 VGKITG07CWS9-6254 VGKIATG07CWS12-4777 VGQITG08CWS5-6214 NKVGQITG08CWS8-5160 NVGKTG08CWS8-5161 NVGKNQG08CWS12-5720 VGQITG08CWS12-5721 VGQITG08CWS12-5722 VGKQITG09CWS5-6256 VGKQTG09CWS5-6257 VGKQTG09CWS5-6258 VGKDQTG09CWS12-9108 LVGTG09CWS12-9109 VGKQITG

93KYR2-9472 VNN94KYR6-10137 VNN94KYR12-9441 VNN95KYR5-815 VGNN00KYR9-9913 VGNN02KYR7-9911 VGNN03KYR1-817 VNN06KYR7-5236 VGNN06KYR11-5249 VGNNS07KYR3-5265 VGNN07KYR3-11387s1 -------------------------------------------------------------------------------------------------07KYR8-4845 VGNN07KYR8-4846 VGNN07KYR8-5166 IVRNKN07KYR8-5167 VGNRN07KYR8-5168 VGNN

94KMH5 EI96KMH11-8534 IAEI97KMH4-9452 ISEI01KMH11-5526 ISIEI03KMH5-687 EI03KMH11-5725 ISKIEI04KMH5 EI05KMH3-5727 ISEI06KMH11-3026 ISKEI07KMH4-3281s1 --------------------------------------------------------------------EI07KMH10-5732 VNEI08KMH5-5165 VGNEI08KMH8-5427s1 ------------------------------------------------------------------------------------------------09KMH5-11475 ISEI09KMH5-11475s2 HQS----------------------10KMH10-9907s1 ------------------------------------------------------------------------------------------------11KMH4-9917 VDI12KMH4-10096 VGNEI

00YJN10-9385 TRGNMS01YJN9-4850 RGNMS03YJN10-840 VVRGNMS05YJN2-1948 VVRGNMS06YJN10-3044 VRGNMS07YJN4-3248 VVRGNMQI07YJN8-5627 VRGNMQS08YJN3-5606 VRGNMQS08YJN8-5408 VRGNPVMQS09YJN2-6041 VRGNPVMQS09YJN12-9156 VRGNVMQS10YJN6-9918 VRGNVMQS11YJN2-9921 VRGNVMS12YJN7-11382 VRGNVMQS13YJN1-11381 VRGNVMSKor consensus MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKEWVYRHHYESTHPRISSEVHIPLGDAKLVITTYWGLHTGEREWHLGQGVSIEWRKKRYNT

1

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ +minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

87-05

89-17

97 120 140 160 180 192QVDPDLADKLIHLHYFDCFSDSAIRHAILGRLVRPKCEYQAGHNKVGSLQYLALTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH

91CSR4-10026 TQRYKHISSTA91CSR11-6922 TQRYKHISSTA92CSR5-9885 TQRYKHISSTA93CSR6-10159 TQRYKHISSTA93CSR6-10162s1 ------------------------------------------------------------------------------------------------03CSR9-805 AQRYEKHISSTA05CSR3 TQRYKHISSTAK06CSR10-5280 TQRYKHISRTA08CSR3-5334 TQRYKHISSTA08CSR8-5279 TQRYKHISSTA10CSR7-8126 TQRYKHISSTIA12CSR2-10141 TQRYKHISSTIA

91KJS12-8538 92KJS3-6819 93KJS7-10155 97KJS3-3814 01KJS2-5667 03KJS3-745 03KJS7-775 05KJS12-3264 08KJS5-5157 10KJS5-10133

minus minusminus minusminus minusminus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ +

Patient Kor consensus KRG HAART

20 60 80 9640

RR

VS

VRE

VR

VRS

VRRES

VRRES

PKVPVIFRHIES

EVRRHES

VRRHES

Figure 2 Continued

8 Evidence-Based Complementary and Alternative Medicine

90-05

90-18

90-50

91-20

92LSK11-9417 YETRQE94LSK6-759 YETRPQES96LSK6-11491 GRYETRQ98LSK4-11495 YENTRPQES00LSK8-9999 YPQE03LSK6-10001s1 -------------------------------------------QRES04LSK7 YETRPQES06LSK1-3236 YEVTRPQRES07LSK2-3232 YEVTRPQES08LSK1-4762 YEVTRPQAESS09LSK4-6143 YEVTRPQES10LSK4-9165 YEVTRPQES10LSK4-9168 YEVTRPQES12LSK4-10131 NYKVTRPQRAES12LSK4-10132 YEVTRPQAESQ13LSK5-11470 PYVRRPTQETESS13LSK5-11471 YVTRPQRES13LSK5-11472 YENRTRQRES

92LSH3-6951 YRDH92LSH11-8548 GYRD93LSH10-1252 YRDR94LSH2-740 YRDR95LSH5-10562 YTRDR96LSH5-10609 YTR98LSH4-10567 YQTRRG---H99LSH1-5776 YQTRRG---RH99LSH1-5777 YQTRRHG---H99LSH1-5778 YQTRG---H99LSH6-5781 YQTRRQG---H99LSH6-5782 YQTRG---H00LSH3-5704 YQTRRG---H00LSH3-5705 YQTRRQSG---H00LSH3-5706 YQTRAG---H02LSH10-5707 YETRQG---H02LSH10-5708 YQTRG---H02LSH10-5710 YQTRRQR---H03LSH5-5650 YQTRRR---03LSH5-5651 YQTRRR---H03LSH5-5652 YQTRRER---03LSH10-5653 YQTRRQRS04LSH7-4848 YQTRRKQG---H04LSH8-3011 YR06LSH8-5647 YQTRRG---H06LSH8-5649 YQTRRG---H06LSH11-3238 YQHTRRQES08LSH5-5644 YQTRRSG---H08LSH5-5645 YQTRQAKS08LSH5-5646 YQTRRR---H08LSH5-5647 YQTRRR---H08LSH11-5694 YQTRRSRKG---H10LSH4-9190 YEQCTRRRG---H10LSH4-9192 YQRTRRRRG---H10LSH4-9193 YQTRRG---H12LSH6-10100 YQTRRG---H12LSH6-10101 YQTRRSRG---H

91KJin7-6723 TT93KJin2-10024 TT93KJin9-8530 VTPAKH01KJin7-5383 ISTT03KJin6-683 ISTPQH04KJin8-1955 ITPTH06KJin1-3030 VTPTH06KJin9-3012 VAPT07KJin3-5148 QEPTN07KJin3-5961 NLEPTAN10KJin4-9162 VTTAH10KJin9-8176 QVTAAH11KJin12-10119 VTPTADH13KJin1-11373 VTPTH

PE94JWK12-5524 VQ97JWK HR99JWK5-11441 HR00JWK3-11453 HR00JWK12-11445 HR01JWK7-5603 NHRQ01JWK7-5603s1 -------------------------------------------------------------------------------02JWK8-11436 NRQQ03JWK8-677 HR05JWK1-5600 NRQQ06JWK7-3042 HRQ

HRQ08JWK1-4772 NRQQ

NRQQ11JWK4-8993 NRQQ12JWK1-10125 NRQQ13JWK1-11366 NRQQ

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ +

10JWK4 9159

06 JWK12 5624

91JWK6 6949

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 9

92-13

93-04

93-60

92CWS5-9398 HSE93CWS3-9925 HE93CWS5-6669 EHTE93CWS7-802 KHTE96CWS11-5341 KHTE99CWS10-5338 HPES01CWS2-10532 HPDE03CWS6-684 HPE03CWS10-5773 HPDE04CWS5-3239 HPTE05CWS5-5783 HPTD----05CWS5-5785 HPTD----06CWS2-3071 HPTDE06CWS2-3203 HPTD----06CWS2-3221 HPTD----06CWS2-3222 VHPTD----06CWS2-3223 HPTD----06CWS8-3038 HPTD----06CWS8-3039 HPTD----07CWS4-3820 HPTATD----07CWS4-3821 HPTATD----07CWS4-10174 HPTATD----07CWS4-10175 HPTATD----07CWS4-10176 HPTATD----07CWS4-10177 VHRD----07CWS9-6252 HPE07CWS9-6253 HPTATD----07CWS9-6254 HPTATD----07CWS12-4777 HPTATD----08CWS5-6214 HPTATD----08CWS8-5160 HPTATD----08CWS8-5161 HPTATD----08CWS12-5720 HPTATD----08CWS12-5721 HPTATD----08CWS12-5722 HPTATD----09CWS5-6256 HPTATD----09CWS5-6257 HPTTATD----09CWS5-6258 HPTAD----09CWS12-9108 HPDE09CWS12-9109 HPTATTD----

93KYR2-9472 NSRIRKE94KYR6-10137 NSRRKE94KYR12-9441 NSRIRKER95KYR5-815 NSRIRKE00KYR9-9913 NSRRRKE02KYR7-9911 NSRRRKE03KYR1-817 NSRIRKE06KYR7-5236 QKPHRIRKE06KYR11-5249 QKPHRIRKE07KYR3-5265 NSHRIRKE07KYR3-11387s1 ---------------QNSHRIRRKER07KYR8-4845 QNSRRIRKER07KYR8-4846 NSRRIRKE07KYR8-5166 KNSHRRRIRKER07KYR8-5167 NKHRRIRRKE07KYR8-5168 IQRKPHRRIRKE

94KMH5 HVPR96KMH11-8534 VRT97KMH4-9452 HVQR01KMH11-5526 HVPR03KMH5-687 HVPR03KMH11-5725 HVQRS04KMH5 VQRS05KMH3-5727 HVPR06KMH11-3026 HVPR07KMH4-3281s1 VHVPRV----------------------------07KMH10-5732 HVQPRVRS08KMH5-5165 HIQPRVRS08KMH8-5427s1 ----------------------------HVQPTRVRS09KMH5-11475 HVQPRVRS09KMH5-11475s2 ------------------------------------------------------------------------------------------------10KMH10-9907s1 -----------------------------------------RVRS11KMH4-9917 HVPRVRS12KMH4-10096 HVPRVRRS

00YJN10-9385 ETVSRE01YJN9-4850 ETVSRE03YJN10-840 ETVSRE05YJN2-1948 ETQVSRE06YJN10-3044 ETQVSRE07YJN4-3248 ETQVSREY07YJN8-5627 ETQVSREY08YJN3-5606 ETQVSREY08YJN8-5408 ETQVSREY09YJN2-6041 ETQVSREY09YJN12-9156 ETQVSREY10YJN6-9918 ETQVSRIEY11YJN2-9921 ETQVSREY12YJN7-11382 ETQVSREY13YJN1-11381 ETQVSREYKor consensus QVDPDLADKLIHLHYFDCFSDSAIRHAILGRLVRPKCEYQAGHNKVGSLQYLALTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH

97 120 140 160 180 192

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ +

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

96-51

Figure 2 Sequential alignment of the amino acids of the Vif protein over 20 years All sequences that demonstrated small in-frame deletionsgross deletions and stop codons are depicted together with the baseline and last sequences of each patient Patients 90-18 demonstrated a9 bp deletion (positions 182ndash184) in April 1998 and patient 92-13 demonstrated a 12 bp deletion (positions 185ndash188) in May 2005 Patient90-05 consistently demonstrated a 9 bp insertion (RQTRAR-RAR-NGASRP) at the same position at the terminus of vpr beginning in August2000 (data not shown) The code 92LSH3-6951 (sampling year initials of the patient sampling month and sequence number) denotes thatthe sampling date is March 1992 and the sequence number is 6951 in patient LSH (90-18) The dot (sdot) hyphen (-) and asterisk (lowast) denotesequence identity deletions and premature stop codons respectively compared with the Korean consensus [20] In the right column theplus (+) and minus (minus) signs denote the presence or absence of the corresponding therapy respectively

10 Evidence-Based Complementary and Alternative Medicine

61 69 112 120 208 216 264 272 520 528Patient WT nucleotide TGGAAAAGT TGGGTCTAT TGGGGTCTG TGGAGGAAA TGGAACAAG Hypermut 20

sequence no P value90-05 10LSK4-9168 G A lt005

12LSK4-10131 A A AA A A lt000113LSK5-11470 A A A 015 13LSK5-11472 A AA lt001

90-18 08LSH5-5645 AA 019 08LSH11-5694 A 05710LSH4-9192 AA 013

90-54 07KJin3-5961 A 00693-04 94KYR12-9441 AA AG 009

07KYR8-5166 A A AA A AG lt000107KYR8-4846 A AA G 00507KYR8-5167 A A A AG 00507KYR8-5168 A A AG 02907KYR8-4845 AA G 005

93-60 03KMH11-5725 AA A 015

WT amino acid W K S W V Y W G L W R K W N K21 22 23 38 39 40 70 71 72 89 90 91 174 175 176

Figure 3 Positions of the premature stop codons in the 15 vif genes from five patients All stop codons resulted from GG rarr GA orGG rarr AG changes APOBEC3G exhibits an intrinsic preference for the second cytosine in a 51015840CC dinucleotide motif leading to 51015840GG-to-AG mutations [38]

In total five of our patients demonstrated in-frame deletionsof the vif gene of the 75 KSB-infected patients who weretreated with KRG Also in our present study cohort fivepatients including 90-18 and 92-13 demonstrated deletedvif genes (small in-frame and gross deletions) during KRGintake whereas all of our 10 LTSP patients demonstrateddeletions in the nef gene (119875 lt 005) [14] In additioncompared with the reported deletions of the nef and 51015840LTRgag genes the locations of the deleted vif gene werepositioned within a narrow range at the terminus [15 16 23]

Regarding gross deletions to date only two patientshave demonstrated gross deletions in the vif gene [17 18]and only one patient has demonstrated the insertion of twoamino acids and these patients were part of a nonprogress-ing mother-child pair [28] However in our present studyof the 10 LTSP patients who were assessed four patientsdemonstrated gross deletion in the vif gene during KRGintake prior to receiving HAART (15) This frequency issignificantly lower than the previously reported incidence of10 of 10 patients demonstrating gross deletions in the nef(187) and 51015840 LTRgag genes (376 119875 lt 001) [27 28]The frequency of deletion in the nef gene was reported tosignificantly decrease during KRG plus HAART (119875 lt 001)[16] In contrast to previous studies on the nef and gag genes[16 29] the detection of in-frame deletions in the vif genewas not suppressed in the patients receiving HAART in ourpresent cohort The reasons for this include the following (1)the proportion and frequency (approximately two-thirds) ofdeleted vif genes after the first occurrence were much higherthan in the nef and 51015840 LTRgag genes (lt20 and 30 resp)(2) small deletions in the vif gene occurred as a single bandwhereas gross deletions in the nef and 51015840 LTRgag genes weremainly detected as two bands (wild type and short) per PCRreaction and (3) the number of amplicons indicating PSCdueto G-to-A hypermutation was significantly higher in patients

receiving KRG plus HAART than patients receiving onlyKRG This phenomenon is consistent with our previous datain 51015840 LTRgag gene [29] However the frequency of G-to-Ahypermutationwas significantly lower in vif gene than that in51015840 LTRgag gene [29] Thus for the vif gene the detection ofsmall deletionsmight be less affected by limit-diluting effectsas has been shown for the nef gene [29] Taken together theseadditional defects in the vif genes in the same patients mightbe related to the replicative impairment in vif defective HIV-1 which ultimately results in defects in the synthesis of viralDNA [30]

In our present study it appears that the frequency ofhypermutation was higher in patients who maintained goodcompliance with HAART during HAART plus KRG (90-0590-18 and 93-04) than in the patients who demonstratedpoor compliance (Figure 1) In our extended data analysis124 (60485) 31 (5161) 30 (19617) and 09 (1108)of vif genes during KRG plus HAART HAART aloneKRG alone and control revealed PSC respectively Thesedata indicate the presence of synergistic effect of KRG plusHAART on PSC (119875 lt 001) There was no case that smalldeletions were induced by HAART alone About 6ndash43 ofthe integrated proviral pol gene is hypermutated by HAART[22 31 32] although hypermutated viral genomes are notpresent in plasma [32]

ApoBec3G-induced hypermutation in LTNPs has beenreported previously [33] Indeed it has been reported alsothat LTNP patients harbor PSC-containing vif genes morefrequently than progressors [25] although the reportedfrequencies of defective genes vary among studies [34] Inour current analyses vif gene-containing PSC due to G-to-A hypermutation was found to be significantly higher duringKRG plus HAART than only KRG intake prior to HAARTThis finding is consistent with our previous data obtainedfrom a cohort of the Korean hemophiliacs (21 incidence

Evidence-Based Complementary and Alternative Medicine 11

of G-to-A hypermutation while receiving only KRG versus98 on KRG plus HAART unpublished data) and otherobservations regarding the pol gene [22]

Previous studies report that certain vif mutations such asL81M R132S S130I and the insertion of two amino acids areassociatedwith slow progression or low viral loads [17 18 28]In our present study L81M [35] and R132S were consistentlydetected in patient 96-51 and patients 87-05 and 96-51respectively although both mutations were found in only in2 and 3 of 169 Korean patients respectively Although thevif gene is highly conserved in HIV-1 genomes the recoveryof hypermutants might be severely underestimated in ourpresent analysis because of the primer mismatching andthe difficulties involved in detection following amplification[36 37] Overall our current data suggest that small in-frame deletion and PSCs are associated with KRG intake andHAART respectively although further studies are needed

5 Conclusions

Our data suggest that HAART and KRG intake might inducePSCs and small in-frame deletions respectively

Conflict of Interests

The authors have no conflict of interests to declare

Acknowledgments

This work was supported by a grant from the Korean Societyof Ginseng (2010ndash2012) The authors thank Professor Seung-Chul Yun the Division of Biostatistics University of UlsanCollege of Medicine Asan Medical Center for the statisticalconsultation

References

[1] E Ernst ldquoPanax ginseng an overview of the clinical evidencerdquoJournal of Ginseng Research vol 34 no 4 pp 259ndash263 2010

[2] K T Choi ldquoBotanical characteristics pharmacological effectsand medicinal components of Korean Panax ginseng C AMeyerrdquoActa Pharmacologica Sinica vol 29 no 9 pp 1109ndash11182008

[3] S J Fulder ldquoGinseng and the hypothalamic-pituitary control ofstressrdquo The American Journal of Chinese Medicine vol 9 no 2pp 112ndash118 1981

[4] V K Singh S S Agarwal and B M Gupta ldquoImmunomodula-tory activity of Panax ginseng extractrdquo Planta Medica vol 50no 6 pp 462ndash465 1984

[5] X Song and S Hu ldquoAdjuvant activities of saponins fromtraditional Chinese medicinal herbsrdquo Vaccine vol 27 no 36pp 4883ndash4890 2009

[6] D G Yoo M C Kim M K Park et al ldquoProtective effectof ginseng polysaccharides on influenza viral infectionrdquo PLoSONE vol 7 no 3 Article ID e33678 2012

[7] H X Sun Y P Ye H J Pan and Y J Pan ldquoAdjuvant effectof Panax notoginseng saponins on the immune responses toovalbumin in micerdquo Vaccine vol 22 no 29-30 pp 3882ndash38892004

[8] J Sun S Hu and X Song ldquoAdjuvant effects of protopanaxadioland protopanaxatriol saponins from ginseng roots on theimmune responses to ovalbumin in micerdquo Vaccine vol 25 no6 pp 1114ndash1120 2007

[9] P W Hunt ldquoHIV and inflammation mechanisms and conse-quencesrdquo Current HIVAIDS Reports vol 9 no 2 pp 139ndash1472012

[10] T Yayeh K H Jung H Y Jeong et al ldquoKorean red ginsengsaponin fraction downregulates proinflammatory mediators inLPS stimulated RAW264 7 cells and protects mice againstendotoxic shockrdquo Journal of Ginseng Research vol 36 no 3 pp263ndash269 2012

[11] A T Smolinski and J J Pestka ldquoModulation of lipopolysaccha-ride-induced proinflammatory cytokine production in vitroand in vivo by the herbal constituents apigenin (chamomile)ginsenoside Rb1 (ginseng) and parthenolide (feverfew)rdquo Foodand Chemical Toxicology vol 41 no 10 pp 1381ndash1390 2003

[12] A Rhule S Navarro J R Smith and D M ShepherdldquoPanax notoginseng attenuates LPS-induced pro-inflammatorymediators in RAW2647 cellsrdquo Journal of Ethnopharmacologyvol 106 no 1 pp 121ndash128 2006

[13] J M Brenchley D A Price T W Schacker et al ldquoMicrobialtranslocation is a cause of systemic immune activation inchronic HIV infectionrdquo Nature Medicine vol 12 no 12 pp1365ndash1371 2006

[14] Y K Cho J Y Lim Y S Jung S K Oh H J Lee and H SungldquoHigh frequency of grossly deleted nef genes in HIV-1 infectedlong-term slow progressors treated with Korean red ginsengrdquoCurrent HIV Research vol 4 no 4 pp 447ndash457 2006

[15] Y K Cho and Y S Jung ldquoHigh frequency of gross deletions inthe 51015840 LTR and gag regions in HIV type 1-infected long-termsurvivors treated with Korean red ginsengrdquo AIDS Research andHuman Retroviruses vol 24 no 2 pp 181ndash193 2008

[16] Y K Cho Y S Jung andH S Sung ldquoFrequent gross deletion intheHIV type 1 nef gene in hemophiliacs treatedwith korean redginseng inhibition of detection by highly active antiretroviraltherapyrdquo AIDS Research and Human Retroviruses vol 25 no 4pp 419ndash424 2009

[17] V Sandonıs C Casado T Alvaro et al ldquoA combination ofdefective DNA and protective host factors are found in a setof HIV-1 ancestral LTNPsrdquo Virology vol 391 no 1 pp 73ndash822009

[18] H R Rangel D Garzaro A K Rodrıguez et al ldquoDeletioninsertion and stop codon mutations in vif genes of HIV-1 infecting slow progressor patientsrdquo Journal of Infection inDeveloping Countries vol 3 no 7 pp 531ndash538 2009

[19] Y K Cho J E Kim and B T Foley ldquoPhylogenetic analysisof near full-length HIV-1 genomic sequences in 21 Koreanindividualsrdquo AIDS Research and Human Retroviruses vol 29no 4 pp 738ndash743 2013

[20] Y K Cho Y S Jung J S Lee and B T Foley ldquoMolecu-lar evidence of HIV-1 transmission in 20 Korean individu-als with haemophilia phylogenetic analysis of the vif generdquoHaemophilia vol 18 no 2 pp 291ndash299 2012

[21] P P Rose and B T Korber ldquoDetecting hypermutations inviral sequences with an emphasis on GrarrA hypermutationrdquoBioinformatics vol 16 no 4 pp 400ndash401 2000

[22] S Fourati S Lambert-Niclot C Soulie et al ldquoHIV-1 genomeis often defective in PBMCs and rectal tissues after long-termHAART as a result of APOBEC3 editing and correlates with thesize of reservoirsrdquo Journal of Antimicrobial Chemotherapy vol67 no 10 pp 2323ndash2326 2012

12 Evidence-Based Complementary and Alternative Medicine

[23] U Wieland A Seelhoff A Hofmann et al ldquoDiversity of thevif gene of human immunodeficiency virus type 1 in UgandardquoJournal of General Virology vol 78 no 2 pp 393ndash400 1997

[24] P Sova M van Ranst P Gupta et al ldquoConservation of an intacthuman immunodeficiency virus type 1 vif gene in vitro and invivordquo Journal of Virology vol 69 no 4 pp 2557ndash2564 1995

[25] T Yamada and A Iwamoto ldquoComparison of proviral accessorygenes between long-term nonprogressors and progressors ofhuman immunodeficiency virus type 1 infectionrdquo Archives ofVirology vol 145 no 5 pp 1021ndash1027 2000

[26] K Tominaga S Kato M Negishi and T Takano ldquoA high fre-quency of defective vif genes in peripheral blood mononuclearcells from HIV type 1-infected individualsrdquo AIDS Research andHuman Retroviruses vol 12 no 16 pp 1543ndash1549 1996

[27] Y K Cho H Sung I G Bae et al ldquoFull sequence of HIV type 1Korean subtype B in anAIDS case with atypical seroconversionTAAAA at TATA boxrdquoAIDS Research andHuman Retrovirusesvol 21 no 11 pp 961ndash964 2005

[28] L Alexander M J Aquino-DeJesus M Chan and W AAndiman ldquoInhibition of human immunodeficiency virus type 1(HIV-1) replication by a two-amino-acid insertion in HIV-1 Viffrom a nonprogressing mother and childrdquo Journal of Virologyvol 76 no 20 pp 10533ndash10539 2002

[29] YK Cho Y S JungH S Sung andCH Joo ldquoFrequent geneticdefects in the HIV-1 51015840LTRgag gene in hemophiliacs treatedwith Korean red ginseng decreased detection of genetic defectsby highly active antiretroviral therapyrdquo Journal of GinsengResearch vol 35 no 4 pp 413ndash420 2011

[30] M Nascimbeni M Bouyac F Rey B Spire and F Clavel ldquoThereplicative impairment of Vif-mutants of human immunodefi-ciency virus type 1 correlates with an overall defect in viral DNAsynthesisrdquo Journal of General Virology vol 79 no 8 pp 1945ndash1950 1998

[31] J P Vartanian M Henry and S Wain-Hobson ldquoSustainedGrarrA hypermutation during reverse transcription of an entirehuman immunodeficiency virus type 1 strain Vau group Ogenomerdquo Journal of General Virology vol 83 no 4 pp 801ndash8052002

[32] T L Kieffer P Kwon R E Nettles Y Han S C Ray andR F Siliciano ldquoGrarrA hypermutation in protease and reversetranscriptase regions of human immunodeficiency virus type 1residing in resting CD4+ T cells in vivordquo Journal of Virology vol79 no 3 pp 1975ndash1980 2005

[33] B Wang M Mikhail W B Dyer J J Zaunders A D Kelleherand N K Saksena ldquoFirst demonstration of a lack of viralsequence evolution in a nonprogressor defining replication-incompetent HIV-1 infectionrdquo Virology vol 312 no 1 pp 135ndash150 2003

[34] G Hassaine I Agostini D Candotti et al ldquoCharacterizationof human immunodeficiency virus type 1 vif gene in long-termasymptomatic individualsrdquoVirology vol 276 no 1 pp 169ndash1802000

[35] F A de Maio C A Rocco P C Aulicino R Bologna AMangano and L Sen ldquoUnusual substitutions in HIV-1 Vif fromchildren infected perinatally without progression to AIDS formore than 8 years without therapyrdquo Journal of Medical Virologyvol 84 no 12 pp 1844ndash1852 2012

[36] M Janini M Rogers D R Birx and F E McCutchan ldquoHumanimmunodeficiency virus type 1 DNA sequences geneticallydamaged by hypermutation are often abundant in patientperipheral blood mononuclear cells and may be generated

during near-simultaneous infection and activation of CD4+ Tcellsrdquo Journal of Virology vol 75 no 17 pp 7973ndash7986 2001

[37] G H Kijak L M Janini S Tovanabutra et al ldquoVariablecontexts and levels of hypermutation inHIV-1 proviral genomesrecovered from primary peripheral blood mononuclear cellsrdquoVirology vol 376 no 1 pp 101ndash111 2008

[38] E W Refsland J F Hultquist and R S Harris ldquoEndogenousorigins of HIV-1 G-to-A hypermutation and restriction in thenonpermissive T cell line CEM2nrdquo PLoS Pathogens vol 8 no7 Article ID e1002800 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 3: Effects of Korean Red Ginseng and HAART on vif Gene in 10 Long

Evidence-Based Complementary and Alternative Medicine 3

CD4+ T-cell count remained low (Figure 1) The remainingnine patients progressed to AIDS demonstrating CD4+ T-cell counts of lt200 cells120583L (Table 1)

32 Effects of HAART on PSC The 10 LTSP patients weincluded in our present study were untreated before Decem-ber 1991 were treated with only KRG between 2004 and2009 and have been treated with KRG plus HAART since2009 (Figure 1) We analyzed 432 vif genes over 20 years inthese 10 LTSP patients Among these 15 vif genes (35)demonstrated PSC In total 275 and 157 vif genes wereobtained during KRG and KRG plus HAART respectivelyEach group demonstrated 3 (11) and 12 (76) vif geneswith PSCs respectively When receiving only KRG threepatients (90-50 93-04 and 93-60) demonstrated PSC at 14years 1 year and 9months and 7 years after starting to receiveKRG respectively The frequency was significantly higherwhen receivingKRGplusHAART thanKRGalone (119875 lt 001Table 2) This suggests that HAART itself might induce G-to-A hypermutation thereby resulting in PSC and possiblylethal hypermutations This is consistent with the findings ofother studies [22] However we found no difference in thefrequency of PSCs between our KRG (3 of 275 genes) andcontrol groups (1 of 106 genes) Interestingly of the 15 vifgenes that demonstrated PSCs in our present analysis elevendid not satisfy the criteria of Hypermut 20 in comparisonwith the earliest sequences obtained from each patient (119875 lt005 Figure 3)

33 Effects of KRG and HAART on Genetic Defects Wedetected 11 gross deletions in 432 amplicons obtained fromour 10 LTSP patients (Table 2 Figure 2) No deleted genesincluding gross deletions were detected in the controlpatients In total five patients demonstrated gross deletionsafter gt19 months of KRG intake Four patients demonstratedgross deletions during KRG intake prior toHAART althoughthe frequency was very low (15) Specifically a sequencecontaining a gross deletion and duplicationrecombination(KC247159) was identified in patient 87-05 A 362-base pair(bp) deletion and 1 bp insertion (KC247195) 309 bp deletion(JQ327719) and 870 bp deletion (JQ327722) were detectedas short bands together with the wild-type genotype after139 months 115 months and 125 months of KRG intakein patients 90-05 91-20 and 93-60 respectively (patient90-05 demonstrated 9 bp insertions in 16 amplicons after104 months of KRG intake including KC247191 JQ248236DQ295192 and JQ248237-50) In addition two patientsdemonstrated gross deletions during KRG plus HAARTSpecifically patient 93-04 demonstrated 386 bp deletionsin one-third of amplicons after 14 years of KRG intakePatient 93-60 demonstrated six deletions (including 374 bp[JQ327723] and 401 bp deletions [KC247314] Figure 2 andTable 2) Each deletion was one (JQ327723) of 3 ampliconsand one (KC247314) of 2 amplicons obtained between August2008 and October 2010 and all 4 amplicons respectively(Figure 1) This frequency is the highest among the relatedstudies to date although we found no significant differencesin terms of the frequencies of gross deletions between patients

who received KRG (15) or KRG plus HAART (45Table 2) in our present study If both PSCs and gross deletionsare only defined as nonfunctional vif genes the proportion ofnonfunctional genes is 25 (7 of 275 genes) during KRG and121 (19 of 157 genes) during KRG plus HAART (119875 lt 001)

34 Position and Frequency of PSCs in the vif Gene Thereare 8 tryptophan residues in the Vif protein In this study15 vif genes from 5 patients (90-05 90-18 90-50 93-04and 93-60 Figure 3) demonstrated PSCs The frequencies ofthe PSCs in 5 tryptophan residues (21 38 70 89 and 174)were 3 7 13 2 and 7 respectively In the control group onepatient demonstrated stop codons at residues 21 70 and 174(JQ066980)

35 Small in-FrameDeletions Are Associated with KRG IntakeWe did not find any specific changes in the nucleotide oramino acid sequences due to KRG intake However inter-estingly two of our patients demonstrated small deletionsduring KRG intake one patient (90-18) demonstrated a 9 bpin-frame deletion (detected in 15 of 26 amplicons) at aminoacid positions 182ndash184 in April 1998 and another patient(92-13) demonstrated a 12 bp deletion (detected in 14 of 16amplicons) at positions 186ndash189 in May 2005 (Figure 2) Thefrequencies of these deletions were similar between patientswho received KRG (105 29 of 275 genes) and KRG plusHAART (159 25 of 157 genes) However the frequencyof these deletions was zero (0 of 52 genes as determinedusing RT-PCR) during pre-KRG and 105 (29 of 275) duringKRG only without HAART (119875 lt 001) These deletionsmanifested after 63 and 120 years of KRG intake in patients90-18 and 92-13 respectively Interestingly patient 90-18 alsodemonstrated a 6 bp deletion in the nef gene after 11 yearsof KRG intake with HAART although we did not categorizethis as a gross deletion [14] Deletions were conservedduring KRG plus HAART therapy (Figure 2) and compriseda higher proportion thanwild-type alleles In particular threeamplicons containing PSC also demonstrated small deletionsin patient 90-18 The presence of both wild-type and mutantalleles in the same samples differed from the findings forthe samples obtained from patients who were not treatedwith KRG in which all amplicons contained deletions in fivepatients (data not shown) In addition there were no suchdeletions in the control group (0 of 106 genes)

36 Overall Rate of Defective Genes Whenwe included smallin-frame deletions of defective genes the overall proportionsof defective genes were 131 (36 of 275 genes) when receivingonly KRG and 268 (42 of 157 genes) when receiving KRGplus HAART (119875 lt 001) In our current study the frequencyof defective genes identified in PBMCs obtained from HIV-1-infected patients prior to receiving HAART was similar tothe results of previous reports byWieland et al (10) [23] andSova et al (13) [24] although exceptional cases have beenreported byYamada and Iwamoto (205) [25] andTominagaet al (31) [26] Overall the proportions of defective genesidentified in our present study were significantly lower thanthe proportions of defective nef (94 of 479 genes including

4 Evidence-Based Complementary and Alternative Medicine

0

1

2

3

4

5

6

7

0100200300400500600700

87-05Korean Red Ginseng (KRG) KRG KRG KRG

Jun

87Se

p 89

Apr

91

Nov

91

Nov

92

Dec

93

Jun

94D

ec 9

4O

ct 9

5O

ct 9

6A

pr 9

7A

pr 9

8O

ct 9

8A

pr 9

9O

ct 9

9M

ar 0

0Fe

b 01

Jul 0

1Se

p 02

Sep

03O

ct 0

4M

ar 0

5M

ay 0

5O

ct 0

5M

ay 0

6O

ct 0

6Ja

n 07

May

07

Aug

07

Oct

07

Feb

08A

ug 0

8D

ec 0

8Ju

n 09

Dec

09

Jul 1

0M

ar 1

1Se

p 11

Feb

12A

ug 1

2D

ec 1

2

CD4

+ T

cells

(120583

L)

CD4+ T cells (120583L)

minus100 0

1

2

3

4

5

6

7

0200400600800

1000120014001600 89-17

KRG KRG HAARTKRGKRG KRG

Aug

89

Jan

90Se

p 90

Apr

91

Dec

91

Jan

92M

ar 9

2Ju

n 92

Nov

92

Feb

93A

pr 9

3Ju

l 93

Oct

93

Apr

94

Oct

94

Sep

95Ju

l 96

Oct

96

Mar

97

Aug

97

Apr

98

Jun

99N

ov 9

9M

ar 0

0Se

p 00

Feb

01Ju

l 01

Jan

02M

ar 0

2Se

p 02

Feb

03M

ay 0

3Ja

n 04

Aug

04

Mar

05

Dec

05

Oct

06

Nov

06

Feb

07A

pr 0

7A

ug 0

7D

ec 0

7Ju

l 08

May

09

May

10

Mar

12

RNA

copy

(log

mL)

minus200

0

1

2

3

4

5

6

7

050

100150200250300350400450 90-05

KRG HAARTKRG

lowast lowast lowast

Jul 9

1N

ov 9

1N

ov 9

2A

pr 9

3O

ct 9

3Ju

n 94

Jan

95M

ay 9

5A

ug 9

6A

ug 9

7A

pr 9

8M

ar 9

9A

ug 0

0Fe

b 01

May

01

Mar

02

Aug

02

Jan

03M

ar 0

3Ju

n 03

Oct

03

Mar

04

Aug

04

Feb

05Ju

l 05

Oct

05

Jan

06Ju

l 06

Oct

06

Feb

07M

ay 0

7A

ug 0

7O

ct 0

7Ja

n 08

Apr

08

May

08

Aug

08

Nov

08

Apr

09

Oct

09

Apr

10

Oct

10

Apr

11

Oct

11

Apr

12

Nov

12

May

13

CD4

+ T

cells

(120583

L)

0

1

2

3

4

5

6

7

0100200300400500600700800900

1000 90-18

HAARTKRGKRG KRG KRGKRGKRGKRG KRG

Aug

90

Feb

91A

ug 9

1D

ec 9

1M

ar 9

2Ju

n 92

Nov

92

Mar

93

May

93

Jun

93O

ct 9

3Fe

b 94

May

94

Aug

94

Nov

94

Feb

95M

ay 9

5O

ct 9

5Fe

b 96

May

96

Dec

96

Mar

97

Mar

97

Jul 9

7M

ar 9

8A

pr 9

8Se

p 98

Feb

99A

pr 9

9Ju

n 99

Nov

99

Mar

00

Jul 0

0A

ug 0

0O

ct 0

0M

ar 0

1Ju

l 01

Dec

01

Apr

02

Oct

02

Jan

03M

ay 0

3O

ct 0

3Ju

l 04

Dec

04

Mar

05

Feb

06Ju

n 06

Jul 0

6A

ug 0

6N

ov 0

6Ja

n 07

Apr

07

Jun

07O

ct 0

7N

ov 0

7D

ec 0

7M

ay 0

8N

ov 0

8Fe

b 09

Apr

09

Oct

09

Apr

10

Oct

10

Oct

11

Jun

12D

ec 1

2

minus100

lowastlowast lowast

RNA

copy

(log

mL)

CD4

+ T

cells

(120583

L)

0

1

2

3

4

5

6

7

0

200

400

600

800

1000

1200 90-50HAARTKRGKRGKRGKRG

Dec

90

Jul 9

1Ja

n 92

Jul 9

2Fe

b 93

Apr

93

Sep

93M

ar 9

4Se

p 94

Apr

95

Oct

95

Apr

96

Nov

96

May

97

Nov

97

Jun

98N

ov 9

8M

ay 9

9N

ov 9

9Se

p 00

Jul 0

1N

ov 0

1Ju

n 02

Dec

02

Mar

03

Jun

03Se

p 03

Mar

04

Aug

04

Jan

05A

ug 0

5Ja

n 06

Jul 0

6Se

p 06

Mar

07

May

07

Jun

07Ju

l 07

Oct

07

Nov

07

Jul 0

9D

ec 0

9A

pr 1

0Ju

l 10

Jan

11A

pr 1

1D

ec 1

1Ju

l 12

Jan

13

lowast

CD4+

T ce

lls (120583

L)

minus200 0

1

2

3

4

5

6

7

0

200

400

600

800

1000

120091-20

KRG HAARTKRGKRG

Jun

91D

ec 9

1M

ar 9

2M

ay 9

2Ja

n 93

May

93

Dec

93

Jul 9

4N

ov 9

4D

ec 9

4Fe

b 95

Mar

95

May

95

Oct

95

Apr

96

Aug

96

Mar

97

Jul 9

7A

pr 9

8N

ov 9

8M

ay 9

9N

ov 9

9M

ar 0

0A

pr 0

0Ju

l 00

Dec

00

Mar

01

Jul 0

1N

ov 0

1M

ar 0

2A

ug 0

2Ja

n 03

Mar

03

Aug

03

Jan

04Ju

l 04

Jan

05Ju

l 05

Jan

06Ju

l 06

Dec

06

May

07

Jul 0

7A

ug 0

7Se

p 07

Nov

07

Jan

08A

pr 0

8Ju

l 08

Dec

08

Oct

10

Apr

11

Jan

12Ju

l 12

Jan

13

minus200

RNA

copy

(log

mL)

CD4+

T ce

lls (120583

L)

0

1

2

3

4

5

6

7

050

100150200250300350400450

92-13KRG HAART HAART

KRGKRGKRGKRGKRG

Expired in 2011

May

92

Nov

92

May

93

Jul 9

3Ja

n 94

Oct

94

Dec

94

Feb

95A

pr 9

5O

ct 9

5A

pr 9

6N

ov 9

6Fe

b 97

Jun

97A

pr 9

8Ja

n 99

Oct

99

Feb

01D

ec 0

1Ju

l 02

Dec

02

Mar

03

Jun

03O

ct 0

3M

ay 0

4N

ov 0

4M

ay 0

5Ju

l 05

Feb

06A

ug 0

6N

ov 0

6A

pr 0

7Ju

n 07

Jul 0

7A

ug 0

7Se

p 07

Dec

07

May

08

Aug

08

Dec

08

May

09

Dec

09

Apr

10

Jun

10Ja

n 11

CD4

+ T

cells

(120583

L)

minus500

1

2

3

4

5

67

0100200300400500600700800900 93-04

KRG HAARTKRG KRG KRGKRG

Feb

93M

ar 9

3Ju

l 93

Dec

93

Feb

94Ju

n 94

Dec

94

May

95

Nov

95

Feb

96Se

p 96

Jan

97Fe

b 97

Jul 9

7A

ug 9

7A

pr 9

8Ju

l 98

Sep

00D

ec 0

0M

ar 0

1Ju

n 01

Sep

01Ju

n 02

Jul 0

2Ja

n 03

May

03

Oct

03

Mar

04

Aug

04

Jan

05M

ay 0

5Ju

n 05

Aug

05

Oct

05

Mar

06

Jul 0

6N

ov 0

6M

ar 0

7A

ug 0

7

lowastlowast

RNA

copy

(log

mL)

CD4

+ T

cells

(120583

L)

RNA

copy

(log

mL)

RNA copy (logmL)

RNA

copy

(log

mL)

RNA

copy

(log

mL)

RNA

copy

(log

mL)

if if

if

if

ifif

ifif

Sampling date Sampling date

Sampling date Sampling date

Sampling date Sampling date

Sampling date Sampling dateCD4+ T cells (120583L)RNA copy (logmL)

CD4

+ T

cells

(120583

L)

Figure 1 Continued

Evidence-Based Complementary and Alternative Medicine 5

0

1

2

3

4

5

6

7

0100200300400500600700800

93-60

HAARTKRGKRG

Apr

94

Nov

95

May

96

Nov

96

Apr

97

Jun

97Ju

n 99

Nov

99

Nov

00

Jul 0

1N

ov 0

1Ja

n 02

Sep

02Fe

b 03

May

03

Nov

03

May

04

Mar

05

Sep

05A

pr 0

6O

ct 0

6N

ov 0

6A

pr 0

7Ju

l 07

Aug

07

Oct

07

Jan

08M

ay 0

8M

ay 0

8Ju

l 08

Aug

08

Dec

08

May

09

Dec

09

Oct

10

Apr

11

Apr

12

lowast

0

1

2

3

4

5

6

7

0

200

400

600

800

1000

120096-51

KRG HAARTKRGKRG

Jul 9

6Ja

n 97

Jul 9

7A

pr 9

8N

ov 9

8M

ay 9

9Ju

n 00

Oct

00

Sep

01A

pr 0

2D

ec 0

2O

ct 0

3Fe

b 04

Aug

04

Feb

05A

ug 0

5N

ov 0

6Ju

l 06

Oct

06

Feb

07A

ug 0

7M

ar 0

8A

ug 0

8Fe

b 09

Jul 0

9D

ec 0

9M

ay 1

0Ju

n 10

Apr

11

Oct

11

Jul 1

2Ja

n 13

RNA

copy

(log

mL)

CD4

+ T

cells

(120583

L)

Sampling dateCD4+ T cells (120583L)RNA copy (logmL)

RNA

copy

(log

mL)

CD4

+ T

cells

(120583

L)

Sampling dateCD4+ T cells (120583L)RNA copy (logmL)

if if

Figure 1 Changes in the CD4+ T-cell count plasma viral load and genetic defects in terms of Korean red ginseng (KRG) intake and highlyactive antiretroviral therapy (HAART) The durations of KRG intake and HAART are indicated by the bars Solid and dotted lines denotegood (gt90) and poor (lt90) compliance according to self-administered responses respectively The upward arrow (uarr) downward arrow(darr) plus sign (+) and asterisk (lowast) denote the sequences of the vif gene gross deletions 3- and- 6-base pair (bp) in-frame insertions and stopcodons respectively

Table 2 Distribution of defective vif gene

Patient On-KRG On-HAARTKRGNo of genes Stop codon gΔ sΔ No of genes Stop codon gΔ sΔ

87-05 20 1 ND ND ND ND89-17 9 9 0 0 090-05 37 1 18 4 0 090-18 35 15 20 3a 0 12a

90-50 14 1 15 0 0 091-20 35 1 20 0 0 092-13 53 14 22 0 0 1393-04 18 1 12 5 1 093-60 28 1 1 20 0 6 096-51 26 21 0 0 0Total 275 3b 4 29 157 12b 7 25gΔ and sΔ denote gross deletion and in-frame small deletion respectivelyHAART highly active antiretroviral therapy ND not determinedaTwo out of three genes contained both stop codon and sΔb119875 lt 001119875 lt 001 for the sum of 3 kinds of defective genes (36275 versus 42157)Fifty-two vif genes at baseline obtained from serum using RT-PCR were all wild types lowastControl patients (119899 = 21) revealed premature stop codon in one outof 106 vif genes

stop codons 119875 lt 005) and 51015840 LTRgag genes (71 of 189 genes119875 lt 0001) identified in the same patients [14 15]

4 Discussion

Todate we have identified the vif gene in 194Korean patientsOf these 145 patients were infected with the Korean subcladeB ofHIV-1 (KSB) and the remaining 49 patients were infectedwith non-KSB Of these 194 patients 10 demonstrated smallin-frame deletions (3ndash15 bp) and all were diagnosed withKSB whereas no such deletions were detected in non-KSBpatients (119875 = 0068) Of the 10 patients with small deletionsthree patients were included in a group of 20 hemophiliacs

who were infected with KSB from plasma donors O and P[16 20] Interestingly plasma donors O and P revealed wild-type vif only between 1991 and 2002 (AY581320-21 JQ248127-33 JF957909 JF957921 JQ248134 and JF957935-37) In otherwords original vif gene was wild type without in-framesmall deletion In our current study only two patients (90-18 and 92-13) demonstrated 9 (119899 = 15) and 12 (119899 = 14)bp in-frame deletions in about two-thirds of the ampliconsobtained after 63 and 120 years of KRG intake respectivelywhereas five patients without KRG intake demonstrated in-frame deletions in all amplicons (data not shown) To ourknowledge these in-frame deletions are very rare and haveonly been reported in two patients in other countries [26 27]

6 Evidence-Based Complementary and Alternative Medicine

87-05

89-17

90-05

90-18

90-50

91-20

1 20 40 60 80 96Kor consensus MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKEWVYRHHYESTHPRISSEVHIPLGDAKLVITTYWGLHTGEREWHLGQGVSIEWRKKRYNT

91CSR4-10026 RGNVVD91CSR11-6922 RGNVVD92CSR5-9885 RGNVVD93CSR6-10159 RGNVVD93CSR6-10162s1 ------------------------------------------------------------------------------------------------03CSR9-805 SGGNVVD05CSR3 RGNIVVDG06CSR10-5280 RGNIVVDRG08CSR3-5334 RGNNVDG08CSR8-5279 RGNIVVD10CSR7-8126 VRGNLVVDG12CSR2-10141 VRGNLVVDG

91KJS12-8538 I92KJS3-6819 GI93KJS7-10155 IAI97KJS3-3814 IGKEI01KJS2-5667 IGKI03KJS2-745 IGAI03KJS7-775 IGAEI05KJS12-3264 YMIVGAEI08KJS5-5157 IVNGAAI10KJS5-10133 IGKAES

92LSK11-9417 K94LSK6-759 KGNPRS96LSK6-11491 AKNPIRS98LSK4-11495 KHN00LSK8-9999 KGNR03LSK6-10001s1 ------------------------------------------------------------------------------04LSK7 KKGNPRST06LSK1-3236 KKGNPVRST07LSK2-3232 KKGNPVRST08LSK1-4762 KKGIPVRST09LSK4-6143 IKNPIGRST10LSK4-9165 KGNPRS10LSK4-9168 KKGNPVRTRST12LSK4-10131 KAVKALKNPEKST 12LSK4-10132 KAKNPGRST13LSK5-11470 KKGNPGRST13LSK5-11471 KKVGNPVRST13LSK5-11472 IKANNRI

92LSH3-6951 K92LSH11-8548 TKK93LSH10-1252 94LSH2-740 95LSH5-10562 V96LSH5-10609 V98LSH4-10567 VG99LSH1-5776 VG99LSH1-5777 VG99LSH1-5778 VG99LSH6-5781 VGE99LSH6-5782 VG00LSH3-5704 KVGR00LSH3-5705 VG00LSH3-5706 VGK02LSH10-5707 VGR02LSH10-5708 VG02LSH10-5710 VG03LSH5-5650 VGG03LSH5-5651 VG03LSH5-5652 VG03LSH10-5653 VG04LSH7-4848 VGKES04LSH8-3011 V06LSH8-5647 VGES06LSH8-5649 VGES06LSH11-3238 VGIS08LSH5-5644 VG08LSH5-5645 VGR08LSH5-5646 KVGE08LSH5-5647 KVGE08LSH11-5694 KVG10LSH4-9190 KVGES10LSH4-9192 VG10LSH4-9193 VGG12LSH6-10100 VGES12LSH6-10101 VG

91KJin7-6723 N93KJin2-10024 N93KJin9-8530 NHVKINVDT01KJin7-5383 KVN03KJin6-683 KVINT04KJin8-1955 KVINDT06KJin1-3030 KVKINDT06KJin9-3012 KVINT07KJin3-5148 VNKINID07KJin3-5961 VNKKINEN10KJin4-9162 NHVKKINVT10KJin9-8176 NHVKKINVDT11KJin12-10119 KVKKINVDT13KJin1-11373 VNNVNDT

K94JWK12-5524 K97JWK K99JWK5-11441 KNE00JWK3-11453 KN00JWK12-11445 KN01JWK7-5603 KVA01JWK7-5603s1 KKNN02JWK8-11435 KVA03JWK8-677 KN05JWK1-5600 KVA06JWK7-3042 KVNAN

KKNVNVVN08JWK1-4772 KVNAN

KVA11JWK4-8993 KVA12JWK1-10125 KVA13JWK1-11366 KVA

minus minusminus minusminus minusminus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ +

Patient KRG HAART

10JWK4 9159

06JWK12 5624

91JWK6 6949

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 7

92-13

93-04

93-60

96-51

92CWS5-9398 G93CWS3-9925 GK93CWS5-6669 GK93CWS7-802 G96CWS11-5341 G99CWS10-5338 KVGE01CWS2-10532 VG03CWS6-684 VGTG03CWS10-5773 VG04CWS5-3239 VGKSTG05CWS5-5783 GTG05CWS5-5785 VGKITG06CWS2-3071 VGTG06CWS2-3203 VGKITG06CWS2-3221 VGTG06CWS2-3222 NVGTG06CWS2-3223 NVGTG06CWS8-3038 NVGKTG06CWS8-3039 NVGKTG07CWS4-3820 VGKIATG07CWS4-3821 VGKIATG07CWS4-10174 HWVGKIATG07CWS4-10175 VGKIATG07CWS4-10176 VGKITG07CWS4-10177 VGKKTG07CWS9-6252 VGTG07CWS9-6253 VGKITG07CWS9-6254 VGKIATG07CWS12-4777 VGQITG08CWS5-6214 NKVGQITG08CWS8-5160 NVGKTG08CWS8-5161 NVGKNQG08CWS12-5720 VGQITG08CWS12-5721 VGQITG08CWS12-5722 VGKQITG09CWS5-6256 VGKQTG09CWS5-6257 VGKQTG09CWS5-6258 VGKDQTG09CWS12-9108 LVGTG09CWS12-9109 VGKQITG

93KYR2-9472 VNN94KYR6-10137 VNN94KYR12-9441 VNN95KYR5-815 VGNN00KYR9-9913 VGNN02KYR7-9911 VGNN03KYR1-817 VNN06KYR7-5236 VGNN06KYR11-5249 VGNNS07KYR3-5265 VGNN07KYR3-11387s1 -------------------------------------------------------------------------------------------------07KYR8-4845 VGNN07KYR8-4846 VGNN07KYR8-5166 IVRNKN07KYR8-5167 VGNRN07KYR8-5168 VGNN

94KMH5 EI96KMH11-8534 IAEI97KMH4-9452 ISEI01KMH11-5526 ISIEI03KMH5-687 EI03KMH11-5725 ISKIEI04KMH5 EI05KMH3-5727 ISEI06KMH11-3026 ISKEI07KMH4-3281s1 --------------------------------------------------------------------EI07KMH10-5732 VNEI08KMH5-5165 VGNEI08KMH8-5427s1 ------------------------------------------------------------------------------------------------09KMH5-11475 ISEI09KMH5-11475s2 HQS----------------------10KMH10-9907s1 ------------------------------------------------------------------------------------------------11KMH4-9917 VDI12KMH4-10096 VGNEI

00YJN10-9385 TRGNMS01YJN9-4850 RGNMS03YJN10-840 VVRGNMS05YJN2-1948 VVRGNMS06YJN10-3044 VRGNMS07YJN4-3248 VVRGNMQI07YJN8-5627 VRGNMQS08YJN3-5606 VRGNMQS08YJN8-5408 VRGNPVMQS09YJN2-6041 VRGNPVMQS09YJN12-9156 VRGNVMQS10YJN6-9918 VRGNVMQS11YJN2-9921 VRGNVMS12YJN7-11382 VRGNVMQS13YJN1-11381 VRGNVMSKor consensus MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKEWVYRHHYESTHPRISSEVHIPLGDAKLVITTYWGLHTGEREWHLGQGVSIEWRKKRYNT

1

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ +minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

87-05

89-17

97 120 140 160 180 192QVDPDLADKLIHLHYFDCFSDSAIRHAILGRLVRPKCEYQAGHNKVGSLQYLALTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH

91CSR4-10026 TQRYKHISSTA91CSR11-6922 TQRYKHISSTA92CSR5-9885 TQRYKHISSTA93CSR6-10159 TQRYKHISSTA93CSR6-10162s1 ------------------------------------------------------------------------------------------------03CSR9-805 AQRYEKHISSTA05CSR3 TQRYKHISSTAK06CSR10-5280 TQRYKHISRTA08CSR3-5334 TQRYKHISSTA08CSR8-5279 TQRYKHISSTA10CSR7-8126 TQRYKHISSTIA12CSR2-10141 TQRYKHISSTIA

91KJS12-8538 92KJS3-6819 93KJS7-10155 97KJS3-3814 01KJS2-5667 03KJS3-745 03KJS7-775 05KJS12-3264 08KJS5-5157 10KJS5-10133

minus minusminus minusminus minusminus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ +

Patient Kor consensus KRG HAART

20 60 80 9640

RR

VS

VRE

VR

VRS

VRRES

VRRES

PKVPVIFRHIES

EVRRHES

VRRHES

Figure 2 Continued

8 Evidence-Based Complementary and Alternative Medicine

90-05

90-18

90-50

91-20

92LSK11-9417 YETRQE94LSK6-759 YETRPQES96LSK6-11491 GRYETRQ98LSK4-11495 YENTRPQES00LSK8-9999 YPQE03LSK6-10001s1 -------------------------------------------QRES04LSK7 YETRPQES06LSK1-3236 YEVTRPQRES07LSK2-3232 YEVTRPQES08LSK1-4762 YEVTRPQAESS09LSK4-6143 YEVTRPQES10LSK4-9165 YEVTRPQES10LSK4-9168 YEVTRPQES12LSK4-10131 NYKVTRPQRAES12LSK4-10132 YEVTRPQAESQ13LSK5-11470 PYVRRPTQETESS13LSK5-11471 YVTRPQRES13LSK5-11472 YENRTRQRES

92LSH3-6951 YRDH92LSH11-8548 GYRD93LSH10-1252 YRDR94LSH2-740 YRDR95LSH5-10562 YTRDR96LSH5-10609 YTR98LSH4-10567 YQTRRG---H99LSH1-5776 YQTRRG---RH99LSH1-5777 YQTRRHG---H99LSH1-5778 YQTRG---H99LSH6-5781 YQTRRQG---H99LSH6-5782 YQTRG---H00LSH3-5704 YQTRRG---H00LSH3-5705 YQTRRQSG---H00LSH3-5706 YQTRAG---H02LSH10-5707 YETRQG---H02LSH10-5708 YQTRG---H02LSH10-5710 YQTRRQR---H03LSH5-5650 YQTRRR---03LSH5-5651 YQTRRR---H03LSH5-5652 YQTRRER---03LSH10-5653 YQTRRQRS04LSH7-4848 YQTRRKQG---H04LSH8-3011 YR06LSH8-5647 YQTRRG---H06LSH8-5649 YQTRRG---H06LSH11-3238 YQHTRRQES08LSH5-5644 YQTRRSG---H08LSH5-5645 YQTRQAKS08LSH5-5646 YQTRRR---H08LSH5-5647 YQTRRR---H08LSH11-5694 YQTRRSRKG---H10LSH4-9190 YEQCTRRRG---H10LSH4-9192 YQRTRRRRG---H10LSH4-9193 YQTRRG---H12LSH6-10100 YQTRRG---H12LSH6-10101 YQTRRSRG---H

91KJin7-6723 TT93KJin2-10024 TT93KJin9-8530 VTPAKH01KJin7-5383 ISTT03KJin6-683 ISTPQH04KJin8-1955 ITPTH06KJin1-3030 VTPTH06KJin9-3012 VAPT07KJin3-5148 QEPTN07KJin3-5961 NLEPTAN10KJin4-9162 VTTAH10KJin9-8176 QVTAAH11KJin12-10119 VTPTADH13KJin1-11373 VTPTH

PE94JWK12-5524 VQ97JWK HR99JWK5-11441 HR00JWK3-11453 HR00JWK12-11445 HR01JWK7-5603 NHRQ01JWK7-5603s1 -------------------------------------------------------------------------------02JWK8-11436 NRQQ03JWK8-677 HR05JWK1-5600 NRQQ06JWK7-3042 HRQ

HRQ08JWK1-4772 NRQQ

NRQQ11JWK4-8993 NRQQ12JWK1-10125 NRQQ13JWK1-11366 NRQQ

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ +

10JWK4 9159

06 JWK12 5624

91JWK6 6949

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 9

92-13

93-04

93-60

92CWS5-9398 HSE93CWS3-9925 HE93CWS5-6669 EHTE93CWS7-802 KHTE96CWS11-5341 KHTE99CWS10-5338 HPES01CWS2-10532 HPDE03CWS6-684 HPE03CWS10-5773 HPDE04CWS5-3239 HPTE05CWS5-5783 HPTD----05CWS5-5785 HPTD----06CWS2-3071 HPTDE06CWS2-3203 HPTD----06CWS2-3221 HPTD----06CWS2-3222 VHPTD----06CWS2-3223 HPTD----06CWS8-3038 HPTD----06CWS8-3039 HPTD----07CWS4-3820 HPTATD----07CWS4-3821 HPTATD----07CWS4-10174 HPTATD----07CWS4-10175 HPTATD----07CWS4-10176 HPTATD----07CWS4-10177 VHRD----07CWS9-6252 HPE07CWS9-6253 HPTATD----07CWS9-6254 HPTATD----07CWS12-4777 HPTATD----08CWS5-6214 HPTATD----08CWS8-5160 HPTATD----08CWS8-5161 HPTATD----08CWS12-5720 HPTATD----08CWS12-5721 HPTATD----08CWS12-5722 HPTATD----09CWS5-6256 HPTATD----09CWS5-6257 HPTTATD----09CWS5-6258 HPTAD----09CWS12-9108 HPDE09CWS12-9109 HPTATTD----

93KYR2-9472 NSRIRKE94KYR6-10137 NSRRKE94KYR12-9441 NSRIRKER95KYR5-815 NSRIRKE00KYR9-9913 NSRRRKE02KYR7-9911 NSRRRKE03KYR1-817 NSRIRKE06KYR7-5236 QKPHRIRKE06KYR11-5249 QKPHRIRKE07KYR3-5265 NSHRIRKE07KYR3-11387s1 ---------------QNSHRIRRKER07KYR8-4845 QNSRRIRKER07KYR8-4846 NSRRIRKE07KYR8-5166 KNSHRRRIRKER07KYR8-5167 NKHRRIRRKE07KYR8-5168 IQRKPHRRIRKE

94KMH5 HVPR96KMH11-8534 VRT97KMH4-9452 HVQR01KMH11-5526 HVPR03KMH5-687 HVPR03KMH11-5725 HVQRS04KMH5 VQRS05KMH3-5727 HVPR06KMH11-3026 HVPR07KMH4-3281s1 VHVPRV----------------------------07KMH10-5732 HVQPRVRS08KMH5-5165 HIQPRVRS08KMH8-5427s1 ----------------------------HVQPTRVRS09KMH5-11475 HVQPRVRS09KMH5-11475s2 ------------------------------------------------------------------------------------------------10KMH10-9907s1 -----------------------------------------RVRS11KMH4-9917 HVPRVRS12KMH4-10096 HVPRVRRS

00YJN10-9385 ETVSRE01YJN9-4850 ETVSRE03YJN10-840 ETVSRE05YJN2-1948 ETQVSRE06YJN10-3044 ETQVSRE07YJN4-3248 ETQVSREY07YJN8-5627 ETQVSREY08YJN3-5606 ETQVSREY08YJN8-5408 ETQVSREY09YJN2-6041 ETQVSREY09YJN12-9156 ETQVSREY10YJN6-9918 ETQVSRIEY11YJN2-9921 ETQVSREY12YJN7-11382 ETQVSREY13YJN1-11381 ETQVSREYKor consensus QVDPDLADKLIHLHYFDCFSDSAIRHAILGRLVRPKCEYQAGHNKVGSLQYLALTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH

97 120 140 160 180 192

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ +

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

96-51

Figure 2 Sequential alignment of the amino acids of the Vif protein over 20 years All sequences that demonstrated small in-frame deletionsgross deletions and stop codons are depicted together with the baseline and last sequences of each patient Patients 90-18 demonstrated a9 bp deletion (positions 182ndash184) in April 1998 and patient 92-13 demonstrated a 12 bp deletion (positions 185ndash188) in May 2005 Patient90-05 consistently demonstrated a 9 bp insertion (RQTRAR-RAR-NGASRP) at the same position at the terminus of vpr beginning in August2000 (data not shown) The code 92LSH3-6951 (sampling year initials of the patient sampling month and sequence number) denotes thatthe sampling date is March 1992 and the sequence number is 6951 in patient LSH (90-18) The dot (sdot) hyphen (-) and asterisk (lowast) denotesequence identity deletions and premature stop codons respectively compared with the Korean consensus [20] In the right column theplus (+) and minus (minus) signs denote the presence or absence of the corresponding therapy respectively

10 Evidence-Based Complementary and Alternative Medicine

61 69 112 120 208 216 264 272 520 528Patient WT nucleotide TGGAAAAGT TGGGTCTAT TGGGGTCTG TGGAGGAAA TGGAACAAG Hypermut 20

sequence no P value90-05 10LSK4-9168 G A lt005

12LSK4-10131 A A AA A A lt000113LSK5-11470 A A A 015 13LSK5-11472 A AA lt001

90-18 08LSH5-5645 AA 019 08LSH11-5694 A 05710LSH4-9192 AA 013

90-54 07KJin3-5961 A 00693-04 94KYR12-9441 AA AG 009

07KYR8-5166 A A AA A AG lt000107KYR8-4846 A AA G 00507KYR8-5167 A A A AG 00507KYR8-5168 A A AG 02907KYR8-4845 AA G 005

93-60 03KMH11-5725 AA A 015

WT amino acid W K S W V Y W G L W R K W N K21 22 23 38 39 40 70 71 72 89 90 91 174 175 176

Figure 3 Positions of the premature stop codons in the 15 vif genes from five patients All stop codons resulted from GG rarr GA orGG rarr AG changes APOBEC3G exhibits an intrinsic preference for the second cytosine in a 51015840CC dinucleotide motif leading to 51015840GG-to-AG mutations [38]

In total five of our patients demonstrated in-frame deletionsof the vif gene of the 75 KSB-infected patients who weretreated with KRG Also in our present study cohort fivepatients including 90-18 and 92-13 demonstrated deletedvif genes (small in-frame and gross deletions) during KRGintake whereas all of our 10 LTSP patients demonstrateddeletions in the nef gene (119875 lt 005) [14] In additioncompared with the reported deletions of the nef and 51015840LTRgag genes the locations of the deleted vif gene werepositioned within a narrow range at the terminus [15 16 23]

Regarding gross deletions to date only two patientshave demonstrated gross deletions in the vif gene [17 18]and only one patient has demonstrated the insertion of twoamino acids and these patients were part of a nonprogress-ing mother-child pair [28] However in our present studyof the 10 LTSP patients who were assessed four patientsdemonstrated gross deletion in the vif gene during KRGintake prior to receiving HAART (15) This frequency issignificantly lower than the previously reported incidence of10 of 10 patients demonstrating gross deletions in the nef(187) and 51015840 LTRgag genes (376 119875 lt 001) [27 28]The frequency of deletion in the nef gene was reported tosignificantly decrease during KRG plus HAART (119875 lt 001)[16] In contrast to previous studies on the nef and gag genes[16 29] the detection of in-frame deletions in the vif genewas not suppressed in the patients receiving HAART in ourpresent cohort The reasons for this include the following (1)the proportion and frequency (approximately two-thirds) ofdeleted vif genes after the first occurrence were much higherthan in the nef and 51015840 LTRgag genes (lt20 and 30 resp)(2) small deletions in the vif gene occurred as a single bandwhereas gross deletions in the nef and 51015840 LTRgag genes weremainly detected as two bands (wild type and short) per PCRreaction and (3) the number of amplicons indicating PSCdueto G-to-A hypermutation was significantly higher in patients

receiving KRG plus HAART than patients receiving onlyKRG This phenomenon is consistent with our previous datain 51015840 LTRgag gene [29] However the frequency of G-to-Ahypermutationwas significantly lower in vif gene than that in51015840 LTRgag gene [29] Thus for the vif gene the detection ofsmall deletionsmight be less affected by limit-diluting effectsas has been shown for the nef gene [29] Taken together theseadditional defects in the vif genes in the same patients mightbe related to the replicative impairment in vif defective HIV-1 which ultimately results in defects in the synthesis of viralDNA [30]

In our present study it appears that the frequency ofhypermutation was higher in patients who maintained goodcompliance with HAART during HAART plus KRG (90-0590-18 and 93-04) than in the patients who demonstratedpoor compliance (Figure 1) In our extended data analysis124 (60485) 31 (5161) 30 (19617) and 09 (1108)of vif genes during KRG plus HAART HAART aloneKRG alone and control revealed PSC respectively Thesedata indicate the presence of synergistic effect of KRG plusHAART on PSC (119875 lt 001) There was no case that smalldeletions were induced by HAART alone About 6ndash43 ofthe integrated proviral pol gene is hypermutated by HAART[22 31 32] although hypermutated viral genomes are notpresent in plasma [32]

ApoBec3G-induced hypermutation in LTNPs has beenreported previously [33] Indeed it has been reported alsothat LTNP patients harbor PSC-containing vif genes morefrequently than progressors [25] although the reportedfrequencies of defective genes vary among studies [34] Inour current analyses vif gene-containing PSC due to G-to-A hypermutation was found to be significantly higher duringKRG plus HAART than only KRG intake prior to HAARTThis finding is consistent with our previous data obtainedfrom a cohort of the Korean hemophiliacs (21 incidence

Evidence-Based Complementary and Alternative Medicine 11

of G-to-A hypermutation while receiving only KRG versus98 on KRG plus HAART unpublished data) and otherobservations regarding the pol gene [22]

Previous studies report that certain vif mutations such asL81M R132S S130I and the insertion of two amino acids areassociatedwith slow progression or low viral loads [17 18 28]In our present study L81M [35] and R132S were consistentlydetected in patient 96-51 and patients 87-05 and 96-51respectively although both mutations were found in only in2 and 3 of 169 Korean patients respectively Although thevif gene is highly conserved in HIV-1 genomes the recoveryof hypermutants might be severely underestimated in ourpresent analysis because of the primer mismatching andthe difficulties involved in detection following amplification[36 37] Overall our current data suggest that small in-frame deletion and PSCs are associated with KRG intake andHAART respectively although further studies are needed

5 Conclusions

Our data suggest that HAART and KRG intake might inducePSCs and small in-frame deletions respectively

Conflict of Interests

The authors have no conflict of interests to declare

Acknowledgments

This work was supported by a grant from the Korean Societyof Ginseng (2010ndash2012) The authors thank Professor Seung-Chul Yun the Division of Biostatistics University of UlsanCollege of Medicine Asan Medical Center for the statisticalconsultation

References

[1] E Ernst ldquoPanax ginseng an overview of the clinical evidencerdquoJournal of Ginseng Research vol 34 no 4 pp 259ndash263 2010

[2] K T Choi ldquoBotanical characteristics pharmacological effectsand medicinal components of Korean Panax ginseng C AMeyerrdquoActa Pharmacologica Sinica vol 29 no 9 pp 1109ndash11182008

[3] S J Fulder ldquoGinseng and the hypothalamic-pituitary control ofstressrdquo The American Journal of Chinese Medicine vol 9 no 2pp 112ndash118 1981

[4] V K Singh S S Agarwal and B M Gupta ldquoImmunomodula-tory activity of Panax ginseng extractrdquo Planta Medica vol 50no 6 pp 462ndash465 1984

[5] X Song and S Hu ldquoAdjuvant activities of saponins fromtraditional Chinese medicinal herbsrdquo Vaccine vol 27 no 36pp 4883ndash4890 2009

[6] D G Yoo M C Kim M K Park et al ldquoProtective effectof ginseng polysaccharides on influenza viral infectionrdquo PLoSONE vol 7 no 3 Article ID e33678 2012

[7] H X Sun Y P Ye H J Pan and Y J Pan ldquoAdjuvant effectof Panax notoginseng saponins on the immune responses toovalbumin in micerdquo Vaccine vol 22 no 29-30 pp 3882ndash38892004

[8] J Sun S Hu and X Song ldquoAdjuvant effects of protopanaxadioland protopanaxatriol saponins from ginseng roots on theimmune responses to ovalbumin in micerdquo Vaccine vol 25 no6 pp 1114ndash1120 2007

[9] P W Hunt ldquoHIV and inflammation mechanisms and conse-quencesrdquo Current HIVAIDS Reports vol 9 no 2 pp 139ndash1472012

[10] T Yayeh K H Jung H Y Jeong et al ldquoKorean red ginsengsaponin fraction downregulates proinflammatory mediators inLPS stimulated RAW264 7 cells and protects mice againstendotoxic shockrdquo Journal of Ginseng Research vol 36 no 3 pp263ndash269 2012

[11] A T Smolinski and J J Pestka ldquoModulation of lipopolysaccha-ride-induced proinflammatory cytokine production in vitroand in vivo by the herbal constituents apigenin (chamomile)ginsenoside Rb1 (ginseng) and parthenolide (feverfew)rdquo Foodand Chemical Toxicology vol 41 no 10 pp 1381ndash1390 2003

[12] A Rhule S Navarro J R Smith and D M ShepherdldquoPanax notoginseng attenuates LPS-induced pro-inflammatorymediators in RAW2647 cellsrdquo Journal of Ethnopharmacologyvol 106 no 1 pp 121ndash128 2006

[13] J M Brenchley D A Price T W Schacker et al ldquoMicrobialtranslocation is a cause of systemic immune activation inchronic HIV infectionrdquo Nature Medicine vol 12 no 12 pp1365ndash1371 2006

[14] Y K Cho J Y Lim Y S Jung S K Oh H J Lee and H SungldquoHigh frequency of grossly deleted nef genes in HIV-1 infectedlong-term slow progressors treated with Korean red ginsengrdquoCurrent HIV Research vol 4 no 4 pp 447ndash457 2006

[15] Y K Cho and Y S Jung ldquoHigh frequency of gross deletions inthe 51015840 LTR and gag regions in HIV type 1-infected long-termsurvivors treated with Korean red ginsengrdquo AIDS Research andHuman Retroviruses vol 24 no 2 pp 181ndash193 2008

[16] Y K Cho Y S Jung andH S Sung ldquoFrequent gross deletion intheHIV type 1 nef gene in hemophiliacs treatedwith korean redginseng inhibition of detection by highly active antiretroviraltherapyrdquo AIDS Research and Human Retroviruses vol 25 no 4pp 419ndash424 2009

[17] V Sandonıs C Casado T Alvaro et al ldquoA combination ofdefective DNA and protective host factors are found in a setof HIV-1 ancestral LTNPsrdquo Virology vol 391 no 1 pp 73ndash822009

[18] H R Rangel D Garzaro A K Rodrıguez et al ldquoDeletioninsertion and stop codon mutations in vif genes of HIV-1 infecting slow progressor patientsrdquo Journal of Infection inDeveloping Countries vol 3 no 7 pp 531ndash538 2009

[19] Y K Cho J E Kim and B T Foley ldquoPhylogenetic analysisof near full-length HIV-1 genomic sequences in 21 Koreanindividualsrdquo AIDS Research and Human Retroviruses vol 29no 4 pp 738ndash743 2013

[20] Y K Cho Y S Jung J S Lee and B T Foley ldquoMolecu-lar evidence of HIV-1 transmission in 20 Korean individu-als with haemophilia phylogenetic analysis of the vif generdquoHaemophilia vol 18 no 2 pp 291ndash299 2012

[21] P P Rose and B T Korber ldquoDetecting hypermutations inviral sequences with an emphasis on GrarrA hypermutationrdquoBioinformatics vol 16 no 4 pp 400ndash401 2000

[22] S Fourati S Lambert-Niclot C Soulie et al ldquoHIV-1 genomeis often defective in PBMCs and rectal tissues after long-termHAART as a result of APOBEC3 editing and correlates with thesize of reservoirsrdquo Journal of Antimicrobial Chemotherapy vol67 no 10 pp 2323ndash2326 2012

12 Evidence-Based Complementary and Alternative Medicine

[23] U Wieland A Seelhoff A Hofmann et al ldquoDiversity of thevif gene of human immunodeficiency virus type 1 in UgandardquoJournal of General Virology vol 78 no 2 pp 393ndash400 1997

[24] P Sova M van Ranst P Gupta et al ldquoConservation of an intacthuman immunodeficiency virus type 1 vif gene in vitro and invivordquo Journal of Virology vol 69 no 4 pp 2557ndash2564 1995

[25] T Yamada and A Iwamoto ldquoComparison of proviral accessorygenes between long-term nonprogressors and progressors ofhuman immunodeficiency virus type 1 infectionrdquo Archives ofVirology vol 145 no 5 pp 1021ndash1027 2000

[26] K Tominaga S Kato M Negishi and T Takano ldquoA high fre-quency of defective vif genes in peripheral blood mononuclearcells from HIV type 1-infected individualsrdquo AIDS Research andHuman Retroviruses vol 12 no 16 pp 1543ndash1549 1996

[27] Y K Cho H Sung I G Bae et al ldquoFull sequence of HIV type 1Korean subtype B in anAIDS case with atypical seroconversionTAAAA at TATA boxrdquoAIDS Research andHuman Retrovirusesvol 21 no 11 pp 961ndash964 2005

[28] L Alexander M J Aquino-DeJesus M Chan and W AAndiman ldquoInhibition of human immunodeficiency virus type 1(HIV-1) replication by a two-amino-acid insertion in HIV-1 Viffrom a nonprogressing mother and childrdquo Journal of Virologyvol 76 no 20 pp 10533ndash10539 2002

[29] YK Cho Y S JungH S Sung andCH Joo ldquoFrequent geneticdefects in the HIV-1 51015840LTRgag gene in hemophiliacs treatedwith Korean red ginseng decreased detection of genetic defectsby highly active antiretroviral therapyrdquo Journal of GinsengResearch vol 35 no 4 pp 413ndash420 2011

[30] M Nascimbeni M Bouyac F Rey B Spire and F Clavel ldquoThereplicative impairment of Vif-mutants of human immunodefi-ciency virus type 1 correlates with an overall defect in viral DNAsynthesisrdquo Journal of General Virology vol 79 no 8 pp 1945ndash1950 1998

[31] J P Vartanian M Henry and S Wain-Hobson ldquoSustainedGrarrA hypermutation during reverse transcription of an entirehuman immunodeficiency virus type 1 strain Vau group Ogenomerdquo Journal of General Virology vol 83 no 4 pp 801ndash8052002

[32] T L Kieffer P Kwon R E Nettles Y Han S C Ray andR F Siliciano ldquoGrarrA hypermutation in protease and reversetranscriptase regions of human immunodeficiency virus type 1residing in resting CD4+ T cells in vivordquo Journal of Virology vol79 no 3 pp 1975ndash1980 2005

[33] B Wang M Mikhail W B Dyer J J Zaunders A D Kelleherand N K Saksena ldquoFirst demonstration of a lack of viralsequence evolution in a nonprogressor defining replication-incompetent HIV-1 infectionrdquo Virology vol 312 no 1 pp 135ndash150 2003

[34] G Hassaine I Agostini D Candotti et al ldquoCharacterizationof human immunodeficiency virus type 1 vif gene in long-termasymptomatic individualsrdquoVirology vol 276 no 1 pp 169ndash1802000

[35] F A de Maio C A Rocco P C Aulicino R Bologna AMangano and L Sen ldquoUnusual substitutions in HIV-1 Vif fromchildren infected perinatally without progression to AIDS formore than 8 years without therapyrdquo Journal of Medical Virologyvol 84 no 12 pp 1844ndash1852 2012

[36] M Janini M Rogers D R Birx and F E McCutchan ldquoHumanimmunodeficiency virus type 1 DNA sequences geneticallydamaged by hypermutation are often abundant in patientperipheral blood mononuclear cells and may be generated

during near-simultaneous infection and activation of CD4+ Tcellsrdquo Journal of Virology vol 75 no 17 pp 7973ndash7986 2001

[37] G H Kijak L M Janini S Tovanabutra et al ldquoVariablecontexts and levels of hypermutation inHIV-1 proviral genomesrecovered from primary peripheral blood mononuclear cellsrdquoVirology vol 376 no 1 pp 101ndash111 2008

[38] E W Refsland J F Hultquist and R S Harris ldquoEndogenousorigins of HIV-1 G-to-A hypermutation and restriction in thenonpermissive T cell line CEM2nrdquo PLoS Pathogens vol 8 no7 Article ID e1002800 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 4: Effects of Korean Red Ginseng and HAART on vif Gene in 10 Long

4 Evidence-Based Complementary and Alternative Medicine

0

1

2

3

4

5

6

7

0100200300400500600700

87-05Korean Red Ginseng (KRG) KRG KRG KRG

Jun

87Se

p 89

Apr

91

Nov

91

Nov

92

Dec

93

Jun

94D

ec 9

4O

ct 9

5O

ct 9

6A

pr 9

7A

pr 9

8O

ct 9

8A

pr 9

9O

ct 9

9M

ar 0

0Fe

b 01

Jul 0

1Se

p 02

Sep

03O

ct 0

4M

ar 0

5M

ay 0

5O

ct 0

5M

ay 0

6O

ct 0

6Ja

n 07

May

07

Aug

07

Oct

07

Feb

08A

ug 0

8D

ec 0

8Ju

n 09

Dec

09

Jul 1

0M

ar 1

1Se

p 11

Feb

12A

ug 1

2D

ec 1

2

CD4

+ T

cells

(120583

L)

CD4+ T cells (120583L)

minus100 0

1

2

3

4

5

6

7

0200400600800

1000120014001600 89-17

KRG KRG HAARTKRGKRG KRG

Aug

89

Jan

90Se

p 90

Apr

91

Dec

91

Jan

92M

ar 9

2Ju

n 92

Nov

92

Feb

93A

pr 9

3Ju

l 93

Oct

93

Apr

94

Oct

94

Sep

95Ju

l 96

Oct

96

Mar

97

Aug

97

Apr

98

Jun

99N

ov 9

9M

ar 0

0Se

p 00

Feb

01Ju

l 01

Jan

02M

ar 0

2Se

p 02

Feb

03M

ay 0

3Ja

n 04

Aug

04

Mar

05

Dec

05

Oct

06

Nov

06

Feb

07A

pr 0

7A

ug 0

7D

ec 0

7Ju

l 08

May

09

May

10

Mar

12

RNA

copy

(log

mL)

minus200

0

1

2

3

4

5

6

7

050

100150200250300350400450 90-05

KRG HAARTKRG

lowast lowast lowast

Jul 9

1N

ov 9

1N

ov 9

2A

pr 9

3O

ct 9

3Ju

n 94

Jan

95M

ay 9

5A

ug 9

6A

ug 9

7A

pr 9

8M

ar 9

9A

ug 0

0Fe

b 01

May

01

Mar

02

Aug

02

Jan

03M

ar 0

3Ju

n 03

Oct

03

Mar

04

Aug

04

Feb

05Ju

l 05

Oct

05

Jan

06Ju

l 06

Oct

06

Feb

07M

ay 0

7A

ug 0

7O

ct 0

7Ja

n 08

Apr

08

May

08

Aug

08

Nov

08

Apr

09

Oct

09

Apr

10

Oct

10

Apr

11

Oct

11

Apr

12

Nov

12

May

13

CD4

+ T

cells

(120583

L)

0

1

2

3

4

5

6

7

0100200300400500600700800900

1000 90-18

HAARTKRGKRG KRG KRGKRGKRGKRG KRG

Aug

90

Feb

91A

ug 9

1D

ec 9

1M

ar 9

2Ju

n 92

Nov

92

Mar

93

May

93

Jun

93O

ct 9

3Fe

b 94

May

94

Aug

94

Nov

94

Feb

95M

ay 9

5O

ct 9

5Fe

b 96

May

96

Dec

96

Mar

97

Mar

97

Jul 9

7M

ar 9

8A

pr 9

8Se

p 98

Feb

99A

pr 9

9Ju

n 99

Nov

99

Mar

00

Jul 0

0A

ug 0

0O

ct 0

0M

ar 0

1Ju

l 01

Dec

01

Apr

02

Oct

02

Jan

03M

ay 0

3O

ct 0

3Ju

l 04

Dec

04

Mar

05

Feb

06Ju

n 06

Jul 0

6A

ug 0

6N

ov 0

6Ja

n 07

Apr

07

Jun

07O

ct 0

7N

ov 0

7D

ec 0

7M

ay 0

8N

ov 0

8Fe

b 09

Apr

09

Oct

09

Apr

10

Oct

10

Oct

11

Jun

12D

ec 1

2

minus100

lowastlowast lowast

RNA

copy

(log

mL)

CD4

+ T

cells

(120583

L)

0

1

2

3

4

5

6

7

0

200

400

600

800

1000

1200 90-50HAARTKRGKRGKRGKRG

Dec

90

Jul 9

1Ja

n 92

Jul 9

2Fe

b 93

Apr

93

Sep

93M

ar 9

4Se

p 94

Apr

95

Oct

95

Apr

96

Nov

96

May

97

Nov

97

Jun

98N

ov 9

8M

ay 9

9N

ov 9

9Se

p 00

Jul 0

1N

ov 0

1Ju

n 02

Dec

02

Mar

03

Jun

03Se

p 03

Mar

04

Aug

04

Jan

05A

ug 0

5Ja

n 06

Jul 0

6Se

p 06

Mar

07

May

07

Jun

07Ju

l 07

Oct

07

Nov

07

Jul 0

9D

ec 0

9A

pr 1

0Ju

l 10

Jan

11A

pr 1

1D

ec 1

1Ju

l 12

Jan

13

lowast

CD4+

T ce

lls (120583

L)

minus200 0

1

2

3

4

5

6

7

0

200

400

600

800

1000

120091-20

KRG HAARTKRGKRG

Jun

91D

ec 9

1M

ar 9

2M

ay 9

2Ja

n 93

May

93

Dec

93

Jul 9

4N

ov 9

4D

ec 9

4Fe

b 95

Mar

95

May

95

Oct

95

Apr

96

Aug

96

Mar

97

Jul 9

7A

pr 9

8N

ov 9

8M

ay 9

9N

ov 9

9M

ar 0

0A

pr 0

0Ju

l 00

Dec

00

Mar

01

Jul 0

1N

ov 0

1M

ar 0

2A

ug 0

2Ja

n 03

Mar

03

Aug

03

Jan

04Ju

l 04

Jan

05Ju

l 05

Jan

06Ju

l 06

Dec

06

May

07

Jul 0

7A

ug 0

7Se

p 07

Nov

07

Jan

08A

pr 0

8Ju

l 08

Dec

08

Oct

10

Apr

11

Jan

12Ju

l 12

Jan

13

minus200

RNA

copy

(log

mL)

CD4+

T ce

lls (120583

L)

0

1

2

3

4

5

6

7

050

100150200250300350400450

92-13KRG HAART HAART

KRGKRGKRGKRGKRG

Expired in 2011

May

92

Nov

92

May

93

Jul 9

3Ja

n 94

Oct

94

Dec

94

Feb

95A

pr 9

5O

ct 9

5A

pr 9

6N

ov 9

6Fe

b 97

Jun

97A

pr 9

8Ja

n 99

Oct

99

Feb

01D

ec 0

1Ju

l 02

Dec

02

Mar

03

Jun

03O

ct 0

3M

ay 0

4N

ov 0

4M

ay 0

5Ju

l 05

Feb

06A

ug 0

6N

ov 0

6A

pr 0

7Ju

n 07

Jul 0

7A

ug 0

7Se

p 07

Dec

07

May

08

Aug

08

Dec

08

May

09

Dec

09

Apr

10

Jun

10Ja

n 11

CD4

+ T

cells

(120583

L)

minus500

1

2

3

4

5

67

0100200300400500600700800900 93-04

KRG HAARTKRG KRG KRGKRG

Feb

93M

ar 9

3Ju

l 93

Dec

93

Feb

94Ju

n 94

Dec

94

May

95

Nov

95

Feb

96Se

p 96

Jan

97Fe

b 97

Jul 9

7A

ug 9

7A

pr 9

8Ju

l 98

Sep

00D

ec 0

0M

ar 0

1Ju

n 01

Sep

01Ju

n 02

Jul 0

2Ja

n 03

May

03

Oct

03

Mar

04

Aug

04

Jan

05M

ay 0

5Ju

n 05

Aug

05

Oct

05

Mar

06

Jul 0

6N

ov 0

6M

ar 0

7A

ug 0

7

lowastlowast

RNA

copy

(log

mL)

CD4

+ T

cells

(120583

L)

RNA

copy

(log

mL)

RNA copy (logmL)

RNA

copy

(log

mL)

RNA

copy

(log

mL)

RNA

copy

(log

mL)

if if

if

if

ifif

ifif

Sampling date Sampling date

Sampling date Sampling date

Sampling date Sampling date

Sampling date Sampling dateCD4+ T cells (120583L)RNA copy (logmL)

CD4

+ T

cells

(120583

L)

Figure 1 Continued

Evidence-Based Complementary and Alternative Medicine 5

0

1

2

3

4

5

6

7

0100200300400500600700800

93-60

HAARTKRGKRG

Apr

94

Nov

95

May

96

Nov

96

Apr

97

Jun

97Ju

n 99

Nov

99

Nov

00

Jul 0

1N

ov 0

1Ja

n 02

Sep

02Fe

b 03

May

03

Nov

03

May

04

Mar

05

Sep

05A

pr 0

6O

ct 0

6N

ov 0

6A

pr 0

7Ju

l 07

Aug

07

Oct

07

Jan

08M

ay 0

8M

ay 0

8Ju

l 08

Aug

08

Dec

08

May

09

Dec

09

Oct

10

Apr

11

Apr

12

lowast

0

1

2

3

4

5

6

7

0

200

400

600

800

1000

120096-51

KRG HAARTKRGKRG

Jul 9

6Ja

n 97

Jul 9

7A

pr 9

8N

ov 9

8M

ay 9

9Ju

n 00

Oct

00

Sep

01A

pr 0

2D

ec 0

2O

ct 0

3Fe

b 04

Aug

04

Feb

05A

ug 0

5N

ov 0

6Ju

l 06

Oct

06

Feb

07A

ug 0

7M

ar 0

8A

ug 0

8Fe

b 09

Jul 0

9D

ec 0

9M

ay 1

0Ju

n 10

Apr

11

Oct

11

Jul 1

2Ja

n 13

RNA

copy

(log

mL)

CD4

+ T

cells

(120583

L)

Sampling dateCD4+ T cells (120583L)RNA copy (logmL)

RNA

copy

(log

mL)

CD4

+ T

cells

(120583

L)

Sampling dateCD4+ T cells (120583L)RNA copy (logmL)

if if

Figure 1 Changes in the CD4+ T-cell count plasma viral load and genetic defects in terms of Korean red ginseng (KRG) intake and highlyactive antiretroviral therapy (HAART) The durations of KRG intake and HAART are indicated by the bars Solid and dotted lines denotegood (gt90) and poor (lt90) compliance according to self-administered responses respectively The upward arrow (uarr) downward arrow(darr) plus sign (+) and asterisk (lowast) denote the sequences of the vif gene gross deletions 3- and- 6-base pair (bp) in-frame insertions and stopcodons respectively

Table 2 Distribution of defective vif gene

Patient On-KRG On-HAARTKRGNo of genes Stop codon gΔ sΔ No of genes Stop codon gΔ sΔ

87-05 20 1 ND ND ND ND89-17 9 9 0 0 090-05 37 1 18 4 0 090-18 35 15 20 3a 0 12a

90-50 14 1 15 0 0 091-20 35 1 20 0 0 092-13 53 14 22 0 0 1393-04 18 1 12 5 1 093-60 28 1 1 20 0 6 096-51 26 21 0 0 0Total 275 3b 4 29 157 12b 7 25gΔ and sΔ denote gross deletion and in-frame small deletion respectivelyHAART highly active antiretroviral therapy ND not determinedaTwo out of three genes contained both stop codon and sΔb119875 lt 001119875 lt 001 for the sum of 3 kinds of defective genes (36275 versus 42157)Fifty-two vif genes at baseline obtained from serum using RT-PCR were all wild types lowastControl patients (119899 = 21) revealed premature stop codon in one outof 106 vif genes

stop codons 119875 lt 005) and 51015840 LTRgag genes (71 of 189 genes119875 lt 0001) identified in the same patients [14 15]

4 Discussion

Todate we have identified the vif gene in 194Korean patientsOf these 145 patients were infected with the Korean subcladeB ofHIV-1 (KSB) and the remaining 49 patients were infectedwith non-KSB Of these 194 patients 10 demonstrated smallin-frame deletions (3ndash15 bp) and all were diagnosed withKSB whereas no such deletions were detected in non-KSBpatients (119875 = 0068) Of the 10 patients with small deletionsthree patients were included in a group of 20 hemophiliacs

who were infected with KSB from plasma donors O and P[16 20] Interestingly plasma donors O and P revealed wild-type vif only between 1991 and 2002 (AY581320-21 JQ248127-33 JF957909 JF957921 JQ248134 and JF957935-37) In otherwords original vif gene was wild type without in-framesmall deletion In our current study only two patients (90-18 and 92-13) demonstrated 9 (119899 = 15) and 12 (119899 = 14)bp in-frame deletions in about two-thirds of the ampliconsobtained after 63 and 120 years of KRG intake respectivelywhereas five patients without KRG intake demonstrated in-frame deletions in all amplicons (data not shown) To ourknowledge these in-frame deletions are very rare and haveonly been reported in two patients in other countries [26 27]

6 Evidence-Based Complementary and Alternative Medicine

87-05

89-17

90-05

90-18

90-50

91-20

1 20 40 60 80 96Kor consensus MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKEWVYRHHYESTHPRISSEVHIPLGDAKLVITTYWGLHTGEREWHLGQGVSIEWRKKRYNT

91CSR4-10026 RGNVVD91CSR11-6922 RGNVVD92CSR5-9885 RGNVVD93CSR6-10159 RGNVVD93CSR6-10162s1 ------------------------------------------------------------------------------------------------03CSR9-805 SGGNVVD05CSR3 RGNIVVDG06CSR10-5280 RGNIVVDRG08CSR3-5334 RGNNVDG08CSR8-5279 RGNIVVD10CSR7-8126 VRGNLVVDG12CSR2-10141 VRGNLVVDG

91KJS12-8538 I92KJS3-6819 GI93KJS7-10155 IAI97KJS3-3814 IGKEI01KJS2-5667 IGKI03KJS2-745 IGAI03KJS7-775 IGAEI05KJS12-3264 YMIVGAEI08KJS5-5157 IVNGAAI10KJS5-10133 IGKAES

92LSK11-9417 K94LSK6-759 KGNPRS96LSK6-11491 AKNPIRS98LSK4-11495 KHN00LSK8-9999 KGNR03LSK6-10001s1 ------------------------------------------------------------------------------04LSK7 KKGNPRST06LSK1-3236 KKGNPVRST07LSK2-3232 KKGNPVRST08LSK1-4762 KKGIPVRST09LSK4-6143 IKNPIGRST10LSK4-9165 KGNPRS10LSK4-9168 KKGNPVRTRST12LSK4-10131 KAVKALKNPEKST 12LSK4-10132 KAKNPGRST13LSK5-11470 KKGNPGRST13LSK5-11471 KKVGNPVRST13LSK5-11472 IKANNRI

92LSH3-6951 K92LSH11-8548 TKK93LSH10-1252 94LSH2-740 95LSH5-10562 V96LSH5-10609 V98LSH4-10567 VG99LSH1-5776 VG99LSH1-5777 VG99LSH1-5778 VG99LSH6-5781 VGE99LSH6-5782 VG00LSH3-5704 KVGR00LSH3-5705 VG00LSH3-5706 VGK02LSH10-5707 VGR02LSH10-5708 VG02LSH10-5710 VG03LSH5-5650 VGG03LSH5-5651 VG03LSH5-5652 VG03LSH10-5653 VG04LSH7-4848 VGKES04LSH8-3011 V06LSH8-5647 VGES06LSH8-5649 VGES06LSH11-3238 VGIS08LSH5-5644 VG08LSH5-5645 VGR08LSH5-5646 KVGE08LSH5-5647 KVGE08LSH11-5694 KVG10LSH4-9190 KVGES10LSH4-9192 VG10LSH4-9193 VGG12LSH6-10100 VGES12LSH6-10101 VG

91KJin7-6723 N93KJin2-10024 N93KJin9-8530 NHVKINVDT01KJin7-5383 KVN03KJin6-683 KVINT04KJin8-1955 KVINDT06KJin1-3030 KVKINDT06KJin9-3012 KVINT07KJin3-5148 VNKINID07KJin3-5961 VNKKINEN10KJin4-9162 NHVKKINVT10KJin9-8176 NHVKKINVDT11KJin12-10119 KVKKINVDT13KJin1-11373 VNNVNDT

K94JWK12-5524 K97JWK K99JWK5-11441 KNE00JWK3-11453 KN00JWK12-11445 KN01JWK7-5603 KVA01JWK7-5603s1 KKNN02JWK8-11435 KVA03JWK8-677 KN05JWK1-5600 KVA06JWK7-3042 KVNAN

KKNVNVVN08JWK1-4772 KVNAN

KVA11JWK4-8993 KVA12JWK1-10125 KVA13JWK1-11366 KVA

minus minusminus minusminus minusminus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ +

Patient KRG HAART

10JWK4 9159

06JWK12 5624

91JWK6 6949

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 7

92-13

93-04

93-60

96-51

92CWS5-9398 G93CWS3-9925 GK93CWS5-6669 GK93CWS7-802 G96CWS11-5341 G99CWS10-5338 KVGE01CWS2-10532 VG03CWS6-684 VGTG03CWS10-5773 VG04CWS5-3239 VGKSTG05CWS5-5783 GTG05CWS5-5785 VGKITG06CWS2-3071 VGTG06CWS2-3203 VGKITG06CWS2-3221 VGTG06CWS2-3222 NVGTG06CWS2-3223 NVGTG06CWS8-3038 NVGKTG06CWS8-3039 NVGKTG07CWS4-3820 VGKIATG07CWS4-3821 VGKIATG07CWS4-10174 HWVGKIATG07CWS4-10175 VGKIATG07CWS4-10176 VGKITG07CWS4-10177 VGKKTG07CWS9-6252 VGTG07CWS9-6253 VGKITG07CWS9-6254 VGKIATG07CWS12-4777 VGQITG08CWS5-6214 NKVGQITG08CWS8-5160 NVGKTG08CWS8-5161 NVGKNQG08CWS12-5720 VGQITG08CWS12-5721 VGQITG08CWS12-5722 VGKQITG09CWS5-6256 VGKQTG09CWS5-6257 VGKQTG09CWS5-6258 VGKDQTG09CWS12-9108 LVGTG09CWS12-9109 VGKQITG

93KYR2-9472 VNN94KYR6-10137 VNN94KYR12-9441 VNN95KYR5-815 VGNN00KYR9-9913 VGNN02KYR7-9911 VGNN03KYR1-817 VNN06KYR7-5236 VGNN06KYR11-5249 VGNNS07KYR3-5265 VGNN07KYR3-11387s1 -------------------------------------------------------------------------------------------------07KYR8-4845 VGNN07KYR8-4846 VGNN07KYR8-5166 IVRNKN07KYR8-5167 VGNRN07KYR8-5168 VGNN

94KMH5 EI96KMH11-8534 IAEI97KMH4-9452 ISEI01KMH11-5526 ISIEI03KMH5-687 EI03KMH11-5725 ISKIEI04KMH5 EI05KMH3-5727 ISEI06KMH11-3026 ISKEI07KMH4-3281s1 --------------------------------------------------------------------EI07KMH10-5732 VNEI08KMH5-5165 VGNEI08KMH8-5427s1 ------------------------------------------------------------------------------------------------09KMH5-11475 ISEI09KMH5-11475s2 HQS----------------------10KMH10-9907s1 ------------------------------------------------------------------------------------------------11KMH4-9917 VDI12KMH4-10096 VGNEI

00YJN10-9385 TRGNMS01YJN9-4850 RGNMS03YJN10-840 VVRGNMS05YJN2-1948 VVRGNMS06YJN10-3044 VRGNMS07YJN4-3248 VVRGNMQI07YJN8-5627 VRGNMQS08YJN3-5606 VRGNMQS08YJN8-5408 VRGNPVMQS09YJN2-6041 VRGNPVMQS09YJN12-9156 VRGNVMQS10YJN6-9918 VRGNVMQS11YJN2-9921 VRGNVMS12YJN7-11382 VRGNVMQS13YJN1-11381 VRGNVMSKor consensus MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKEWVYRHHYESTHPRISSEVHIPLGDAKLVITTYWGLHTGEREWHLGQGVSIEWRKKRYNT

1

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ +minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

87-05

89-17

97 120 140 160 180 192QVDPDLADKLIHLHYFDCFSDSAIRHAILGRLVRPKCEYQAGHNKVGSLQYLALTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH

91CSR4-10026 TQRYKHISSTA91CSR11-6922 TQRYKHISSTA92CSR5-9885 TQRYKHISSTA93CSR6-10159 TQRYKHISSTA93CSR6-10162s1 ------------------------------------------------------------------------------------------------03CSR9-805 AQRYEKHISSTA05CSR3 TQRYKHISSTAK06CSR10-5280 TQRYKHISRTA08CSR3-5334 TQRYKHISSTA08CSR8-5279 TQRYKHISSTA10CSR7-8126 TQRYKHISSTIA12CSR2-10141 TQRYKHISSTIA

91KJS12-8538 92KJS3-6819 93KJS7-10155 97KJS3-3814 01KJS2-5667 03KJS3-745 03KJS7-775 05KJS12-3264 08KJS5-5157 10KJS5-10133

minus minusminus minusminus minusminus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ +

Patient Kor consensus KRG HAART

20 60 80 9640

RR

VS

VRE

VR

VRS

VRRES

VRRES

PKVPVIFRHIES

EVRRHES

VRRHES

Figure 2 Continued

8 Evidence-Based Complementary and Alternative Medicine

90-05

90-18

90-50

91-20

92LSK11-9417 YETRQE94LSK6-759 YETRPQES96LSK6-11491 GRYETRQ98LSK4-11495 YENTRPQES00LSK8-9999 YPQE03LSK6-10001s1 -------------------------------------------QRES04LSK7 YETRPQES06LSK1-3236 YEVTRPQRES07LSK2-3232 YEVTRPQES08LSK1-4762 YEVTRPQAESS09LSK4-6143 YEVTRPQES10LSK4-9165 YEVTRPQES10LSK4-9168 YEVTRPQES12LSK4-10131 NYKVTRPQRAES12LSK4-10132 YEVTRPQAESQ13LSK5-11470 PYVRRPTQETESS13LSK5-11471 YVTRPQRES13LSK5-11472 YENRTRQRES

92LSH3-6951 YRDH92LSH11-8548 GYRD93LSH10-1252 YRDR94LSH2-740 YRDR95LSH5-10562 YTRDR96LSH5-10609 YTR98LSH4-10567 YQTRRG---H99LSH1-5776 YQTRRG---RH99LSH1-5777 YQTRRHG---H99LSH1-5778 YQTRG---H99LSH6-5781 YQTRRQG---H99LSH6-5782 YQTRG---H00LSH3-5704 YQTRRG---H00LSH3-5705 YQTRRQSG---H00LSH3-5706 YQTRAG---H02LSH10-5707 YETRQG---H02LSH10-5708 YQTRG---H02LSH10-5710 YQTRRQR---H03LSH5-5650 YQTRRR---03LSH5-5651 YQTRRR---H03LSH5-5652 YQTRRER---03LSH10-5653 YQTRRQRS04LSH7-4848 YQTRRKQG---H04LSH8-3011 YR06LSH8-5647 YQTRRG---H06LSH8-5649 YQTRRG---H06LSH11-3238 YQHTRRQES08LSH5-5644 YQTRRSG---H08LSH5-5645 YQTRQAKS08LSH5-5646 YQTRRR---H08LSH5-5647 YQTRRR---H08LSH11-5694 YQTRRSRKG---H10LSH4-9190 YEQCTRRRG---H10LSH4-9192 YQRTRRRRG---H10LSH4-9193 YQTRRG---H12LSH6-10100 YQTRRG---H12LSH6-10101 YQTRRSRG---H

91KJin7-6723 TT93KJin2-10024 TT93KJin9-8530 VTPAKH01KJin7-5383 ISTT03KJin6-683 ISTPQH04KJin8-1955 ITPTH06KJin1-3030 VTPTH06KJin9-3012 VAPT07KJin3-5148 QEPTN07KJin3-5961 NLEPTAN10KJin4-9162 VTTAH10KJin9-8176 QVTAAH11KJin12-10119 VTPTADH13KJin1-11373 VTPTH

PE94JWK12-5524 VQ97JWK HR99JWK5-11441 HR00JWK3-11453 HR00JWK12-11445 HR01JWK7-5603 NHRQ01JWK7-5603s1 -------------------------------------------------------------------------------02JWK8-11436 NRQQ03JWK8-677 HR05JWK1-5600 NRQQ06JWK7-3042 HRQ

HRQ08JWK1-4772 NRQQ

NRQQ11JWK4-8993 NRQQ12JWK1-10125 NRQQ13JWK1-11366 NRQQ

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ +

10JWK4 9159

06 JWK12 5624

91JWK6 6949

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 9

92-13

93-04

93-60

92CWS5-9398 HSE93CWS3-9925 HE93CWS5-6669 EHTE93CWS7-802 KHTE96CWS11-5341 KHTE99CWS10-5338 HPES01CWS2-10532 HPDE03CWS6-684 HPE03CWS10-5773 HPDE04CWS5-3239 HPTE05CWS5-5783 HPTD----05CWS5-5785 HPTD----06CWS2-3071 HPTDE06CWS2-3203 HPTD----06CWS2-3221 HPTD----06CWS2-3222 VHPTD----06CWS2-3223 HPTD----06CWS8-3038 HPTD----06CWS8-3039 HPTD----07CWS4-3820 HPTATD----07CWS4-3821 HPTATD----07CWS4-10174 HPTATD----07CWS4-10175 HPTATD----07CWS4-10176 HPTATD----07CWS4-10177 VHRD----07CWS9-6252 HPE07CWS9-6253 HPTATD----07CWS9-6254 HPTATD----07CWS12-4777 HPTATD----08CWS5-6214 HPTATD----08CWS8-5160 HPTATD----08CWS8-5161 HPTATD----08CWS12-5720 HPTATD----08CWS12-5721 HPTATD----08CWS12-5722 HPTATD----09CWS5-6256 HPTATD----09CWS5-6257 HPTTATD----09CWS5-6258 HPTAD----09CWS12-9108 HPDE09CWS12-9109 HPTATTD----

93KYR2-9472 NSRIRKE94KYR6-10137 NSRRKE94KYR12-9441 NSRIRKER95KYR5-815 NSRIRKE00KYR9-9913 NSRRRKE02KYR7-9911 NSRRRKE03KYR1-817 NSRIRKE06KYR7-5236 QKPHRIRKE06KYR11-5249 QKPHRIRKE07KYR3-5265 NSHRIRKE07KYR3-11387s1 ---------------QNSHRIRRKER07KYR8-4845 QNSRRIRKER07KYR8-4846 NSRRIRKE07KYR8-5166 KNSHRRRIRKER07KYR8-5167 NKHRRIRRKE07KYR8-5168 IQRKPHRRIRKE

94KMH5 HVPR96KMH11-8534 VRT97KMH4-9452 HVQR01KMH11-5526 HVPR03KMH5-687 HVPR03KMH11-5725 HVQRS04KMH5 VQRS05KMH3-5727 HVPR06KMH11-3026 HVPR07KMH4-3281s1 VHVPRV----------------------------07KMH10-5732 HVQPRVRS08KMH5-5165 HIQPRVRS08KMH8-5427s1 ----------------------------HVQPTRVRS09KMH5-11475 HVQPRVRS09KMH5-11475s2 ------------------------------------------------------------------------------------------------10KMH10-9907s1 -----------------------------------------RVRS11KMH4-9917 HVPRVRS12KMH4-10096 HVPRVRRS

00YJN10-9385 ETVSRE01YJN9-4850 ETVSRE03YJN10-840 ETVSRE05YJN2-1948 ETQVSRE06YJN10-3044 ETQVSRE07YJN4-3248 ETQVSREY07YJN8-5627 ETQVSREY08YJN3-5606 ETQVSREY08YJN8-5408 ETQVSREY09YJN2-6041 ETQVSREY09YJN12-9156 ETQVSREY10YJN6-9918 ETQVSRIEY11YJN2-9921 ETQVSREY12YJN7-11382 ETQVSREY13YJN1-11381 ETQVSREYKor consensus QVDPDLADKLIHLHYFDCFSDSAIRHAILGRLVRPKCEYQAGHNKVGSLQYLALTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH

97 120 140 160 180 192

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ +

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

96-51

Figure 2 Sequential alignment of the amino acids of the Vif protein over 20 years All sequences that demonstrated small in-frame deletionsgross deletions and stop codons are depicted together with the baseline and last sequences of each patient Patients 90-18 demonstrated a9 bp deletion (positions 182ndash184) in April 1998 and patient 92-13 demonstrated a 12 bp deletion (positions 185ndash188) in May 2005 Patient90-05 consistently demonstrated a 9 bp insertion (RQTRAR-RAR-NGASRP) at the same position at the terminus of vpr beginning in August2000 (data not shown) The code 92LSH3-6951 (sampling year initials of the patient sampling month and sequence number) denotes thatthe sampling date is March 1992 and the sequence number is 6951 in patient LSH (90-18) The dot (sdot) hyphen (-) and asterisk (lowast) denotesequence identity deletions and premature stop codons respectively compared with the Korean consensus [20] In the right column theplus (+) and minus (minus) signs denote the presence or absence of the corresponding therapy respectively

10 Evidence-Based Complementary and Alternative Medicine

61 69 112 120 208 216 264 272 520 528Patient WT nucleotide TGGAAAAGT TGGGTCTAT TGGGGTCTG TGGAGGAAA TGGAACAAG Hypermut 20

sequence no P value90-05 10LSK4-9168 G A lt005

12LSK4-10131 A A AA A A lt000113LSK5-11470 A A A 015 13LSK5-11472 A AA lt001

90-18 08LSH5-5645 AA 019 08LSH11-5694 A 05710LSH4-9192 AA 013

90-54 07KJin3-5961 A 00693-04 94KYR12-9441 AA AG 009

07KYR8-5166 A A AA A AG lt000107KYR8-4846 A AA G 00507KYR8-5167 A A A AG 00507KYR8-5168 A A AG 02907KYR8-4845 AA G 005

93-60 03KMH11-5725 AA A 015

WT amino acid W K S W V Y W G L W R K W N K21 22 23 38 39 40 70 71 72 89 90 91 174 175 176

Figure 3 Positions of the premature stop codons in the 15 vif genes from five patients All stop codons resulted from GG rarr GA orGG rarr AG changes APOBEC3G exhibits an intrinsic preference for the second cytosine in a 51015840CC dinucleotide motif leading to 51015840GG-to-AG mutations [38]

In total five of our patients demonstrated in-frame deletionsof the vif gene of the 75 KSB-infected patients who weretreated with KRG Also in our present study cohort fivepatients including 90-18 and 92-13 demonstrated deletedvif genes (small in-frame and gross deletions) during KRGintake whereas all of our 10 LTSP patients demonstrateddeletions in the nef gene (119875 lt 005) [14] In additioncompared with the reported deletions of the nef and 51015840LTRgag genes the locations of the deleted vif gene werepositioned within a narrow range at the terminus [15 16 23]

Regarding gross deletions to date only two patientshave demonstrated gross deletions in the vif gene [17 18]and only one patient has demonstrated the insertion of twoamino acids and these patients were part of a nonprogress-ing mother-child pair [28] However in our present studyof the 10 LTSP patients who were assessed four patientsdemonstrated gross deletion in the vif gene during KRGintake prior to receiving HAART (15) This frequency issignificantly lower than the previously reported incidence of10 of 10 patients demonstrating gross deletions in the nef(187) and 51015840 LTRgag genes (376 119875 lt 001) [27 28]The frequency of deletion in the nef gene was reported tosignificantly decrease during KRG plus HAART (119875 lt 001)[16] In contrast to previous studies on the nef and gag genes[16 29] the detection of in-frame deletions in the vif genewas not suppressed in the patients receiving HAART in ourpresent cohort The reasons for this include the following (1)the proportion and frequency (approximately two-thirds) ofdeleted vif genes after the first occurrence were much higherthan in the nef and 51015840 LTRgag genes (lt20 and 30 resp)(2) small deletions in the vif gene occurred as a single bandwhereas gross deletions in the nef and 51015840 LTRgag genes weremainly detected as two bands (wild type and short) per PCRreaction and (3) the number of amplicons indicating PSCdueto G-to-A hypermutation was significantly higher in patients

receiving KRG plus HAART than patients receiving onlyKRG This phenomenon is consistent with our previous datain 51015840 LTRgag gene [29] However the frequency of G-to-Ahypermutationwas significantly lower in vif gene than that in51015840 LTRgag gene [29] Thus for the vif gene the detection ofsmall deletionsmight be less affected by limit-diluting effectsas has been shown for the nef gene [29] Taken together theseadditional defects in the vif genes in the same patients mightbe related to the replicative impairment in vif defective HIV-1 which ultimately results in defects in the synthesis of viralDNA [30]

In our present study it appears that the frequency ofhypermutation was higher in patients who maintained goodcompliance with HAART during HAART plus KRG (90-0590-18 and 93-04) than in the patients who demonstratedpoor compliance (Figure 1) In our extended data analysis124 (60485) 31 (5161) 30 (19617) and 09 (1108)of vif genes during KRG plus HAART HAART aloneKRG alone and control revealed PSC respectively Thesedata indicate the presence of synergistic effect of KRG plusHAART on PSC (119875 lt 001) There was no case that smalldeletions were induced by HAART alone About 6ndash43 ofthe integrated proviral pol gene is hypermutated by HAART[22 31 32] although hypermutated viral genomes are notpresent in plasma [32]

ApoBec3G-induced hypermutation in LTNPs has beenreported previously [33] Indeed it has been reported alsothat LTNP patients harbor PSC-containing vif genes morefrequently than progressors [25] although the reportedfrequencies of defective genes vary among studies [34] Inour current analyses vif gene-containing PSC due to G-to-A hypermutation was found to be significantly higher duringKRG plus HAART than only KRG intake prior to HAARTThis finding is consistent with our previous data obtainedfrom a cohort of the Korean hemophiliacs (21 incidence

Evidence-Based Complementary and Alternative Medicine 11

of G-to-A hypermutation while receiving only KRG versus98 on KRG plus HAART unpublished data) and otherobservations regarding the pol gene [22]

Previous studies report that certain vif mutations such asL81M R132S S130I and the insertion of two amino acids areassociatedwith slow progression or low viral loads [17 18 28]In our present study L81M [35] and R132S were consistentlydetected in patient 96-51 and patients 87-05 and 96-51respectively although both mutations were found in only in2 and 3 of 169 Korean patients respectively Although thevif gene is highly conserved in HIV-1 genomes the recoveryof hypermutants might be severely underestimated in ourpresent analysis because of the primer mismatching andthe difficulties involved in detection following amplification[36 37] Overall our current data suggest that small in-frame deletion and PSCs are associated with KRG intake andHAART respectively although further studies are needed

5 Conclusions

Our data suggest that HAART and KRG intake might inducePSCs and small in-frame deletions respectively

Conflict of Interests

The authors have no conflict of interests to declare

Acknowledgments

This work was supported by a grant from the Korean Societyof Ginseng (2010ndash2012) The authors thank Professor Seung-Chul Yun the Division of Biostatistics University of UlsanCollege of Medicine Asan Medical Center for the statisticalconsultation

References

[1] E Ernst ldquoPanax ginseng an overview of the clinical evidencerdquoJournal of Ginseng Research vol 34 no 4 pp 259ndash263 2010

[2] K T Choi ldquoBotanical characteristics pharmacological effectsand medicinal components of Korean Panax ginseng C AMeyerrdquoActa Pharmacologica Sinica vol 29 no 9 pp 1109ndash11182008

[3] S J Fulder ldquoGinseng and the hypothalamic-pituitary control ofstressrdquo The American Journal of Chinese Medicine vol 9 no 2pp 112ndash118 1981

[4] V K Singh S S Agarwal and B M Gupta ldquoImmunomodula-tory activity of Panax ginseng extractrdquo Planta Medica vol 50no 6 pp 462ndash465 1984

[5] X Song and S Hu ldquoAdjuvant activities of saponins fromtraditional Chinese medicinal herbsrdquo Vaccine vol 27 no 36pp 4883ndash4890 2009

[6] D G Yoo M C Kim M K Park et al ldquoProtective effectof ginseng polysaccharides on influenza viral infectionrdquo PLoSONE vol 7 no 3 Article ID e33678 2012

[7] H X Sun Y P Ye H J Pan and Y J Pan ldquoAdjuvant effectof Panax notoginseng saponins on the immune responses toovalbumin in micerdquo Vaccine vol 22 no 29-30 pp 3882ndash38892004

[8] J Sun S Hu and X Song ldquoAdjuvant effects of protopanaxadioland protopanaxatriol saponins from ginseng roots on theimmune responses to ovalbumin in micerdquo Vaccine vol 25 no6 pp 1114ndash1120 2007

[9] P W Hunt ldquoHIV and inflammation mechanisms and conse-quencesrdquo Current HIVAIDS Reports vol 9 no 2 pp 139ndash1472012

[10] T Yayeh K H Jung H Y Jeong et al ldquoKorean red ginsengsaponin fraction downregulates proinflammatory mediators inLPS stimulated RAW264 7 cells and protects mice againstendotoxic shockrdquo Journal of Ginseng Research vol 36 no 3 pp263ndash269 2012

[11] A T Smolinski and J J Pestka ldquoModulation of lipopolysaccha-ride-induced proinflammatory cytokine production in vitroand in vivo by the herbal constituents apigenin (chamomile)ginsenoside Rb1 (ginseng) and parthenolide (feverfew)rdquo Foodand Chemical Toxicology vol 41 no 10 pp 1381ndash1390 2003

[12] A Rhule S Navarro J R Smith and D M ShepherdldquoPanax notoginseng attenuates LPS-induced pro-inflammatorymediators in RAW2647 cellsrdquo Journal of Ethnopharmacologyvol 106 no 1 pp 121ndash128 2006

[13] J M Brenchley D A Price T W Schacker et al ldquoMicrobialtranslocation is a cause of systemic immune activation inchronic HIV infectionrdquo Nature Medicine vol 12 no 12 pp1365ndash1371 2006

[14] Y K Cho J Y Lim Y S Jung S K Oh H J Lee and H SungldquoHigh frequency of grossly deleted nef genes in HIV-1 infectedlong-term slow progressors treated with Korean red ginsengrdquoCurrent HIV Research vol 4 no 4 pp 447ndash457 2006

[15] Y K Cho and Y S Jung ldquoHigh frequency of gross deletions inthe 51015840 LTR and gag regions in HIV type 1-infected long-termsurvivors treated with Korean red ginsengrdquo AIDS Research andHuman Retroviruses vol 24 no 2 pp 181ndash193 2008

[16] Y K Cho Y S Jung andH S Sung ldquoFrequent gross deletion intheHIV type 1 nef gene in hemophiliacs treatedwith korean redginseng inhibition of detection by highly active antiretroviraltherapyrdquo AIDS Research and Human Retroviruses vol 25 no 4pp 419ndash424 2009

[17] V Sandonıs C Casado T Alvaro et al ldquoA combination ofdefective DNA and protective host factors are found in a setof HIV-1 ancestral LTNPsrdquo Virology vol 391 no 1 pp 73ndash822009

[18] H R Rangel D Garzaro A K Rodrıguez et al ldquoDeletioninsertion and stop codon mutations in vif genes of HIV-1 infecting slow progressor patientsrdquo Journal of Infection inDeveloping Countries vol 3 no 7 pp 531ndash538 2009

[19] Y K Cho J E Kim and B T Foley ldquoPhylogenetic analysisof near full-length HIV-1 genomic sequences in 21 Koreanindividualsrdquo AIDS Research and Human Retroviruses vol 29no 4 pp 738ndash743 2013

[20] Y K Cho Y S Jung J S Lee and B T Foley ldquoMolecu-lar evidence of HIV-1 transmission in 20 Korean individu-als with haemophilia phylogenetic analysis of the vif generdquoHaemophilia vol 18 no 2 pp 291ndash299 2012

[21] P P Rose and B T Korber ldquoDetecting hypermutations inviral sequences with an emphasis on GrarrA hypermutationrdquoBioinformatics vol 16 no 4 pp 400ndash401 2000

[22] S Fourati S Lambert-Niclot C Soulie et al ldquoHIV-1 genomeis often defective in PBMCs and rectal tissues after long-termHAART as a result of APOBEC3 editing and correlates with thesize of reservoirsrdquo Journal of Antimicrobial Chemotherapy vol67 no 10 pp 2323ndash2326 2012

12 Evidence-Based Complementary and Alternative Medicine

[23] U Wieland A Seelhoff A Hofmann et al ldquoDiversity of thevif gene of human immunodeficiency virus type 1 in UgandardquoJournal of General Virology vol 78 no 2 pp 393ndash400 1997

[24] P Sova M van Ranst P Gupta et al ldquoConservation of an intacthuman immunodeficiency virus type 1 vif gene in vitro and invivordquo Journal of Virology vol 69 no 4 pp 2557ndash2564 1995

[25] T Yamada and A Iwamoto ldquoComparison of proviral accessorygenes between long-term nonprogressors and progressors ofhuman immunodeficiency virus type 1 infectionrdquo Archives ofVirology vol 145 no 5 pp 1021ndash1027 2000

[26] K Tominaga S Kato M Negishi and T Takano ldquoA high fre-quency of defective vif genes in peripheral blood mononuclearcells from HIV type 1-infected individualsrdquo AIDS Research andHuman Retroviruses vol 12 no 16 pp 1543ndash1549 1996

[27] Y K Cho H Sung I G Bae et al ldquoFull sequence of HIV type 1Korean subtype B in anAIDS case with atypical seroconversionTAAAA at TATA boxrdquoAIDS Research andHuman Retrovirusesvol 21 no 11 pp 961ndash964 2005

[28] L Alexander M J Aquino-DeJesus M Chan and W AAndiman ldquoInhibition of human immunodeficiency virus type 1(HIV-1) replication by a two-amino-acid insertion in HIV-1 Viffrom a nonprogressing mother and childrdquo Journal of Virologyvol 76 no 20 pp 10533ndash10539 2002

[29] YK Cho Y S JungH S Sung andCH Joo ldquoFrequent geneticdefects in the HIV-1 51015840LTRgag gene in hemophiliacs treatedwith Korean red ginseng decreased detection of genetic defectsby highly active antiretroviral therapyrdquo Journal of GinsengResearch vol 35 no 4 pp 413ndash420 2011

[30] M Nascimbeni M Bouyac F Rey B Spire and F Clavel ldquoThereplicative impairment of Vif-mutants of human immunodefi-ciency virus type 1 correlates with an overall defect in viral DNAsynthesisrdquo Journal of General Virology vol 79 no 8 pp 1945ndash1950 1998

[31] J P Vartanian M Henry and S Wain-Hobson ldquoSustainedGrarrA hypermutation during reverse transcription of an entirehuman immunodeficiency virus type 1 strain Vau group Ogenomerdquo Journal of General Virology vol 83 no 4 pp 801ndash8052002

[32] T L Kieffer P Kwon R E Nettles Y Han S C Ray andR F Siliciano ldquoGrarrA hypermutation in protease and reversetranscriptase regions of human immunodeficiency virus type 1residing in resting CD4+ T cells in vivordquo Journal of Virology vol79 no 3 pp 1975ndash1980 2005

[33] B Wang M Mikhail W B Dyer J J Zaunders A D Kelleherand N K Saksena ldquoFirst demonstration of a lack of viralsequence evolution in a nonprogressor defining replication-incompetent HIV-1 infectionrdquo Virology vol 312 no 1 pp 135ndash150 2003

[34] G Hassaine I Agostini D Candotti et al ldquoCharacterizationof human immunodeficiency virus type 1 vif gene in long-termasymptomatic individualsrdquoVirology vol 276 no 1 pp 169ndash1802000

[35] F A de Maio C A Rocco P C Aulicino R Bologna AMangano and L Sen ldquoUnusual substitutions in HIV-1 Vif fromchildren infected perinatally without progression to AIDS formore than 8 years without therapyrdquo Journal of Medical Virologyvol 84 no 12 pp 1844ndash1852 2012

[36] M Janini M Rogers D R Birx and F E McCutchan ldquoHumanimmunodeficiency virus type 1 DNA sequences geneticallydamaged by hypermutation are often abundant in patientperipheral blood mononuclear cells and may be generated

during near-simultaneous infection and activation of CD4+ Tcellsrdquo Journal of Virology vol 75 no 17 pp 7973ndash7986 2001

[37] G H Kijak L M Janini S Tovanabutra et al ldquoVariablecontexts and levels of hypermutation inHIV-1 proviral genomesrecovered from primary peripheral blood mononuclear cellsrdquoVirology vol 376 no 1 pp 101ndash111 2008

[38] E W Refsland J F Hultquist and R S Harris ldquoEndogenousorigins of HIV-1 G-to-A hypermutation and restriction in thenonpermissive T cell line CEM2nrdquo PLoS Pathogens vol 8 no7 Article ID e1002800 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 5: Effects of Korean Red Ginseng and HAART on vif Gene in 10 Long

Evidence-Based Complementary and Alternative Medicine 5

0

1

2

3

4

5

6

7

0100200300400500600700800

93-60

HAARTKRGKRG

Apr

94

Nov

95

May

96

Nov

96

Apr

97

Jun

97Ju

n 99

Nov

99

Nov

00

Jul 0

1N

ov 0

1Ja

n 02

Sep

02Fe

b 03

May

03

Nov

03

May

04

Mar

05

Sep

05A

pr 0

6O

ct 0

6N

ov 0

6A

pr 0

7Ju

l 07

Aug

07

Oct

07

Jan

08M

ay 0

8M

ay 0

8Ju

l 08

Aug

08

Dec

08

May

09

Dec

09

Oct

10

Apr

11

Apr

12

lowast

0

1

2

3

4

5

6

7

0

200

400

600

800

1000

120096-51

KRG HAARTKRGKRG

Jul 9

6Ja

n 97

Jul 9

7A

pr 9

8N

ov 9

8M

ay 9

9Ju

n 00

Oct

00

Sep

01A

pr 0

2D

ec 0

2O

ct 0

3Fe

b 04

Aug

04

Feb

05A

ug 0

5N

ov 0

6Ju

l 06

Oct

06

Feb

07A

ug 0

7M

ar 0

8A

ug 0

8Fe

b 09

Jul 0

9D

ec 0

9M

ay 1

0Ju

n 10

Apr

11

Oct

11

Jul 1

2Ja

n 13

RNA

copy

(log

mL)

CD4

+ T

cells

(120583

L)

Sampling dateCD4+ T cells (120583L)RNA copy (logmL)

RNA

copy

(log

mL)

CD4

+ T

cells

(120583

L)

Sampling dateCD4+ T cells (120583L)RNA copy (logmL)

if if

Figure 1 Changes in the CD4+ T-cell count plasma viral load and genetic defects in terms of Korean red ginseng (KRG) intake and highlyactive antiretroviral therapy (HAART) The durations of KRG intake and HAART are indicated by the bars Solid and dotted lines denotegood (gt90) and poor (lt90) compliance according to self-administered responses respectively The upward arrow (uarr) downward arrow(darr) plus sign (+) and asterisk (lowast) denote the sequences of the vif gene gross deletions 3- and- 6-base pair (bp) in-frame insertions and stopcodons respectively

Table 2 Distribution of defective vif gene

Patient On-KRG On-HAARTKRGNo of genes Stop codon gΔ sΔ No of genes Stop codon gΔ sΔ

87-05 20 1 ND ND ND ND89-17 9 9 0 0 090-05 37 1 18 4 0 090-18 35 15 20 3a 0 12a

90-50 14 1 15 0 0 091-20 35 1 20 0 0 092-13 53 14 22 0 0 1393-04 18 1 12 5 1 093-60 28 1 1 20 0 6 096-51 26 21 0 0 0Total 275 3b 4 29 157 12b 7 25gΔ and sΔ denote gross deletion and in-frame small deletion respectivelyHAART highly active antiretroviral therapy ND not determinedaTwo out of three genes contained both stop codon and sΔb119875 lt 001119875 lt 001 for the sum of 3 kinds of defective genes (36275 versus 42157)Fifty-two vif genes at baseline obtained from serum using RT-PCR were all wild types lowastControl patients (119899 = 21) revealed premature stop codon in one outof 106 vif genes

stop codons 119875 lt 005) and 51015840 LTRgag genes (71 of 189 genes119875 lt 0001) identified in the same patients [14 15]

4 Discussion

Todate we have identified the vif gene in 194Korean patientsOf these 145 patients were infected with the Korean subcladeB ofHIV-1 (KSB) and the remaining 49 patients were infectedwith non-KSB Of these 194 patients 10 demonstrated smallin-frame deletions (3ndash15 bp) and all were diagnosed withKSB whereas no such deletions were detected in non-KSBpatients (119875 = 0068) Of the 10 patients with small deletionsthree patients were included in a group of 20 hemophiliacs

who were infected with KSB from plasma donors O and P[16 20] Interestingly plasma donors O and P revealed wild-type vif only between 1991 and 2002 (AY581320-21 JQ248127-33 JF957909 JF957921 JQ248134 and JF957935-37) In otherwords original vif gene was wild type without in-framesmall deletion In our current study only two patients (90-18 and 92-13) demonstrated 9 (119899 = 15) and 12 (119899 = 14)bp in-frame deletions in about two-thirds of the ampliconsobtained after 63 and 120 years of KRG intake respectivelywhereas five patients without KRG intake demonstrated in-frame deletions in all amplicons (data not shown) To ourknowledge these in-frame deletions are very rare and haveonly been reported in two patients in other countries [26 27]

6 Evidence-Based Complementary and Alternative Medicine

87-05

89-17

90-05

90-18

90-50

91-20

1 20 40 60 80 96Kor consensus MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKEWVYRHHYESTHPRISSEVHIPLGDAKLVITTYWGLHTGEREWHLGQGVSIEWRKKRYNT

91CSR4-10026 RGNVVD91CSR11-6922 RGNVVD92CSR5-9885 RGNVVD93CSR6-10159 RGNVVD93CSR6-10162s1 ------------------------------------------------------------------------------------------------03CSR9-805 SGGNVVD05CSR3 RGNIVVDG06CSR10-5280 RGNIVVDRG08CSR3-5334 RGNNVDG08CSR8-5279 RGNIVVD10CSR7-8126 VRGNLVVDG12CSR2-10141 VRGNLVVDG

91KJS12-8538 I92KJS3-6819 GI93KJS7-10155 IAI97KJS3-3814 IGKEI01KJS2-5667 IGKI03KJS2-745 IGAI03KJS7-775 IGAEI05KJS12-3264 YMIVGAEI08KJS5-5157 IVNGAAI10KJS5-10133 IGKAES

92LSK11-9417 K94LSK6-759 KGNPRS96LSK6-11491 AKNPIRS98LSK4-11495 KHN00LSK8-9999 KGNR03LSK6-10001s1 ------------------------------------------------------------------------------04LSK7 KKGNPRST06LSK1-3236 KKGNPVRST07LSK2-3232 KKGNPVRST08LSK1-4762 KKGIPVRST09LSK4-6143 IKNPIGRST10LSK4-9165 KGNPRS10LSK4-9168 KKGNPVRTRST12LSK4-10131 KAVKALKNPEKST 12LSK4-10132 KAKNPGRST13LSK5-11470 KKGNPGRST13LSK5-11471 KKVGNPVRST13LSK5-11472 IKANNRI

92LSH3-6951 K92LSH11-8548 TKK93LSH10-1252 94LSH2-740 95LSH5-10562 V96LSH5-10609 V98LSH4-10567 VG99LSH1-5776 VG99LSH1-5777 VG99LSH1-5778 VG99LSH6-5781 VGE99LSH6-5782 VG00LSH3-5704 KVGR00LSH3-5705 VG00LSH3-5706 VGK02LSH10-5707 VGR02LSH10-5708 VG02LSH10-5710 VG03LSH5-5650 VGG03LSH5-5651 VG03LSH5-5652 VG03LSH10-5653 VG04LSH7-4848 VGKES04LSH8-3011 V06LSH8-5647 VGES06LSH8-5649 VGES06LSH11-3238 VGIS08LSH5-5644 VG08LSH5-5645 VGR08LSH5-5646 KVGE08LSH5-5647 KVGE08LSH11-5694 KVG10LSH4-9190 KVGES10LSH4-9192 VG10LSH4-9193 VGG12LSH6-10100 VGES12LSH6-10101 VG

91KJin7-6723 N93KJin2-10024 N93KJin9-8530 NHVKINVDT01KJin7-5383 KVN03KJin6-683 KVINT04KJin8-1955 KVINDT06KJin1-3030 KVKINDT06KJin9-3012 KVINT07KJin3-5148 VNKINID07KJin3-5961 VNKKINEN10KJin4-9162 NHVKKINVT10KJin9-8176 NHVKKINVDT11KJin12-10119 KVKKINVDT13KJin1-11373 VNNVNDT

K94JWK12-5524 K97JWK K99JWK5-11441 KNE00JWK3-11453 KN00JWK12-11445 KN01JWK7-5603 KVA01JWK7-5603s1 KKNN02JWK8-11435 KVA03JWK8-677 KN05JWK1-5600 KVA06JWK7-3042 KVNAN

KKNVNVVN08JWK1-4772 KVNAN

KVA11JWK4-8993 KVA12JWK1-10125 KVA13JWK1-11366 KVA

minus minusminus minusminus minusminus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ +

Patient KRG HAART

10JWK4 9159

06JWK12 5624

91JWK6 6949

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 7

92-13

93-04

93-60

96-51

92CWS5-9398 G93CWS3-9925 GK93CWS5-6669 GK93CWS7-802 G96CWS11-5341 G99CWS10-5338 KVGE01CWS2-10532 VG03CWS6-684 VGTG03CWS10-5773 VG04CWS5-3239 VGKSTG05CWS5-5783 GTG05CWS5-5785 VGKITG06CWS2-3071 VGTG06CWS2-3203 VGKITG06CWS2-3221 VGTG06CWS2-3222 NVGTG06CWS2-3223 NVGTG06CWS8-3038 NVGKTG06CWS8-3039 NVGKTG07CWS4-3820 VGKIATG07CWS4-3821 VGKIATG07CWS4-10174 HWVGKIATG07CWS4-10175 VGKIATG07CWS4-10176 VGKITG07CWS4-10177 VGKKTG07CWS9-6252 VGTG07CWS9-6253 VGKITG07CWS9-6254 VGKIATG07CWS12-4777 VGQITG08CWS5-6214 NKVGQITG08CWS8-5160 NVGKTG08CWS8-5161 NVGKNQG08CWS12-5720 VGQITG08CWS12-5721 VGQITG08CWS12-5722 VGKQITG09CWS5-6256 VGKQTG09CWS5-6257 VGKQTG09CWS5-6258 VGKDQTG09CWS12-9108 LVGTG09CWS12-9109 VGKQITG

93KYR2-9472 VNN94KYR6-10137 VNN94KYR12-9441 VNN95KYR5-815 VGNN00KYR9-9913 VGNN02KYR7-9911 VGNN03KYR1-817 VNN06KYR7-5236 VGNN06KYR11-5249 VGNNS07KYR3-5265 VGNN07KYR3-11387s1 -------------------------------------------------------------------------------------------------07KYR8-4845 VGNN07KYR8-4846 VGNN07KYR8-5166 IVRNKN07KYR8-5167 VGNRN07KYR8-5168 VGNN

94KMH5 EI96KMH11-8534 IAEI97KMH4-9452 ISEI01KMH11-5526 ISIEI03KMH5-687 EI03KMH11-5725 ISKIEI04KMH5 EI05KMH3-5727 ISEI06KMH11-3026 ISKEI07KMH4-3281s1 --------------------------------------------------------------------EI07KMH10-5732 VNEI08KMH5-5165 VGNEI08KMH8-5427s1 ------------------------------------------------------------------------------------------------09KMH5-11475 ISEI09KMH5-11475s2 HQS----------------------10KMH10-9907s1 ------------------------------------------------------------------------------------------------11KMH4-9917 VDI12KMH4-10096 VGNEI

00YJN10-9385 TRGNMS01YJN9-4850 RGNMS03YJN10-840 VVRGNMS05YJN2-1948 VVRGNMS06YJN10-3044 VRGNMS07YJN4-3248 VVRGNMQI07YJN8-5627 VRGNMQS08YJN3-5606 VRGNMQS08YJN8-5408 VRGNPVMQS09YJN2-6041 VRGNPVMQS09YJN12-9156 VRGNVMQS10YJN6-9918 VRGNVMQS11YJN2-9921 VRGNVMS12YJN7-11382 VRGNVMQS13YJN1-11381 VRGNVMSKor consensus MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKEWVYRHHYESTHPRISSEVHIPLGDAKLVITTYWGLHTGEREWHLGQGVSIEWRKKRYNT

1

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ +minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

87-05

89-17

97 120 140 160 180 192QVDPDLADKLIHLHYFDCFSDSAIRHAILGRLVRPKCEYQAGHNKVGSLQYLALTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH

91CSR4-10026 TQRYKHISSTA91CSR11-6922 TQRYKHISSTA92CSR5-9885 TQRYKHISSTA93CSR6-10159 TQRYKHISSTA93CSR6-10162s1 ------------------------------------------------------------------------------------------------03CSR9-805 AQRYEKHISSTA05CSR3 TQRYKHISSTAK06CSR10-5280 TQRYKHISRTA08CSR3-5334 TQRYKHISSTA08CSR8-5279 TQRYKHISSTA10CSR7-8126 TQRYKHISSTIA12CSR2-10141 TQRYKHISSTIA

91KJS12-8538 92KJS3-6819 93KJS7-10155 97KJS3-3814 01KJS2-5667 03KJS3-745 03KJS7-775 05KJS12-3264 08KJS5-5157 10KJS5-10133

minus minusminus minusminus minusminus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ +

Patient Kor consensus KRG HAART

20 60 80 9640

RR

VS

VRE

VR

VRS

VRRES

VRRES

PKVPVIFRHIES

EVRRHES

VRRHES

Figure 2 Continued

8 Evidence-Based Complementary and Alternative Medicine

90-05

90-18

90-50

91-20

92LSK11-9417 YETRQE94LSK6-759 YETRPQES96LSK6-11491 GRYETRQ98LSK4-11495 YENTRPQES00LSK8-9999 YPQE03LSK6-10001s1 -------------------------------------------QRES04LSK7 YETRPQES06LSK1-3236 YEVTRPQRES07LSK2-3232 YEVTRPQES08LSK1-4762 YEVTRPQAESS09LSK4-6143 YEVTRPQES10LSK4-9165 YEVTRPQES10LSK4-9168 YEVTRPQES12LSK4-10131 NYKVTRPQRAES12LSK4-10132 YEVTRPQAESQ13LSK5-11470 PYVRRPTQETESS13LSK5-11471 YVTRPQRES13LSK5-11472 YENRTRQRES

92LSH3-6951 YRDH92LSH11-8548 GYRD93LSH10-1252 YRDR94LSH2-740 YRDR95LSH5-10562 YTRDR96LSH5-10609 YTR98LSH4-10567 YQTRRG---H99LSH1-5776 YQTRRG---RH99LSH1-5777 YQTRRHG---H99LSH1-5778 YQTRG---H99LSH6-5781 YQTRRQG---H99LSH6-5782 YQTRG---H00LSH3-5704 YQTRRG---H00LSH3-5705 YQTRRQSG---H00LSH3-5706 YQTRAG---H02LSH10-5707 YETRQG---H02LSH10-5708 YQTRG---H02LSH10-5710 YQTRRQR---H03LSH5-5650 YQTRRR---03LSH5-5651 YQTRRR---H03LSH5-5652 YQTRRER---03LSH10-5653 YQTRRQRS04LSH7-4848 YQTRRKQG---H04LSH8-3011 YR06LSH8-5647 YQTRRG---H06LSH8-5649 YQTRRG---H06LSH11-3238 YQHTRRQES08LSH5-5644 YQTRRSG---H08LSH5-5645 YQTRQAKS08LSH5-5646 YQTRRR---H08LSH5-5647 YQTRRR---H08LSH11-5694 YQTRRSRKG---H10LSH4-9190 YEQCTRRRG---H10LSH4-9192 YQRTRRRRG---H10LSH4-9193 YQTRRG---H12LSH6-10100 YQTRRG---H12LSH6-10101 YQTRRSRG---H

91KJin7-6723 TT93KJin2-10024 TT93KJin9-8530 VTPAKH01KJin7-5383 ISTT03KJin6-683 ISTPQH04KJin8-1955 ITPTH06KJin1-3030 VTPTH06KJin9-3012 VAPT07KJin3-5148 QEPTN07KJin3-5961 NLEPTAN10KJin4-9162 VTTAH10KJin9-8176 QVTAAH11KJin12-10119 VTPTADH13KJin1-11373 VTPTH

PE94JWK12-5524 VQ97JWK HR99JWK5-11441 HR00JWK3-11453 HR00JWK12-11445 HR01JWK7-5603 NHRQ01JWK7-5603s1 -------------------------------------------------------------------------------02JWK8-11436 NRQQ03JWK8-677 HR05JWK1-5600 NRQQ06JWK7-3042 HRQ

HRQ08JWK1-4772 NRQQ

NRQQ11JWK4-8993 NRQQ12JWK1-10125 NRQQ13JWK1-11366 NRQQ

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ +

10JWK4 9159

06 JWK12 5624

91JWK6 6949

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 9

92-13

93-04

93-60

92CWS5-9398 HSE93CWS3-9925 HE93CWS5-6669 EHTE93CWS7-802 KHTE96CWS11-5341 KHTE99CWS10-5338 HPES01CWS2-10532 HPDE03CWS6-684 HPE03CWS10-5773 HPDE04CWS5-3239 HPTE05CWS5-5783 HPTD----05CWS5-5785 HPTD----06CWS2-3071 HPTDE06CWS2-3203 HPTD----06CWS2-3221 HPTD----06CWS2-3222 VHPTD----06CWS2-3223 HPTD----06CWS8-3038 HPTD----06CWS8-3039 HPTD----07CWS4-3820 HPTATD----07CWS4-3821 HPTATD----07CWS4-10174 HPTATD----07CWS4-10175 HPTATD----07CWS4-10176 HPTATD----07CWS4-10177 VHRD----07CWS9-6252 HPE07CWS9-6253 HPTATD----07CWS9-6254 HPTATD----07CWS12-4777 HPTATD----08CWS5-6214 HPTATD----08CWS8-5160 HPTATD----08CWS8-5161 HPTATD----08CWS12-5720 HPTATD----08CWS12-5721 HPTATD----08CWS12-5722 HPTATD----09CWS5-6256 HPTATD----09CWS5-6257 HPTTATD----09CWS5-6258 HPTAD----09CWS12-9108 HPDE09CWS12-9109 HPTATTD----

93KYR2-9472 NSRIRKE94KYR6-10137 NSRRKE94KYR12-9441 NSRIRKER95KYR5-815 NSRIRKE00KYR9-9913 NSRRRKE02KYR7-9911 NSRRRKE03KYR1-817 NSRIRKE06KYR7-5236 QKPHRIRKE06KYR11-5249 QKPHRIRKE07KYR3-5265 NSHRIRKE07KYR3-11387s1 ---------------QNSHRIRRKER07KYR8-4845 QNSRRIRKER07KYR8-4846 NSRRIRKE07KYR8-5166 KNSHRRRIRKER07KYR8-5167 NKHRRIRRKE07KYR8-5168 IQRKPHRRIRKE

94KMH5 HVPR96KMH11-8534 VRT97KMH4-9452 HVQR01KMH11-5526 HVPR03KMH5-687 HVPR03KMH11-5725 HVQRS04KMH5 VQRS05KMH3-5727 HVPR06KMH11-3026 HVPR07KMH4-3281s1 VHVPRV----------------------------07KMH10-5732 HVQPRVRS08KMH5-5165 HIQPRVRS08KMH8-5427s1 ----------------------------HVQPTRVRS09KMH5-11475 HVQPRVRS09KMH5-11475s2 ------------------------------------------------------------------------------------------------10KMH10-9907s1 -----------------------------------------RVRS11KMH4-9917 HVPRVRS12KMH4-10096 HVPRVRRS

00YJN10-9385 ETVSRE01YJN9-4850 ETVSRE03YJN10-840 ETVSRE05YJN2-1948 ETQVSRE06YJN10-3044 ETQVSRE07YJN4-3248 ETQVSREY07YJN8-5627 ETQVSREY08YJN3-5606 ETQVSREY08YJN8-5408 ETQVSREY09YJN2-6041 ETQVSREY09YJN12-9156 ETQVSREY10YJN6-9918 ETQVSRIEY11YJN2-9921 ETQVSREY12YJN7-11382 ETQVSREY13YJN1-11381 ETQVSREYKor consensus QVDPDLADKLIHLHYFDCFSDSAIRHAILGRLVRPKCEYQAGHNKVGSLQYLALTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH

97 120 140 160 180 192

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ +

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

96-51

Figure 2 Sequential alignment of the amino acids of the Vif protein over 20 years All sequences that demonstrated small in-frame deletionsgross deletions and stop codons are depicted together with the baseline and last sequences of each patient Patients 90-18 demonstrated a9 bp deletion (positions 182ndash184) in April 1998 and patient 92-13 demonstrated a 12 bp deletion (positions 185ndash188) in May 2005 Patient90-05 consistently demonstrated a 9 bp insertion (RQTRAR-RAR-NGASRP) at the same position at the terminus of vpr beginning in August2000 (data not shown) The code 92LSH3-6951 (sampling year initials of the patient sampling month and sequence number) denotes thatthe sampling date is March 1992 and the sequence number is 6951 in patient LSH (90-18) The dot (sdot) hyphen (-) and asterisk (lowast) denotesequence identity deletions and premature stop codons respectively compared with the Korean consensus [20] In the right column theplus (+) and minus (minus) signs denote the presence or absence of the corresponding therapy respectively

10 Evidence-Based Complementary and Alternative Medicine

61 69 112 120 208 216 264 272 520 528Patient WT nucleotide TGGAAAAGT TGGGTCTAT TGGGGTCTG TGGAGGAAA TGGAACAAG Hypermut 20

sequence no P value90-05 10LSK4-9168 G A lt005

12LSK4-10131 A A AA A A lt000113LSK5-11470 A A A 015 13LSK5-11472 A AA lt001

90-18 08LSH5-5645 AA 019 08LSH11-5694 A 05710LSH4-9192 AA 013

90-54 07KJin3-5961 A 00693-04 94KYR12-9441 AA AG 009

07KYR8-5166 A A AA A AG lt000107KYR8-4846 A AA G 00507KYR8-5167 A A A AG 00507KYR8-5168 A A AG 02907KYR8-4845 AA G 005

93-60 03KMH11-5725 AA A 015

WT amino acid W K S W V Y W G L W R K W N K21 22 23 38 39 40 70 71 72 89 90 91 174 175 176

Figure 3 Positions of the premature stop codons in the 15 vif genes from five patients All stop codons resulted from GG rarr GA orGG rarr AG changes APOBEC3G exhibits an intrinsic preference for the second cytosine in a 51015840CC dinucleotide motif leading to 51015840GG-to-AG mutations [38]

In total five of our patients demonstrated in-frame deletionsof the vif gene of the 75 KSB-infected patients who weretreated with KRG Also in our present study cohort fivepatients including 90-18 and 92-13 demonstrated deletedvif genes (small in-frame and gross deletions) during KRGintake whereas all of our 10 LTSP patients demonstrateddeletions in the nef gene (119875 lt 005) [14] In additioncompared with the reported deletions of the nef and 51015840LTRgag genes the locations of the deleted vif gene werepositioned within a narrow range at the terminus [15 16 23]

Regarding gross deletions to date only two patientshave demonstrated gross deletions in the vif gene [17 18]and only one patient has demonstrated the insertion of twoamino acids and these patients were part of a nonprogress-ing mother-child pair [28] However in our present studyof the 10 LTSP patients who were assessed four patientsdemonstrated gross deletion in the vif gene during KRGintake prior to receiving HAART (15) This frequency issignificantly lower than the previously reported incidence of10 of 10 patients demonstrating gross deletions in the nef(187) and 51015840 LTRgag genes (376 119875 lt 001) [27 28]The frequency of deletion in the nef gene was reported tosignificantly decrease during KRG plus HAART (119875 lt 001)[16] In contrast to previous studies on the nef and gag genes[16 29] the detection of in-frame deletions in the vif genewas not suppressed in the patients receiving HAART in ourpresent cohort The reasons for this include the following (1)the proportion and frequency (approximately two-thirds) ofdeleted vif genes after the first occurrence were much higherthan in the nef and 51015840 LTRgag genes (lt20 and 30 resp)(2) small deletions in the vif gene occurred as a single bandwhereas gross deletions in the nef and 51015840 LTRgag genes weremainly detected as two bands (wild type and short) per PCRreaction and (3) the number of amplicons indicating PSCdueto G-to-A hypermutation was significantly higher in patients

receiving KRG plus HAART than patients receiving onlyKRG This phenomenon is consistent with our previous datain 51015840 LTRgag gene [29] However the frequency of G-to-Ahypermutationwas significantly lower in vif gene than that in51015840 LTRgag gene [29] Thus for the vif gene the detection ofsmall deletionsmight be less affected by limit-diluting effectsas has been shown for the nef gene [29] Taken together theseadditional defects in the vif genes in the same patients mightbe related to the replicative impairment in vif defective HIV-1 which ultimately results in defects in the synthesis of viralDNA [30]

In our present study it appears that the frequency ofhypermutation was higher in patients who maintained goodcompliance with HAART during HAART plus KRG (90-0590-18 and 93-04) than in the patients who demonstratedpoor compliance (Figure 1) In our extended data analysis124 (60485) 31 (5161) 30 (19617) and 09 (1108)of vif genes during KRG plus HAART HAART aloneKRG alone and control revealed PSC respectively Thesedata indicate the presence of synergistic effect of KRG plusHAART on PSC (119875 lt 001) There was no case that smalldeletions were induced by HAART alone About 6ndash43 ofthe integrated proviral pol gene is hypermutated by HAART[22 31 32] although hypermutated viral genomes are notpresent in plasma [32]

ApoBec3G-induced hypermutation in LTNPs has beenreported previously [33] Indeed it has been reported alsothat LTNP patients harbor PSC-containing vif genes morefrequently than progressors [25] although the reportedfrequencies of defective genes vary among studies [34] Inour current analyses vif gene-containing PSC due to G-to-A hypermutation was found to be significantly higher duringKRG plus HAART than only KRG intake prior to HAARTThis finding is consistent with our previous data obtainedfrom a cohort of the Korean hemophiliacs (21 incidence

Evidence-Based Complementary and Alternative Medicine 11

of G-to-A hypermutation while receiving only KRG versus98 on KRG plus HAART unpublished data) and otherobservations regarding the pol gene [22]

Previous studies report that certain vif mutations such asL81M R132S S130I and the insertion of two amino acids areassociatedwith slow progression or low viral loads [17 18 28]In our present study L81M [35] and R132S were consistentlydetected in patient 96-51 and patients 87-05 and 96-51respectively although both mutations were found in only in2 and 3 of 169 Korean patients respectively Although thevif gene is highly conserved in HIV-1 genomes the recoveryof hypermutants might be severely underestimated in ourpresent analysis because of the primer mismatching andthe difficulties involved in detection following amplification[36 37] Overall our current data suggest that small in-frame deletion and PSCs are associated with KRG intake andHAART respectively although further studies are needed

5 Conclusions

Our data suggest that HAART and KRG intake might inducePSCs and small in-frame deletions respectively

Conflict of Interests

The authors have no conflict of interests to declare

Acknowledgments

This work was supported by a grant from the Korean Societyof Ginseng (2010ndash2012) The authors thank Professor Seung-Chul Yun the Division of Biostatistics University of UlsanCollege of Medicine Asan Medical Center for the statisticalconsultation

References

[1] E Ernst ldquoPanax ginseng an overview of the clinical evidencerdquoJournal of Ginseng Research vol 34 no 4 pp 259ndash263 2010

[2] K T Choi ldquoBotanical characteristics pharmacological effectsand medicinal components of Korean Panax ginseng C AMeyerrdquoActa Pharmacologica Sinica vol 29 no 9 pp 1109ndash11182008

[3] S J Fulder ldquoGinseng and the hypothalamic-pituitary control ofstressrdquo The American Journal of Chinese Medicine vol 9 no 2pp 112ndash118 1981

[4] V K Singh S S Agarwal and B M Gupta ldquoImmunomodula-tory activity of Panax ginseng extractrdquo Planta Medica vol 50no 6 pp 462ndash465 1984

[5] X Song and S Hu ldquoAdjuvant activities of saponins fromtraditional Chinese medicinal herbsrdquo Vaccine vol 27 no 36pp 4883ndash4890 2009

[6] D G Yoo M C Kim M K Park et al ldquoProtective effectof ginseng polysaccharides on influenza viral infectionrdquo PLoSONE vol 7 no 3 Article ID e33678 2012

[7] H X Sun Y P Ye H J Pan and Y J Pan ldquoAdjuvant effectof Panax notoginseng saponins on the immune responses toovalbumin in micerdquo Vaccine vol 22 no 29-30 pp 3882ndash38892004

[8] J Sun S Hu and X Song ldquoAdjuvant effects of protopanaxadioland protopanaxatriol saponins from ginseng roots on theimmune responses to ovalbumin in micerdquo Vaccine vol 25 no6 pp 1114ndash1120 2007

[9] P W Hunt ldquoHIV and inflammation mechanisms and conse-quencesrdquo Current HIVAIDS Reports vol 9 no 2 pp 139ndash1472012

[10] T Yayeh K H Jung H Y Jeong et al ldquoKorean red ginsengsaponin fraction downregulates proinflammatory mediators inLPS stimulated RAW264 7 cells and protects mice againstendotoxic shockrdquo Journal of Ginseng Research vol 36 no 3 pp263ndash269 2012

[11] A T Smolinski and J J Pestka ldquoModulation of lipopolysaccha-ride-induced proinflammatory cytokine production in vitroand in vivo by the herbal constituents apigenin (chamomile)ginsenoside Rb1 (ginseng) and parthenolide (feverfew)rdquo Foodand Chemical Toxicology vol 41 no 10 pp 1381ndash1390 2003

[12] A Rhule S Navarro J R Smith and D M ShepherdldquoPanax notoginseng attenuates LPS-induced pro-inflammatorymediators in RAW2647 cellsrdquo Journal of Ethnopharmacologyvol 106 no 1 pp 121ndash128 2006

[13] J M Brenchley D A Price T W Schacker et al ldquoMicrobialtranslocation is a cause of systemic immune activation inchronic HIV infectionrdquo Nature Medicine vol 12 no 12 pp1365ndash1371 2006

[14] Y K Cho J Y Lim Y S Jung S K Oh H J Lee and H SungldquoHigh frequency of grossly deleted nef genes in HIV-1 infectedlong-term slow progressors treated with Korean red ginsengrdquoCurrent HIV Research vol 4 no 4 pp 447ndash457 2006

[15] Y K Cho and Y S Jung ldquoHigh frequency of gross deletions inthe 51015840 LTR and gag regions in HIV type 1-infected long-termsurvivors treated with Korean red ginsengrdquo AIDS Research andHuman Retroviruses vol 24 no 2 pp 181ndash193 2008

[16] Y K Cho Y S Jung andH S Sung ldquoFrequent gross deletion intheHIV type 1 nef gene in hemophiliacs treatedwith korean redginseng inhibition of detection by highly active antiretroviraltherapyrdquo AIDS Research and Human Retroviruses vol 25 no 4pp 419ndash424 2009

[17] V Sandonıs C Casado T Alvaro et al ldquoA combination ofdefective DNA and protective host factors are found in a setof HIV-1 ancestral LTNPsrdquo Virology vol 391 no 1 pp 73ndash822009

[18] H R Rangel D Garzaro A K Rodrıguez et al ldquoDeletioninsertion and stop codon mutations in vif genes of HIV-1 infecting slow progressor patientsrdquo Journal of Infection inDeveloping Countries vol 3 no 7 pp 531ndash538 2009

[19] Y K Cho J E Kim and B T Foley ldquoPhylogenetic analysisof near full-length HIV-1 genomic sequences in 21 Koreanindividualsrdquo AIDS Research and Human Retroviruses vol 29no 4 pp 738ndash743 2013

[20] Y K Cho Y S Jung J S Lee and B T Foley ldquoMolecu-lar evidence of HIV-1 transmission in 20 Korean individu-als with haemophilia phylogenetic analysis of the vif generdquoHaemophilia vol 18 no 2 pp 291ndash299 2012

[21] P P Rose and B T Korber ldquoDetecting hypermutations inviral sequences with an emphasis on GrarrA hypermutationrdquoBioinformatics vol 16 no 4 pp 400ndash401 2000

[22] S Fourati S Lambert-Niclot C Soulie et al ldquoHIV-1 genomeis often defective in PBMCs and rectal tissues after long-termHAART as a result of APOBEC3 editing and correlates with thesize of reservoirsrdquo Journal of Antimicrobial Chemotherapy vol67 no 10 pp 2323ndash2326 2012

12 Evidence-Based Complementary and Alternative Medicine

[23] U Wieland A Seelhoff A Hofmann et al ldquoDiversity of thevif gene of human immunodeficiency virus type 1 in UgandardquoJournal of General Virology vol 78 no 2 pp 393ndash400 1997

[24] P Sova M van Ranst P Gupta et al ldquoConservation of an intacthuman immunodeficiency virus type 1 vif gene in vitro and invivordquo Journal of Virology vol 69 no 4 pp 2557ndash2564 1995

[25] T Yamada and A Iwamoto ldquoComparison of proviral accessorygenes between long-term nonprogressors and progressors ofhuman immunodeficiency virus type 1 infectionrdquo Archives ofVirology vol 145 no 5 pp 1021ndash1027 2000

[26] K Tominaga S Kato M Negishi and T Takano ldquoA high fre-quency of defective vif genes in peripheral blood mononuclearcells from HIV type 1-infected individualsrdquo AIDS Research andHuman Retroviruses vol 12 no 16 pp 1543ndash1549 1996

[27] Y K Cho H Sung I G Bae et al ldquoFull sequence of HIV type 1Korean subtype B in anAIDS case with atypical seroconversionTAAAA at TATA boxrdquoAIDS Research andHuman Retrovirusesvol 21 no 11 pp 961ndash964 2005

[28] L Alexander M J Aquino-DeJesus M Chan and W AAndiman ldquoInhibition of human immunodeficiency virus type 1(HIV-1) replication by a two-amino-acid insertion in HIV-1 Viffrom a nonprogressing mother and childrdquo Journal of Virologyvol 76 no 20 pp 10533ndash10539 2002

[29] YK Cho Y S JungH S Sung andCH Joo ldquoFrequent geneticdefects in the HIV-1 51015840LTRgag gene in hemophiliacs treatedwith Korean red ginseng decreased detection of genetic defectsby highly active antiretroviral therapyrdquo Journal of GinsengResearch vol 35 no 4 pp 413ndash420 2011

[30] M Nascimbeni M Bouyac F Rey B Spire and F Clavel ldquoThereplicative impairment of Vif-mutants of human immunodefi-ciency virus type 1 correlates with an overall defect in viral DNAsynthesisrdquo Journal of General Virology vol 79 no 8 pp 1945ndash1950 1998

[31] J P Vartanian M Henry and S Wain-Hobson ldquoSustainedGrarrA hypermutation during reverse transcription of an entirehuman immunodeficiency virus type 1 strain Vau group Ogenomerdquo Journal of General Virology vol 83 no 4 pp 801ndash8052002

[32] T L Kieffer P Kwon R E Nettles Y Han S C Ray andR F Siliciano ldquoGrarrA hypermutation in protease and reversetranscriptase regions of human immunodeficiency virus type 1residing in resting CD4+ T cells in vivordquo Journal of Virology vol79 no 3 pp 1975ndash1980 2005

[33] B Wang M Mikhail W B Dyer J J Zaunders A D Kelleherand N K Saksena ldquoFirst demonstration of a lack of viralsequence evolution in a nonprogressor defining replication-incompetent HIV-1 infectionrdquo Virology vol 312 no 1 pp 135ndash150 2003

[34] G Hassaine I Agostini D Candotti et al ldquoCharacterizationof human immunodeficiency virus type 1 vif gene in long-termasymptomatic individualsrdquoVirology vol 276 no 1 pp 169ndash1802000

[35] F A de Maio C A Rocco P C Aulicino R Bologna AMangano and L Sen ldquoUnusual substitutions in HIV-1 Vif fromchildren infected perinatally without progression to AIDS formore than 8 years without therapyrdquo Journal of Medical Virologyvol 84 no 12 pp 1844ndash1852 2012

[36] M Janini M Rogers D R Birx and F E McCutchan ldquoHumanimmunodeficiency virus type 1 DNA sequences geneticallydamaged by hypermutation are often abundant in patientperipheral blood mononuclear cells and may be generated

during near-simultaneous infection and activation of CD4+ Tcellsrdquo Journal of Virology vol 75 no 17 pp 7973ndash7986 2001

[37] G H Kijak L M Janini S Tovanabutra et al ldquoVariablecontexts and levels of hypermutation inHIV-1 proviral genomesrecovered from primary peripheral blood mononuclear cellsrdquoVirology vol 376 no 1 pp 101ndash111 2008

[38] E W Refsland J F Hultquist and R S Harris ldquoEndogenousorigins of HIV-1 G-to-A hypermutation and restriction in thenonpermissive T cell line CEM2nrdquo PLoS Pathogens vol 8 no7 Article ID e1002800 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 6: Effects of Korean Red Ginseng and HAART on vif Gene in 10 Long

6 Evidence-Based Complementary and Alternative Medicine

87-05

89-17

90-05

90-18

90-50

91-20

1 20 40 60 80 96Kor consensus MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKEWVYRHHYESTHPRISSEVHIPLGDAKLVITTYWGLHTGEREWHLGQGVSIEWRKKRYNT

91CSR4-10026 RGNVVD91CSR11-6922 RGNVVD92CSR5-9885 RGNVVD93CSR6-10159 RGNVVD93CSR6-10162s1 ------------------------------------------------------------------------------------------------03CSR9-805 SGGNVVD05CSR3 RGNIVVDG06CSR10-5280 RGNIVVDRG08CSR3-5334 RGNNVDG08CSR8-5279 RGNIVVD10CSR7-8126 VRGNLVVDG12CSR2-10141 VRGNLVVDG

91KJS12-8538 I92KJS3-6819 GI93KJS7-10155 IAI97KJS3-3814 IGKEI01KJS2-5667 IGKI03KJS2-745 IGAI03KJS7-775 IGAEI05KJS12-3264 YMIVGAEI08KJS5-5157 IVNGAAI10KJS5-10133 IGKAES

92LSK11-9417 K94LSK6-759 KGNPRS96LSK6-11491 AKNPIRS98LSK4-11495 KHN00LSK8-9999 KGNR03LSK6-10001s1 ------------------------------------------------------------------------------04LSK7 KKGNPRST06LSK1-3236 KKGNPVRST07LSK2-3232 KKGNPVRST08LSK1-4762 KKGIPVRST09LSK4-6143 IKNPIGRST10LSK4-9165 KGNPRS10LSK4-9168 KKGNPVRTRST12LSK4-10131 KAVKALKNPEKST 12LSK4-10132 KAKNPGRST13LSK5-11470 KKGNPGRST13LSK5-11471 KKVGNPVRST13LSK5-11472 IKANNRI

92LSH3-6951 K92LSH11-8548 TKK93LSH10-1252 94LSH2-740 95LSH5-10562 V96LSH5-10609 V98LSH4-10567 VG99LSH1-5776 VG99LSH1-5777 VG99LSH1-5778 VG99LSH6-5781 VGE99LSH6-5782 VG00LSH3-5704 KVGR00LSH3-5705 VG00LSH3-5706 VGK02LSH10-5707 VGR02LSH10-5708 VG02LSH10-5710 VG03LSH5-5650 VGG03LSH5-5651 VG03LSH5-5652 VG03LSH10-5653 VG04LSH7-4848 VGKES04LSH8-3011 V06LSH8-5647 VGES06LSH8-5649 VGES06LSH11-3238 VGIS08LSH5-5644 VG08LSH5-5645 VGR08LSH5-5646 KVGE08LSH5-5647 KVGE08LSH11-5694 KVG10LSH4-9190 KVGES10LSH4-9192 VG10LSH4-9193 VGG12LSH6-10100 VGES12LSH6-10101 VG

91KJin7-6723 N93KJin2-10024 N93KJin9-8530 NHVKINVDT01KJin7-5383 KVN03KJin6-683 KVINT04KJin8-1955 KVINDT06KJin1-3030 KVKINDT06KJin9-3012 KVINT07KJin3-5148 VNKINID07KJin3-5961 VNKKINEN10KJin4-9162 NHVKKINVT10KJin9-8176 NHVKKINVDT11KJin12-10119 KVKKINVDT13KJin1-11373 VNNVNDT

K94JWK12-5524 K97JWK K99JWK5-11441 KNE00JWK3-11453 KN00JWK12-11445 KN01JWK7-5603 KVA01JWK7-5603s1 KKNN02JWK8-11435 KVA03JWK8-677 KN05JWK1-5600 KVA06JWK7-3042 KVNAN

KKNVNVVN08JWK1-4772 KVNAN

KVA11JWK4-8993 KVA12JWK1-10125 KVA13JWK1-11366 KVA

minus minusminus minusminus minusminus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ +

Patient KRG HAART

10JWK4 9159

06JWK12 5624

91JWK6 6949

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 7

92-13

93-04

93-60

96-51

92CWS5-9398 G93CWS3-9925 GK93CWS5-6669 GK93CWS7-802 G96CWS11-5341 G99CWS10-5338 KVGE01CWS2-10532 VG03CWS6-684 VGTG03CWS10-5773 VG04CWS5-3239 VGKSTG05CWS5-5783 GTG05CWS5-5785 VGKITG06CWS2-3071 VGTG06CWS2-3203 VGKITG06CWS2-3221 VGTG06CWS2-3222 NVGTG06CWS2-3223 NVGTG06CWS8-3038 NVGKTG06CWS8-3039 NVGKTG07CWS4-3820 VGKIATG07CWS4-3821 VGKIATG07CWS4-10174 HWVGKIATG07CWS4-10175 VGKIATG07CWS4-10176 VGKITG07CWS4-10177 VGKKTG07CWS9-6252 VGTG07CWS9-6253 VGKITG07CWS9-6254 VGKIATG07CWS12-4777 VGQITG08CWS5-6214 NKVGQITG08CWS8-5160 NVGKTG08CWS8-5161 NVGKNQG08CWS12-5720 VGQITG08CWS12-5721 VGQITG08CWS12-5722 VGKQITG09CWS5-6256 VGKQTG09CWS5-6257 VGKQTG09CWS5-6258 VGKDQTG09CWS12-9108 LVGTG09CWS12-9109 VGKQITG

93KYR2-9472 VNN94KYR6-10137 VNN94KYR12-9441 VNN95KYR5-815 VGNN00KYR9-9913 VGNN02KYR7-9911 VGNN03KYR1-817 VNN06KYR7-5236 VGNN06KYR11-5249 VGNNS07KYR3-5265 VGNN07KYR3-11387s1 -------------------------------------------------------------------------------------------------07KYR8-4845 VGNN07KYR8-4846 VGNN07KYR8-5166 IVRNKN07KYR8-5167 VGNRN07KYR8-5168 VGNN

94KMH5 EI96KMH11-8534 IAEI97KMH4-9452 ISEI01KMH11-5526 ISIEI03KMH5-687 EI03KMH11-5725 ISKIEI04KMH5 EI05KMH3-5727 ISEI06KMH11-3026 ISKEI07KMH4-3281s1 --------------------------------------------------------------------EI07KMH10-5732 VNEI08KMH5-5165 VGNEI08KMH8-5427s1 ------------------------------------------------------------------------------------------------09KMH5-11475 ISEI09KMH5-11475s2 HQS----------------------10KMH10-9907s1 ------------------------------------------------------------------------------------------------11KMH4-9917 VDI12KMH4-10096 VGNEI

00YJN10-9385 TRGNMS01YJN9-4850 RGNMS03YJN10-840 VVRGNMS05YJN2-1948 VVRGNMS06YJN10-3044 VRGNMS07YJN4-3248 VVRGNMQI07YJN8-5627 VRGNMQS08YJN3-5606 VRGNMQS08YJN8-5408 VRGNPVMQS09YJN2-6041 VRGNPVMQS09YJN12-9156 VRGNVMQS10YJN6-9918 VRGNVMQS11YJN2-9921 VRGNVMS12YJN7-11382 VRGNVMQS13YJN1-11381 VRGNVMSKor consensus MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKEWVYRHHYESTHPRISSEVHIPLGDAKLVITTYWGLHTGEREWHLGQGVSIEWRKKRYNT

1

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ +minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

87-05

89-17

97 120 140 160 180 192QVDPDLADKLIHLHYFDCFSDSAIRHAILGRLVRPKCEYQAGHNKVGSLQYLALTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH

91CSR4-10026 TQRYKHISSTA91CSR11-6922 TQRYKHISSTA92CSR5-9885 TQRYKHISSTA93CSR6-10159 TQRYKHISSTA93CSR6-10162s1 ------------------------------------------------------------------------------------------------03CSR9-805 AQRYEKHISSTA05CSR3 TQRYKHISSTAK06CSR10-5280 TQRYKHISRTA08CSR3-5334 TQRYKHISSTA08CSR8-5279 TQRYKHISSTA10CSR7-8126 TQRYKHISSTIA12CSR2-10141 TQRYKHISSTIA

91KJS12-8538 92KJS3-6819 93KJS7-10155 97KJS3-3814 01KJS2-5667 03KJS3-745 03KJS7-775 05KJS12-3264 08KJS5-5157 10KJS5-10133

minus minusminus minusminus minusminus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ +

Patient Kor consensus KRG HAART

20 60 80 9640

RR

VS

VRE

VR

VRS

VRRES

VRRES

PKVPVIFRHIES

EVRRHES

VRRHES

Figure 2 Continued

8 Evidence-Based Complementary and Alternative Medicine

90-05

90-18

90-50

91-20

92LSK11-9417 YETRQE94LSK6-759 YETRPQES96LSK6-11491 GRYETRQ98LSK4-11495 YENTRPQES00LSK8-9999 YPQE03LSK6-10001s1 -------------------------------------------QRES04LSK7 YETRPQES06LSK1-3236 YEVTRPQRES07LSK2-3232 YEVTRPQES08LSK1-4762 YEVTRPQAESS09LSK4-6143 YEVTRPQES10LSK4-9165 YEVTRPQES10LSK4-9168 YEVTRPQES12LSK4-10131 NYKVTRPQRAES12LSK4-10132 YEVTRPQAESQ13LSK5-11470 PYVRRPTQETESS13LSK5-11471 YVTRPQRES13LSK5-11472 YENRTRQRES

92LSH3-6951 YRDH92LSH11-8548 GYRD93LSH10-1252 YRDR94LSH2-740 YRDR95LSH5-10562 YTRDR96LSH5-10609 YTR98LSH4-10567 YQTRRG---H99LSH1-5776 YQTRRG---RH99LSH1-5777 YQTRRHG---H99LSH1-5778 YQTRG---H99LSH6-5781 YQTRRQG---H99LSH6-5782 YQTRG---H00LSH3-5704 YQTRRG---H00LSH3-5705 YQTRRQSG---H00LSH3-5706 YQTRAG---H02LSH10-5707 YETRQG---H02LSH10-5708 YQTRG---H02LSH10-5710 YQTRRQR---H03LSH5-5650 YQTRRR---03LSH5-5651 YQTRRR---H03LSH5-5652 YQTRRER---03LSH10-5653 YQTRRQRS04LSH7-4848 YQTRRKQG---H04LSH8-3011 YR06LSH8-5647 YQTRRG---H06LSH8-5649 YQTRRG---H06LSH11-3238 YQHTRRQES08LSH5-5644 YQTRRSG---H08LSH5-5645 YQTRQAKS08LSH5-5646 YQTRRR---H08LSH5-5647 YQTRRR---H08LSH11-5694 YQTRRSRKG---H10LSH4-9190 YEQCTRRRG---H10LSH4-9192 YQRTRRRRG---H10LSH4-9193 YQTRRG---H12LSH6-10100 YQTRRG---H12LSH6-10101 YQTRRSRG---H

91KJin7-6723 TT93KJin2-10024 TT93KJin9-8530 VTPAKH01KJin7-5383 ISTT03KJin6-683 ISTPQH04KJin8-1955 ITPTH06KJin1-3030 VTPTH06KJin9-3012 VAPT07KJin3-5148 QEPTN07KJin3-5961 NLEPTAN10KJin4-9162 VTTAH10KJin9-8176 QVTAAH11KJin12-10119 VTPTADH13KJin1-11373 VTPTH

PE94JWK12-5524 VQ97JWK HR99JWK5-11441 HR00JWK3-11453 HR00JWK12-11445 HR01JWK7-5603 NHRQ01JWK7-5603s1 -------------------------------------------------------------------------------02JWK8-11436 NRQQ03JWK8-677 HR05JWK1-5600 NRQQ06JWK7-3042 HRQ

HRQ08JWK1-4772 NRQQ

NRQQ11JWK4-8993 NRQQ12JWK1-10125 NRQQ13JWK1-11366 NRQQ

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ +

10JWK4 9159

06 JWK12 5624

91JWK6 6949

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 9

92-13

93-04

93-60

92CWS5-9398 HSE93CWS3-9925 HE93CWS5-6669 EHTE93CWS7-802 KHTE96CWS11-5341 KHTE99CWS10-5338 HPES01CWS2-10532 HPDE03CWS6-684 HPE03CWS10-5773 HPDE04CWS5-3239 HPTE05CWS5-5783 HPTD----05CWS5-5785 HPTD----06CWS2-3071 HPTDE06CWS2-3203 HPTD----06CWS2-3221 HPTD----06CWS2-3222 VHPTD----06CWS2-3223 HPTD----06CWS8-3038 HPTD----06CWS8-3039 HPTD----07CWS4-3820 HPTATD----07CWS4-3821 HPTATD----07CWS4-10174 HPTATD----07CWS4-10175 HPTATD----07CWS4-10176 HPTATD----07CWS4-10177 VHRD----07CWS9-6252 HPE07CWS9-6253 HPTATD----07CWS9-6254 HPTATD----07CWS12-4777 HPTATD----08CWS5-6214 HPTATD----08CWS8-5160 HPTATD----08CWS8-5161 HPTATD----08CWS12-5720 HPTATD----08CWS12-5721 HPTATD----08CWS12-5722 HPTATD----09CWS5-6256 HPTATD----09CWS5-6257 HPTTATD----09CWS5-6258 HPTAD----09CWS12-9108 HPDE09CWS12-9109 HPTATTD----

93KYR2-9472 NSRIRKE94KYR6-10137 NSRRKE94KYR12-9441 NSRIRKER95KYR5-815 NSRIRKE00KYR9-9913 NSRRRKE02KYR7-9911 NSRRRKE03KYR1-817 NSRIRKE06KYR7-5236 QKPHRIRKE06KYR11-5249 QKPHRIRKE07KYR3-5265 NSHRIRKE07KYR3-11387s1 ---------------QNSHRIRRKER07KYR8-4845 QNSRRIRKER07KYR8-4846 NSRRIRKE07KYR8-5166 KNSHRRRIRKER07KYR8-5167 NKHRRIRRKE07KYR8-5168 IQRKPHRRIRKE

94KMH5 HVPR96KMH11-8534 VRT97KMH4-9452 HVQR01KMH11-5526 HVPR03KMH5-687 HVPR03KMH11-5725 HVQRS04KMH5 VQRS05KMH3-5727 HVPR06KMH11-3026 HVPR07KMH4-3281s1 VHVPRV----------------------------07KMH10-5732 HVQPRVRS08KMH5-5165 HIQPRVRS08KMH8-5427s1 ----------------------------HVQPTRVRS09KMH5-11475 HVQPRVRS09KMH5-11475s2 ------------------------------------------------------------------------------------------------10KMH10-9907s1 -----------------------------------------RVRS11KMH4-9917 HVPRVRS12KMH4-10096 HVPRVRRS

00YJN10-9385 ETVSRE01YJN9-4850 ETVSRE03YJN10-840 ETVSRE05YJN2-1948 ETQVSRE06YJN10-3044 ETQVSRE07YJN4-3248 ETQVSREY07YJN8-5627 ETQVSREY08YJN3-5606 ETQVSREY08YJN8-5408 ETQVSREY09YJN2-6041 ETQVSREY09YJN12-9156 ETQVSREY10YJN6-9918 ETQVSRIEY11YJN2-9921 ETQVSREY12YJN7-11382 ETQVSREY13YJN1-11381 ETQVSREYKor consensus QVDPDLADKLIHLHYFDCFSDSAIRHAILGRLVRPKCEYQAGHNKVGSLQYLALTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH

97 120 140 160 180 192

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ +

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

96-51

Figure 2 Sequential alignment of the amino acids of the Vif protein over 20 years All sequences that demonstrated small in-frame deletionsgross deletions and stop codons are depicted together with the baseline and last sequences of each patient Patients 90-18 demonstrated a9 bp deletion (positions 182ndash184) in April 1998 and patient 92-13 demonstrated a 12 bp deletion (positions 185ndash188) in May 2005 Patient90-05 consistently demonstrated a 9 bp insertion (RQTRAR-RAR-NGASRP) at the same position at the terminus of vpr beginning in August2000 (data not shown) The code 92LSH3-6951 (sampling year initials of the patient sampling month and sequence number) denotes thatthe sampling date is March 1992 and the sequence number is 6951 in patient LSH (90-18) The dot (sdot) hyphen (-) and asterisk (lowast) denotesequence identity deletions and premature stop codons respectively compared with the Korean consensus [20] In the right column theplus (+) and minus (minus) signs denote the presence or absence of the corresponding therapy respectively

10 Evidence-Based Complementary and Alternative Medicine

61 69 112 120 208 216 264 272 520 528Patient WT nucleotide TGGAAAAGT TGGGTCTAT TGGGGTCTG TGGAGGAAA TGGAACAAG Hypermut 20

sequence no P value90-05 10LSK4-9168 G A lt005

12LSK4-10131 A A AA A A lt000113LSK5-11470 A A A 015 13LSK5-11472 A AA lt001

90-18 08LSH5-5645 AA 019 08LSH11-5694 A 05710LSH4-9192 AA 013

90-54 07KJin3-5961 A 00693-04 94KYR12-9441 AA AG 009

07KYR8-5166 A A AA A AG lt000107KYR8-4846 A AA G 00507KYR8-5167 A A A AG 00507KYR8-5168 A A AG 02907KYR8-4845 AA G 005

93-60 03KMH11-5725 AA A 015

WT amino acid W K S W V Y W G L W R K W N K21 22 23 38 39 40 70 71 72 89 90 91 174 175 176

Figure 3 Positions of the premature stop codons in the 15 vif genes from five patients All stop codons resulted from GG rarr GA orGG rarr AG changes APOBEC3G exhibits an intrinsic preference for the second cytosine in a 51015840CC dinucleotide motif leading to 51015840GG-to-AG mutations [38]

In total five of our patients demonstrated in-frame deletionsof the vif gene of the 75 KSB-infected patients who weretreated with KRG Also in our present study cohort fivepatients including 90-18 and 92-13 demonstrated deletedvif genes (small in-frame and gross deletions) during KRGintake whereas all of our 10 LTSP patients demonstrateddeletions in the nef gene (119875 lt 005) [14] In additioncompared with the reported deletions of the nef and 51015840LTRgag genes the locations of the deleted vif gene werepositioned within a narrow range at the terminus [15 16 23]

Regarding gross deletions to date only two patientshave demonstrated gross deletions in the vif gene [17 18]and only one patient has demonstrated the insertion of twoamino acids and these patients were part of a nonprogress-ing mother-child pair [28] However in our present studyof the 10 LTSP patients who were assessed four patientsdemonstrated gross deletion in the vif gene during KRGintake prior to receiving HAART (15) This frequency issignificantly lower than the previously reported incidence of10 of 10 patients demonstrating gross deletions in the nef(187) and 51015840 LTRgag genes (376 119875 lt 001) [27 28]The frequency of deletion in the nef gene was reported tosignificantly decrease during KRG plus HAART (119875 lt 001)[16] In contrast to previous studies on the nef and gag genes[16 29] the detection of in-frame deletions in the vif genewas not suppressed in the patients receiving HAART in ourpresent cohort The reasons for this include the following (1)the proportion and frequency (approximately two-thirds) ofdeleted vif genes after the first occurrence were much higherthan in the nef and 51015840 LTRgag genes (lt20 and 30 resp)(2) small deletions in the vif gene occurred as a single bandwhereas gross deletions in the nef and 51015840 LTRgag genes weremainly detected as two bands (wild type and short) per PCRreaction and (3) the number of amplicons indicating PSCdueto G-to-A hypermutation was significantly higher in patients

receiving KRG plus HAART than patients receiving onlyKRG This phenomenon is consistent with our previous datain 51015840 LTRgag gene [29] However the frequency of G-to-Ahypermutationwas significantly lower in vif gene than that in51015840 LTRgag gene [29] Thus for the vif gene the detection ofsmall deletionsmight be less affected by limit-diluting effectsas has been shown for the nef gene [29] Taken together theseadditional defects in the vif genes in the same patients mightbe related to the replicative impairment in vif defective HIV-1 which ultimately results in defects in the synthesis of viralDNA [30]

In our present study it appears that the frequency ofhypermutation was higher in patients who maintained goodcompliance with HAART during HAART plus KRG (90-0590-18 and 93-04) than in the patients who demonstratedpoor compliance (Figure 1) In our extended data analysis124 (60485) 31 (5161) 30 (19617) and 09 (1108)of vif genes during KRG plus HAART HAART aloneKRG alone and control revealed PSC respectively Thesedata indicate the presence of synergistic effect of KRG plusHAART on PSC (119875 lt 001) There was no case that smalldeletions were induced by HAART alone About 6ndash43 ofthe integrated proviral pol gene is hypermutated by HAART[22 31 32] although hypermutated viral genomes are notpresent in plasma [32]

ApoBec3G-induced hypermutation in LTNPs has beenreported previously [33] Indeed it has been reported alsothat LTNP patients harbor PSC-containing vif genes morefrequently than progressors [25] although the reportedfrequencies of defective genes vary among studies [34] Inour current analyses vif gene-containing PSC due to G-to-A hypermutation was found to be significantly higher duringKRG plus HAART than only KRG intake prior to HAARTThis finding is consistent with our previous data obtainedfrom a cohort of the Korean hemophiliacs (21 incidence

Evidence-Based Complementary and Alternative Medicine 11

of G-to-A hypermutation while receiving only KRG versus98 on KRG plus HAART unpublished data) and otherobservations regarding the pol gene [22]

Previous studies report that certain vif mutations such asL81M R132S S130I and the insertion of two amino acids areassociatedwith slow progression or low viral loads [17 18 28]In our present study L81M [35] and R132S were consistentlydetected in patient 96-51 and patients 87-05 and 96-51respectively although both mutations were found in only in2 and 3 of 169 Korean patients respectively Although thevif gene is highly conserved in HIV-1 genomes the recoveryof hypermutants might be severely underestimated in ourpresent analysis because of the primer mismatching andthe difficulties involved in detection following amplification[36 37] Overall our current data suggest that small in-frame deletion and PSCs are associated with KRG intake andHAART respectively although further studies are needed

5 Conclusions

Our data suggest that HAART and KRG intake might inducePSCs and small in-frame deletions respectively

Conflict of Interests

The authors have no conflict of interests to declare

Acknowledgments

This work was supported by a grant from the Korean Societyof Ginseng (2010ndash2012) The authors thank Professor Seung-Chul Yun the Division of Biostatistics University of UlsanCollege of Medicine Asan Medical Center for the statisticalconsultation

References

[1] E Ernst ldquoPanax ginseng an overview of the clinical evidencerdquoJournal of Ginseng Research vol 34 no 4 pp 259ndash263 2010

[2] K T Choi ldquoBotanical characteristics pharmacological effectsand medicinal components of Korean Panax ginseng C AMeyerrdquoActa Pharmacologica Sinica vol 29 no 9 pp 1109ndash11182008

[3] S J Fulder ldquoGinseng and the hypothalamic-pituitary control ofstressrdquo The American Journal of Chinese Medicine vol 9 no 2pp 112ndash118 1981

[4] V K Singh S S Agarwal and B M Gupta ldquoImmunomodula-tory activity of Panax ginseng extractrdquo Planta Medica vol 50no 6 pp 462ndash465 1984

[5] X Song and S Hu ldquoAdjuvant activities of saponins fromtraditional Chinese medicinal herbsrdquo Vaccine vol 27 no 36pp 4883ndash4890 2009

[6] D G Yoo M C Kim M K Park et al ldquoProtective effectof ginseng polysaccharides on influenza viral infectionrdquo PLoSONE vol 7 no 3 Article ID e33678 2012

[7] H X Sun Y P Ye H J Pan and Y J Pan ldquoAdjuvant effectof Panax notoginseng saponins on the immune responses toovalbumin in micerdquo Vaccine vol 22 no 29-30 pp 3882ndash38892004

[8] J Sun S Hu and X Song ldquoAdjuvant effects of protopanaxadioland protopanaxatriol saponins from ginseng roots on theimmune responses to ovalbumin in micerdquo Vaccine vol 25 no6 pp 1114ndash1120 2007

[9] P W Hunt ldquoHIV and inflammation mechanisms and conse-quencesrdquo Current HIVAIDS Reports vol 9 no 2 pp 139ndash1472012

[10] T Yayeh K H Jung H Y Jeong et al ldquoKorean red ginsengsaponin fraction downregulates proinflammatory mediators inLPS stimulated RAW264 7 cells and protects mice againstendotoxic shockrdquo Journal of Ginseng Research vol 36 no 3 pp263ndash269 2012

[11] A T Smolinski and J J Pestka ldquoModulation of lipopolysaccha-ride-induced proinflammatory cytokine production in vitroand in vivo by the herbal constituents apigenin (chamomile)ginsenoside Rb1 (ginseng) and parthenolide (feverfew)rdquo Foodand Chemical Toxicology vol 41 no 10 pp 1381ndash1390 2003

[12] A Rhule S Navarro J R Smith and D M ShepherdldquoPanax notoginseng attenuates LPS-induced pro-inflammatorymediators in RAW2647 cellsrdquo Journal of Ethnopharmacologyvol 106 no 1 pp 121ndash128 2006

[13] J M Brenchley D A Price T W Schacker et al ldquoMicrobialtranslocation is a cause of systemic immune activation inchronic HIV infectionrdquo Nature Medicine vol 12 no 12 pp1365ndash1371 2006

[14] Y K Cho J Y Lim Y S Jung S K Oh H J Lee and H SungldquoHigh frequency of grossly deleted nef genes in HIV-1 infectedlong-term slow progressors treated with Korean red ginsengrdquoCurrent HIV Research vol 4 no 4 pp 447ndash457 2006

[15] Y K Cho and Y S Jung ldquoHigh frequency of gross deletions inthe 51015840 LTR and gag regions in HIV type 1-infected long-termsurvivors treated with Korean red ginsengrdquo AIDS Research andHuman Retroviruses vol 24 no 2 pp 181ndash193 2008

[16] Y K Cho Y S Jung andH S Sung ldquoFrequent gross deletion intheHIV type 1 nef gene in hemophiliacs treatedwith korean redginseng inhibition of detection by highly active antiretroviraltherapyrdquo AIDS Research and Human Retroviruses vol 25 no 4pp 419ndash424 2009

[17] V Sandonıs C Casado T Alvaro et al ldquoA combination ofdefective DNA and protective host factors are found in a setof HIV-1 ancestral LTNPsrdquo Virology vol 391 no 1 pp 73ndash822009

[18] H R Rangel D Garzaro A K Rodrıguez et al ldquoDeletioninsertion and stop codon mutations in vif genes of HIV-1 infecting slow progressor patientsrdquo Journal of Infection inDeveloping Countries vol 3 no 7 pp 531ndash538 2009

[19] Y K Cho J E Kim and B T Foley ldquoPhylogenetic analysisof near full-length HIV-1 genomic sequences in 21 Koreanindividualsrdquo AIDS Research and Human Retroviruses vol 29no 4 pp 738ndash743 2013

[20] Y K Cho Y S Jung J S Lee and B T Foley ldquoMolecu-lar evidence of HIV-1 transmission in 20 Korean individu-als with haemophilia phylogenetic analysis of the vif generdquoHaemophilia vol 18 no 2 pp 291ndash299 2012

[21] P P Rose and B T Korber ldquoDetecting hypermutations inviral sequences with an emphasis on GrarrA hypermutationrdquoBioinformatics vol 16 no 4 pp 400ndash401 2000

[22] S Fourati S Lambert-Niclot C Soulie et al ldquoHIV-1 genomeis often defective in PBMCs and rectal tissues after long-termHAART as a result of APOBEC3 editing and correlates with thesize of reservoirsrdquo Journal of Antimicrobial Chemotherapy vol67 no 10 pp 2323ndash2326 2012

12 Evidence-Based Complementary and Alternative Medicine

[23] U Wieland A Seelhoff A Hofmann et al ldquoDiversity of thevif gene of human immunodeficiency virus type 1 in UgandardquoJournal of General Virology vol 78 no 2 pp 393ndash400 1997

[24] P Sova M van Ranst P Gupta et al ldquoConservation of an intacthuman immunodeficiency virus type 1 vif gene in vitro and invivordquo Journal of Virology vol 69 no 4 pp 2557ndash2564 1995

[25] T Yamada and A Iwamoto ldquoComparison of proviral accessorygenes between long-term nonprogressors and progressors ofhuman immunodeficiency virus type 1 infectionrdquo Archives ofVirology vol 145 no 5 pp 1021ndash1027 2000

[26] K Tominaga S Kato M Negishi and T Takano ldquoA high fre-quency of defective vif genes in peripheral blood mononuclearcells from HIV type 1-infected individualsrdquo AIDS Research andHuman Retroviruses vol 12 no 16 pp 1543ndash1549 1996

[27] Y K Cho H Sung I G Bae et al ldquoFull sequence of HIV type 1Korean subtype B in anAIDS case with atypical seroconversionTAAAA at TATA boxrdquoAIDS Research andHuman Retrovirusesvol 21 no 11 pp 961ndash964 2005

[28] L Alexander M J Aquino-DeJesus M Chan and W AAndiman ldquoInhibition of human immunodeficiency virus type 1(HIV-1) replication by a two-amino-acid insertion in HIV-1 Viffrom a nonprogressing mother and childrdquo Journal of Virologyvol 76 no 20 pp 10533ndash10539 2002

[29] YK Cho Y S JungH S Sung andCH Joo ldquoFrequent geneticdefects in the HIV-1 51015840LTRgag gene in hemophiliacs treatedwith Korean red ginseng decreased detection of genetic defectsby highly active antiretroviral therapyrdquo Journal of GinsengResearch vol 35 no 4 pp 413ndash420 2011

[30] M Nascimbeni M Bouyac F Rey B Spire and F Clavel ldquoThereplicative impairment of Vif-mutants of human immunodefi-ciency virus type 1 correlates with an overall defect in viral DNAsynthesisrdquo Journal of General Virology vol 79 no 8 pp 1945ndash1950 1998

[31] J P Vartanian M Henry and S Wain-Hobson ldquoSustainedGrarrA hypermutation during reverse transcription of an entirehuman immunodeficiency virus type 1 strain Vau group Ogenomerdquo Journal of General Virology vol 83 no 4 pp 801ndash8052002

[32] T L Kieffer P Kwon R E Nettles Y Han S C Ray andR F Siliciano ldquoGrarrA hypermutation in protease and reversetranscriptase regions of human immunodeficiency virus type 1residing in resting CD4+ T cells in vivordquo Journal of Virology vol79 no 3 pp 1975ndash1980 2005

[33] B Wang M Mikhail W B Dyer J J Zaunders A D Kelleherand N K Saksena ldquoFirst demonstration of a lack of viralsequence evolution in a nonprogressor defining replication-incompetent HIV-1 infectionrdquo Virology vol 312 no 1 pp 135ndash150 2003

[34] G Hassaine I Agostini D Candotti et al ldquoCharacterizationof human immunodeficiency virus type 1 vif gene in long-termasymptomatic individualsrdquoVirology vol 276 no 1 pp 169ndash1802000

[35] F A de Maio C A Rocco P C Aulicino R Bologna AMangano and L Sen ldquoUnusual substitutions in HIV-1 Vif fromchildren infected perinatally without progression to AIDS formore than 8 years without therapyrdquo Journal of Medical Virologyvol 84 no 12 pp 1844ndash1852 2012

[36] M Janini M Rogers D R Birx and F E McCutchan ldquoHumanimmunodeficiency virus type 1 DNA sequences geneticallydamaged by hypermutation are often abundant in patientperipheral blood mononuclear cells and may be generated

during near-simultaneous infection and activation of CD4+ Tcellsrdquo Journal of Virology vol 75 no 17 pp 7973ndash7986 2001

[37] G H Kijak L M Janini S Tovanabutra et al ldquoVariablecontexts and levels of hypermutation inHIV-1 proviral genomesrecovered from primary peripheral blood mononuclear cellsrdquoVirology vol 376 no 1 pp 101ndash111 2008

[38] E W Refsland J F Hultquist and R S Harris ldquoEndogenousorigins of HIV-1 G-to-A hypermutation and restriction in thenonpermissive T cell line CEM2nrdquo PLoS Pathogens vol 8 no7 Article ID e1002800 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 7: Effects of Korean Red Ginseng and HAART on vif Gene in 10 Long

Evidence-Based Complementary and Alternative Medicine 7

92-13

93-04

93-60

96-51

92CWS5-9398 G93CWS3-9925 GK93CWS5-6669 GK93CWS7-802 G96CWS11-5341 G99CWS10-5338 KVGE01CWS2-10532 VG03CWS6-684 VGTG03CWS10-5773 VG04CWS5-3239 VGKSTG05CWS5-5783 GTG05CWS5-5785 VGKITG06CWS2-3071 VGTG06CWS2-3203 VGKITG06CWS2-3221 VGTG06CWS2-3222 NVGTG06CWS2-3223 NVGTG06CWS8-3038 NVGKTG06CWS8-3039 NVGKTG07CWS4-3820 VGKIATG07CWS4-3821 VGKIATG07CWS4-10174 HWVGKIATG07CWS4-10175 VGKIATG07CWS4-10176 VGKITG07CWS4-10177 VGKKTG07CWS9-6252 VGTG07CWS9-6253 VGKITG07CWS9-6254 VGKIATG07CWS12-4777 VGQITG08CWS5-6214 NKVGQITG08CWS8-5160 NVGKTG08CWS8-5161 NVGKNQG08CWS12-5720 VGQITG08CWS12-5721 VGQITG08CWS12-5722 VGKQITG09CWS5-6256 VGKQTG09CWS5-6257 VGKQTG09CWS5-6258 VGKDQTG09CWS12-9108 LVGTG09CWS12-9109 VGKQITG

93KYR2-9472 VNN94KYR6-10137 VNN94KYR12-9441 VNN95KYR5-815 VGNN00KYR9-9913 VGNN02KYR7-9911 VGNN03KYR1-817 VNN06KYR7-5236 VGNN06KYR11-5249 VGNNS07KYR3-5265 VGNN07KYR3-11387s1 -------------------------------------------------------------------------------------------------07KYR8-4845 VGNN07KYR8-4846 VGNN07KYR8-5166 IVRNKN07KYR8-5167 VGNRN07KYR8-5168 VGNN

94KMH5 EI96KMH11-8534 IAEI97KMH4-9452 ISEI01KMH11-5526 ISIEI03KMH5-687 EI03KMH11-5725 ISKIEI04KMH5 EI05KMH3-5727 ISEI06KMH11-3026 ISKEI07KMH4-3281s1 --------------------------------------------------------------------EI07KMH10-5732 VNEI08KMH5-5165 VGNEI08KMH8-5427s1 ------------------------------------------------------------------------------------------------09KMH5-11475 ISEI09KMH5-11475s2 HQS----------------------10KMH10-9907s1 ------------------------------------------------------------------------------------------------11KMH4-9917 VDI12KMH4-10096 VGNEI

00YJN10-9385 TRGNMS01YJN9-4850 RGNMS03YJN10-840 VVRGNMS05YJN2-1948 VVRGNMS06YJN10-3044 VRGNMS07YJN4-3248 VVRGNMQI07YJN8-5627 VRGNMQS08YJN3-5606 VRGNMQS08YJN8-5408 VRGNPVMQS09YJN2-6041 VRGNPVMQS09YJN12-9156 VRGNVMQS10YJN6-9918 VRGNVMQS11YJN2-9921 VRGNVMS12YJN7-11382 VRGNVMQS13YJN1-11381 VRGNVMSKor consensus MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKEWVYRHHYESTHPRISSEVHIPLGDAKLVITTYWGLHTGEREWHLGQGVSIEWRKKRYNT

1

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ +minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

87-05

89-17

97 120 140 160 180 192QVDPDLADKLIHLHYFDCFSDSAIRHAILGRLVRPKCEYQAGHNKVGSLQYLALTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH

91CSR4-10026 TQRYKHISSTA91CSR11-6922 TQRYKHISSTA92CSR5-9885 TQRYKHISSTA93CSR6-10159 TQRYKHISSTA93CSR6-10162s1 ------------------------------------------------------------------------------------------------03CSR9-805 AQRYEKHISSTA05CSR3 TQRYKHISSTAK06CSR10-5280 TQRYKHISRTA08CSR3-5334 TQRYKHISSTA08CSR8-5279 TQRYKHISSTA10CSR7-8126 TQRYKHISSTIA12CSR2-10141 TQRYKHISSTIA

91KJS12-8538 92KJS3-6819 93KJS7-10155 97KJS3-3814 01KJS2-5667 03KJS3-745 03KJS7-775 05KJS12-3264 08KJS5-5157 10KJS5-10133

minus minusminus minusminus minusminus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ +

Patient Kor consensus KRG HAART

20 60 80 9640

RR

VS

VRE

VR

VRS

VRRES

VRRES

PKVPVIFRHIES

EVRRHES

VRRHES

Figure 2 Continued

8 Evidence-Based Complementary and Alternative Medicine

90-05

90-18

90-50

91-20

92LSK11-9417 YETRQE94LSK6-759 YETRPQES96LSK6-11491 GRYETRQ98LSK4-11495 YENTRPQES00LSK8-9999 YPQE03LSK6-10001s1 -------------------------------------------QRES04LSK7 YETRPQES06LSK1-3236 YEVTRPQRES07LSK2-3232 YEVTRPQES08LSK1-4762 YEVTRPQAESS09LSK4-6143 YEVTRPQES10LSK4-9165 YEVTRPQES10LSK4-9168 YEVTRPQES12LSK4-10131 NYKVTRPQRAES12LSK4-10132 YEVTRPQAESQ13LSK5-11470 PYVRRPTQETESS13LSK5-11471 YVTRPQRES13LSK5-11472 YENRTRQRES

92LSH3-6951 YRDH92LSH11-8548 GYRD93LSH10-1252 YRDR94LSH2-740 YRDR95LSH5-10562 YTRDR96LSH5-10609 YTR98LSH4-10567 YQTRRG---H99LSH1-5776 YQTRRG---RH99LSH1-5777 YQTRRHG---H99LSH1-5778 YQTRG---H99LSH6-5781 YQTRRQG---H99LSH6-5782 YQTRG---H00LSH3-5704 YQTRRG---H00LSH3-5705 YQTRRQSG---H00LSH3-5706 YQTRAG---H02LSH10-5707 YETRQG---H02LSH10-5708 YQTRG---H02LSH10-5710 YQTRRQR---H03LSH5-5650 YQTRRR---03LSH5-5651 YQTRRR---H03LSH5-5652 YQTRRER---03LSH10-5653 YQTRRQRS04LSH7-4848 YQTRRKQG---H04LSH8-3011 YR06LSH8-5647 YQTRRG---H06LSH8-5649 YQTRRG---H06LSH11-3238 YQHTRRQES08LSH5-5644 YQTRRSG---H08LSH5-5645 YQTRQAKS08LSH5-5646 YQTRRR---H08LSH5-5647 YQTRRR---H08LSH11-5694 YQTRRSRKG---H10LSH4-9190 YEQCTRRRG---H10LSH4-9192 YQRTRRRRG---H10LSH4-9193 YQTRRG---H12LSH6-10100 YQTRRG---H12LSH6-10101 YQTRRSRG---H

91KJin7-6723 TT93KJin2-10024 TT93KJin9-8530 VTPAKH01KJin7-5383 ISTT03KJin6-683 ISTPQH04KJin8-1955 ITPTH06KJin1-3030 VTPTH06KJin9-3012 VAPT07KJin3-5148 QEPTN07KJin3-5961 NLEPTAN10KJin4-9162 VTTAH10KJin9-8176 QVTAAH11KJin12-10119 VTPTADH13KJin1-11373 VTPTH

PE94JWK12-5524 VQ97JWK HR99JWK5-11441 HR00JWK3-11453 HR00JWK12-11445 HR01JWK7-5603 NHRQ01JWK7-5603s1 -------------------------------------------------------------------------------02JWK8-11436 NRQQ03JWK8-677 HR05JWK1-5600 NRQQ06JWK7-3042 HRQ

HRQ08JWK1-4772 NRQQ

NRQQ11JWK4-8993 NRQQ12JWK1-10125 NRQQ13JWK1-11366 NRQQ

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ +

10JWK4 9159

06 JWK12 5624

91JWK6 6949

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 9

92-13

93-04

93-60

92CWS5-9398 HSE93CWS3-9925 HE93CWS5-6669 EHTE93CWS7-802 KHTE96CWS11-5341 KHTE99CWS10-5338 HPES01CWS2-10532 HPDE03CWS6-684 HPE03CWS10-5773 HPDE04CWS5-3239 HPTE05CWS5-5783 HPTD----05CWS5-5785 HPTD----06CWS2-3071 HPTDE06CWS2-3203 HPTD----06CWS2-3221 HPTD----06CWS2-3222 VHPTD----06CWS2-3223 HPTD----06CWS8-3038 HPTD----06CWS8-3039 HPTD----07CWS4-3820 HPTATD----07CWS4-3821 HPTATD----07CWS4-10174 HPTATD----07CWS4-10175 HPTATD----07CWS4-10176 HPTATD----07CWS4-10177 VHRD----07CWS9-6252 HPE07CWS9-6253 HPTATD----07CWS9-6254 HPTATD----07CWS12-4777 HPTATD----08CWS5-6214 HPTATD----08CWS8-5160 HPTATD----08CWS8-5161 HPTATD----08CWS12-5720 HPTATD----08CWS12-5721 HPTATD----08CWS12-5722 HPTATD----09CWS5-6256 HPTATD----09CWS5-6257 HPTTATD----09CWS5-6258 HPTAD----09CWS12-9108 HPDE09CWS12-9109 HPTATTD----

93KYR2-9472 NSRIRKE94KYR6-10137 NSRRKE94KYR12-9441 NSRIRKER95KYR5-815 NSRIRKE00KYR9-9913 NSRRRKE02KYR7-9911 NSRRRKE03KYR1-817 NSRIRKE06KYR7-5236 QKPHRIRKE06KYR11-5249 QKPHRIRKE07KYR3-5265 NSHRIRKE07KYR3-11387s1 ---------------QNSHRIRRKER07KYR8-4845 QNSRRIRKER07KYR8-4846 NSRRIRKE07KYR8-5166 KNSHRRRIRKER07KYR8-5167 NKHRRIRRKE07KYR8-5168 IQRKPHRRIRKE

94KMH5 HVPR96KMH11-8534 VRT97KMH4-9452 HVQR01KMH11-5526 HVPR03KMH5-687 HVPR03KMH11-5725 HVQRS04KMH5 VQRS05KMH3-5727 HVPR06KMH11-3026 HVPR07KMH4-3281s1 VHVPRV----------------------------07KMH10-5732 HVQPRVRS08KMH5-5165 HIQPRVRS08KMH8-5427s1 ----------------------------HVQPTRVRS09KMH5-11475 HVQPRVRS09KMH5-11475s2 ------------------------------------------------------------------------------------------------10KMH10-9907s1 -----------------------------------------RVRS11KMH4-9917 HVPRVRS12KMH4-10096 HVPRVRRS

00YJN10-9385 ETVSRE01YJN9-4850 ETVSRE03YJN10-840 ETVSRE05YJN2-1948 ETQVSRE06YJN10-3044 ETQVSRE07YJN4-3248 ETQVSREY07YJN8-5627 ETQVSREY08YJN3-5606 ETQVSREY08YJN8-5408 ETQVSREY09YJN2-6041 ETQVSREY09YJN12-9156 ETQVSREY10YJN6-9918 ETQVSRIEY11YJN2-9921 ETQVSREY12YJN7-11382 ETQVSREY13YJN1-11381 ETQVSREYKor consensus QVDPDLADKLIHLHYFDCFSDSAIRHAILGRLVRPKCEYQAGHNKVGSLQYLALTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH

97 120 140 160 180 192

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ +

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

96-51

Figure 2 Sequential alignment of the amino acids of the Vif protein over 20 years All sequences that demonstrated small in-frame deletionsgross deletions and stop codons are depicted together with the baseline and last sequences of each patient Patients 90-18 demonstrated a9 bp deletion (positions 182ndash184) in April 1998 and patient 92-13 demonstrated a 12 bp deletion (positions 185ndash188) in May 2005 Patient90-05 consistently demonstrated a 9 bp insertion (RQTRAR-RAR-NGASRP) at the same position at the terminus of vpr beginning in August2000 (data not shown) The code 92LSH3-6951 (sampling year initials of the patient sampling month and sequence number) denotes thatthe sampling date is March 1992 and the sequence number is 6951 in patient LSH (90-18) The dot (sdot) hyphen (-) and asterisk (lowast) denotesequence identity deletions and premature stop codons respectively compared with the Korean consensus [20] In the right column theplus (+) and minus (minus) signs denote the presence or absence of the corresponding therapy respectively

10 Evidence-Based Complementary and Alternative Medicine

61 69 112 120 208 216 264 272 520 528Patient WT nucleotide TGGAAAAGT TGGGTCTAT TGGGGTCTG TGGAGGAAA TGGAACAAG Hypermut 20

sequence no P value90-05 10LSK4-9168 G A lt005

12LSK4-10131 A A AA A A lt000113LSK5-11470 A A A 015 13LSK5-11472 A AA lt001

90-18 08LSH5-5645 AA 019 08LSH11-5694 A 05710LSH4-9192 AA 013

90-54 07KJin3-5961 A 00693-04 94KYR12-9441 AA AG 009

07KYR8-5166 A A AA A AG lt000107KYR8-4846 A AA G 00507KYR8-5167 A A A AG 00507KYR8-5168 A A AG 02907KYR8-4845 AA G 005

93-60 03KMH11-5725 AA A 015

WT amino acid W K S W V Y W G L W R K W N K21 22 23 38 39 40 70 71 72 89 90 91 174 175 176

Figure 3 Positions of the premature stop codons in the 15 vif genes from five patients All stop codons resulted from GG rarr GA orGG rarr AG changes APOBEC3G exhibits an intrinsic preference for the second cytosine in a 51015840CC dinucleotide motif leading to 51015840GG-to-AG mutations [38]

In total five of our patients demonstrated in-frame deletionsof the vif gene of the 75 KSB-infected patients who weretreated with KRG Also in our present study cohort fivepatients including 90-18 and 92-13 demonstrated deletedvif genes (small in-frame and gross deletions) during KRGintake whereas all of our 10 LTSP patients demonstrateddeletions in the nef gene (119875 lt 005) [14] In additioncompared with the reported deletions of the nef and 51015840LTRgag genes the locations of the deleted vif gene werepositioned within a narrow range at the terminus [15 16 23]

Regarding gross deletions to date only two patientshave demonstrated gross deletions in the vif gene [17 18]and only one patient has demonstrated the insertion of twoamino acids and these patients were part of a nonprogress-ing mother-child pair [28] However in our present studyof the 10 LTSP patients who were assessed four patientsdemonstrated gross deletion in the vif gene during KRGintake prior to receiving HAART (15) This frequency issignificantly lower than the previously reported incidence of10 of 10 patients demonstrating gross deletions in the nef(187) and 51015840 LTRgag genes (376 119875 lt 001) [27 28]The frequency of deletion in the nef gene was reported tosignificantly decrease during KRG plus HAART (119875 lt 001)[16] In contrast to previous studies on the nef and gag genes[16 29] the detection of in-frame deletions in the vif genewas not suppressed in the patients receiving HAART in ourpresent cohort The reasons for this include the following (1)the proportion and frequency (approximately two-thirds) ofdeleted vif genes after the first occurrence were much higherthan in the nef and 51015840 LTRgag genes (lt20 and 30 resp)(2) small deletions in the vif gene occurred as a single bandwhereas gross deletions in the nef and 51015840 LTRgag genes weremainly detected as two bands (wild type and short) per PCRreaction and (3) the number of amplicons indicating PSCdueto G-to-A hypermutation was significantly higher in patients

receiving KRG plus HAART than patients receiving onlyKRG This phenomenon is consistent with our previous datain 51015840 LTRgag gene [29] However the frequency of G-to-Ahypermutationwas significantly lower in vif gene than that in51015840 LTRgag gene [29] Thus for the vif gene the detection ofsmall deletionsmight be less affected by limit-diluting effectsas has been shown for the nef gene [29] Taken together theseadditional defects in the vif genes in the same patients mightbe related to the replicative impairment in vif defective HIV-1 which ultimately results in defects in the synthesis of viralDNA [30]

In our present study it appears that the frequency ofhypermutation was higher in patients who maintained goodcompliance with HAART during HAART plus KRG (90-0590-18 and 93-04) than in the patients who demonstratedpoor compliance (Figure 1) In our extended data analysis124 (60485) 31 (5161) 30 (19617) and 09 (1108)of vif genes during KRG plus HAART HAART aloneKRG alone and control revealed PSC respectively Thesedata indicate the presence of synergistic effect of KRG plusHAART on PSC (119875 lt 001) There was no case that smalldeletions were induced by HAART alone About 6ndash43 ofthe integrated proviral pol gene is hypermutated by HAART[22 31 32] although hypermutated viral genomes are notpresent in plasma [32]

ApoBec3G-induced hypermutation in LTNPs has beenreported previously [33] Indeed it has been reported alsothat LTNP patients harbor PSC-containing vif genes morefrequently than progressors [25] although the reportedfrequencies of defective genes vary among studies [34] Inour current analyses vif gene-containing PSC due to G-to-A hypermutation was found to be significantly higher duringKRG plus HAART than only KRG intake prior to HAARTThis finding is consistent with our previous data obtainedfrom a cohort of the Korean hemophiliacs (21 incidence

Evidence-Based Complementary and Alternative Medicine 11

of G-to-A hypermutation while receiving only KRG versus98 on KRG plus HAART unpublished data) and otherobservations regarding the pol gene [22]

Previous studies report that certain vif mutations such asL81M R132S S130I and the insertion of two amino acids areassociatedwith slow progression or low viral loads [17 18 28]In our present study L81M [35] and R132S were consistentlydetected in patient 96-51 and patients 87-05 and 96-51respectively although both mutations were found in only in2 and 3 of 169 Korean patients respectively Although thevif gene is highly conserved in HIV-1 genomes the recoveryof hypermutants might be severely underestimated in ourpresent analysis because of the primer mismatching andthe difficulties involved in detection following amplification[36 37] Overall our current data suggest that small in-frame deletion and PSCs are associated with KRG intake andHAART respectively although further studies are needed

5 Conclusions

Our data suggest that HAART and KRG intake might inducePSCs and small in-frame deletions respectively

Conflict of Interests

The authors have no conflict of interests to declare

Acknowledgments

This work was supported by a grant from the Korean Societyof Ginseng (2010ndash2012) The authors thank Professor Seung-Chul Yun the Division of Biostatistics University of UlsanCollege of Medicine Asan Medical Center for the statisticalconsultation

References

[1] E Ernst ldquoPanax ginseng an overview of the clinical evidencerdquoJournal of Ginseng Research vol 34 no 4 pp 259ndash263 2010

[2] K T Choi ldquoBotanical characteristics pharmacological effectsand medicinal components of Korean Panax ginseng C AMeyerrdquoActa Pharmacologica Sinica vol 29 no 9 pp 1109ndash11182008

[3] S J Fulder ldquoGinseng and the hypothalamic-pituitary control ofstressrdquo The American Journal of Chinese Medicine vol 9 no 2pp 112ndash118 1981

[4] V K Singh S S Agarwal and B M Gupta ldquoImmunomodula-tory activity of Panax ginseng extractrdquo Planta Medica vol 50no 6 pp 462ndash465 1984

[5] X Song and S Hu ldquoAdjuvant activities of saponins fromtraditional Chinese medicinal herbsrdquo Vaccine vol 27 no 36pp 4883ndash4890 2009

[6] D G Yoo M C Kim M K Park et al ldquoProtective effectof ginseng polysaccharides on influenza viral infectionrdquo PLoSONE vol 7 no 3 Article ID e33678 2012

[7] H X Sun Y P Ye H J Pan and Y J Pan ldquoAdjuvant effectof Panax notoginseng saponins on the immune responses toovalbumin in micerdquo Vaccine vol 22 no 29-30 pp 3882ndash38892004

[8] J Sun S Hu and X Song ldquoAdjuvant effects of protopanaxadioland protopanaxatriol saponins from ginseng roots on theimmune responses to ovalbumin in micerdquo Vaccine vol 25 no6 pp 1114ndash1120 2007

[9] P W Hunt ldquoHIV and inflammation mechanisms and conse-quencesrdquo Current HIVAIDS Reports vol 9 no 2 pp 139ndash1472012

[10] T Yayeh K H Jung H Y Jeong et al ldquoKorean red ginsengsaponin fraction downregulates proinflammatory mediators inLPS stimulated RAW264 7 cells and protects mice againstendotoxic shockrdquo Journal of Ginseng Research vol 36 no 3 pp263ndash269 2012

[11] A T Smolinski and J J Pestka ldquoModulation of lipopolysaccha-ride-induced proinflammatory cytokine production in vitroand in vivo by the herbal constituents apigenin (chamomile)ginsenoside Rb1 (ginseng) and parthenolide (feverfew)rdquo Foodand Chemical Toxicology vol 41 no 10 pp 1381ndash1390 2003

[12] A Rhule S Navarro J R Smith and D M ShepherdldquoPanax notoginseng attenuates LPS-induced pro-inflammatorymediators in RAW2647 cellsrdquo Journal of Ethnopharmacologyvol 106 no 1 pp 121ndash128 2006

[13] J M Brenchley D A Price T W Schacker et al ldquoMicrobialtranslocation is a cause of systemic immune activation inchronic HIV infectionrdquo Nature Medicine vol 12 no 12 pp1365ndash1371 2006

[14] Y K Cho J Y Lim Y S Jung S K Oh H J Lee and H SungldquoHigh frequency of grossly deleted nef genes in HIV-1 infectedlong-term slow progressors treated with Korean red ginsengrdquoCurrent HIV Research vol 4 no 4 pp 447ndash457 2006

[15] Y K Cho and Y S Jung ldquoHigh frequency of gross deletions inthe 51015840 LTR and gag regions in HIV type 1-infected long-termsurvivors treated with Korean red ginsengrdquo AIDS Research andHuman Retroviruses vol 24 no 2 pp 181ndash193 2008

[16] Y K Cho Y S Jung andH S Sung ldquoFrequent gross deletion intheHIV type 1 nef gene in hemophiliacs treatedwith korean redginseng inhibition of detection by highly active antiretroviraltherapyrdquo AIDS Research and Human Retroviruses vol 25 no 4pp 419ndash424 2009

[17] V Sandonıs C Casado T Alvaro et al ldquoA combination ofdefective DNA and protective host factors are found in a setof HIV-1 ancestral LTNPsrdquo Virology vol 391 no 1 pp 73ndash822009

[18] H R Rangel D Garzaro A K Rodrıguez et al ldquoDeletioninsertion and stop codon mutations in vif genes of HIV-1 infecting slow progressor patientsrdquo Journal of Infection inDeveloping Countries vol 3 no 7 pp 531ndash538 2009

[19] Y K Cho J E Kim and B T Foley ldquoPhylogenetic analysisof near full-length HIV-1 genomic sequences in 21 Koreanindividualsrdquo AIDS Research and Human Retroviruses vol 29no 4 pp 738ndash743 2013

[20] Y K Cho Y S Jung J S Lee and B T Foley ldquoMolecu-lar evidence of HIV-1 transmission in 20 Korean individu-als with haemophilia phylogenetic analysis of the vif generdquoHaemophilia vol 18 no 2 pp 291ndash299 2012

[21] P P Rose and B T Korber ldquoDetecting hypermutations inviral sequences with an emphasis on GrarrA hypermutationrdquoBioinformatics vol 16 no 4 pp 400ndash401 2000

[22] S Fourati S Lambert-Niclot C Soulie et al ldquoHIV-1 genomeis often defective in PBMCs and rectal tissues after long-termHAART as a result of APOBEC3 editing and correlates with thesize of reservoirsrdquo Journal of Antimicrobial Chemotherapy vol67 no 10 pp 2323ndash2326 2012

12 Evidence-Based Complementary and Alternative Medicine

[23] U Wieland A Seelhoff A Hofmann et al ldquoDiversity of thevif gene of human immunodeficiency virus type 1 in UgandardquoJournal of General Virology vol 78 no 2 pp 393ndash400 1997

[24] P Sova M van Ranst P Gupta et al ldquoConservation of an intacthuman immunodeficiency virus type 1 vif gene in vitro and invivordquo Journal of Virology vol 69 no 4 pp 2557ndash2564 1995

[25] T Yamada and A Iwamoto ldquoComparison of proviral accessorygenes between long-term nonprogressors and progressors ofhuman immunodeficiency virus type 1 infectionrdquo Archives ofVirology vol 145 no 5 pp 1021ndash1027 2000

[26] K Tominaga S Kato M Negishi and T Takano ldquoA high fre-quency of defective vif genes in peripheral blood mononuclearcells from HIV type 1-infected individualsrdquo AIDS Research andHuman Retroviruses vol 12 no 16 pp 1543ndash1549 1996

[27] Y K Cho H Sung I G Bae et al ldquoFull sequence of HIV type 1Korean subtype B in anAIDS case with atypical seroconversionTAAAA at TATA boxrdquoAIDS Research andHuman Retrovirusesvol 21 no 11 pp 961ndash964 2005

[28] L Alexander M J Aquino-DeJesus M Chan and W AAndiman ldquoInhibition of human immunodeficiency virus type 1(HIV-1) replication by a two-amino-acid insertion in HIV-1 Viffrom a nonprogressing mother and childrdquo Journal of Virologyvol 76 no 20 pp 10533ndash10539 2002

[29] YK Cho Y S JungH S Sung andCH Joo ldquoFrequent geneticdefects in the HIV-1 51015840LTRgag gene in hemophiliacs treatedwith Korean red ginseng decreased detection of genetic defectsby highly active antiretroviral therapyrdquo Journal of GinsengResearch vol 35 no 4 pp 413ndash420 2011

[30] M Nascimbeni M Bouyac F Rey B Spire and F Clavel ldquoThereplicative impairment of Vif-mutants of human immunodefi-ciency virus type 1 correlates with an overall defect in viral DNAsynthesisrdquo Journal of General Virology vol 79 no 8 pp 1945ndash1950 1998

[31] J P Vartanian M Henry and S Wain-Hobson ldquoSustainedGrarrA hypermutation during reverse transcription of an entirehuman immunodeficiency virus type 1 strain Vau group Ogenomerdquo Journal of General Virology vol 83 no 4 pp 801ndash8052002

[32] T L Kieffer P Kwon R E Nettles Y Han S C Ray andR F Siliciano ldquoGrarrA hypermutation in protease and reversetranscriptase regions of human immunodeficiency virus type 1residing in resting CD4+ T cells in vivordquo Journal of Virology vol79 no 3 pp 1975ndash1980 2005

[33] B Wang M Mikhail W B Dyer J J Zaunders A D Kelleherand N K Saksena ldquoFirst demonstration of a lack of viralsequence evolution in a nonprogressor defining replication-incompetent HIV-1 infectionrdquo Virology vol 312 no 1 pp 135ndash150 2003

[34] G Hassaine I Agostini D Candotti et al ldquoCharacterizationof human immunodeficiency virus type 1 vif gene in long-termasymptomatic individualsrdquoVirology vol 276 no 1 pp 169ndash1802000

[35] F A de Maio C A Rocco P C Aulicino R Bologna AMangano and L Sen ldquoUnusual substitutions in HIV-1 Vif fromchildren infected perinatally without progression to AIDS formore than 8 years without therapyrdquo Journal of Medical Virologyvol 84 no 12 pp 1844ndash1852 2012

[36] M Janini M Rogers D R Birx and F E McCutchan ldquoHumanimmunodeficiency virus type 1 DNA sequences geneticallydamaged by hypermutation are often abundant in patientperipheral blood mononuclear cells and may be generated

during near-simultaneous infection and activation of CD4+ Tcellsrdquo Journal of Virology vol 75 no 17 pp 7973ndash7986 2001

[37] G H Kijak L M Janini S Tovanabutra et al ldquoVariablecontexts and levels of hypermutation inHIV-1 proviral genomesrecovered from primary peripheral blood mononuclear cellsrdquoVirology vol 376 no 1 pp 101ndash111 2008

[38] E W Refsland J F Hultquist and R S Harris ldquoEndogenousorigins of HIV-1 G-to-A hypermutation and restriction in thenonpermissive T cell line CEM2nrdquo PLoS Pathogens vol 8 no7 Article ID e1002800 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 8: Effects of Korean Red Ginseng and HAART on vif Gene in 10 Long

8 Evidence-Based Complementary and Alternative Medicine

90-05

90-18

90-50

91-20

92LSK11-9417 YETRQE94LSK6-759 YETRPQES96LSK6-11491 GRYETRQ98LSK4-11495 YENTRPQES00LSK8-9999 YPQE03LSK6-10001s1 -------------------------------------------QRES04LSK7 YETRPQES06LSK1-3236 YEVTRPQRES07LSK2-3232 YEVTRPQES08LSK1-4762 YEVTRPQAESS09LSK4-6143 YEVTRPQES10LSK4-9165 YEVTRPQES10LSK4-9168 YEVTRPQES12LSK4-10131 NYKVTRPQRAES12LSK4-10132 YEVTRPQAESQ13LSK5-11470 PYVRRPTQETESS13LSK5-11471 YVTRPQRES13LSK5-11472 YENRTRQRES

92LSH3-6951 YRDH92LSH11-8548 GYRD93LSH10-1252 YRDR94LSH2-740 YRDR95LSH5-10562 YTRDR96LSH5-10609 YTR98LSH4-10567 YQTRRG---H99LSH1-5776 YQTRRG---RH99LSH1-5777 YQTRRHG---H99LSH1-5778 YQTRG---H99LSH6-5781 YQTRRQG---H99LSH6-5782 YQTRG---H00LSH3-5704 YQTRRG---H00LSH3-5705 YQTRRQSG---H00LSH3-5706 YQTRAG---H02LSH10-5707 YETRQG---H02LSH10-5708 YQTRG---H02LSH10-5710 YQTRRQR---H03LSH5-5650 YQTRRR---03LSH5-5651 YQTRRR---H03LSH5-5652 YQTRRER---03LSH10-5653 YQTRRQRS04LSH7-4848 YQTRRKQG---H04LSH8-3011 YR06LSH8-5647 YQTRRG---H06LSH8-5649 YQTRRG---H06LSH11-3238 YQHTRRQES08LSH5-5644 YQTRRSG---H08LSH5-5645 YQTRQAKS08LSH5-5646 YQTRRR---H08LSH5-5647 YQTRRR---H08LSH11-5694 YQTRRSRKG---H10LSH4-9190 YEQCTRRRG---H10LSH4-9192 YQRTRRRRG---H10LSH4-9193 YQTRRG---H12LSH6-10100 YQTRRG---H12LSH6-10101 YQTRRSRG---H

91KJin7-6723 TT93KJin2-10024 TT93KJin9-8530 VTPAKH01KJin7-5383 ISTT03KJin6-683 ISTPQH04KJin8-1955 ITPTH06KJin1-3030 VTPTH06KJin9-3012 VAPT07KJin3-5148 QEPTN07KJin3-5961 NLEPTAN10KJin4-9162 VTTAH10KJin9-8176 QVTAAH11KJin12-10119 VTPTADH13KJin1-11373 VTPTH

PE94JWK12-5524 VQ97JWK HR99JWK5-11441 HR00JWK3-11453 HR00JWK12-11445 HR01JWK7-5603 NHRQ01JWK7-5603s1 -------------------------------------------------------------------------------02JWK8-11436 NRQQ03JWK8-677 HR05JWK1-5600 NRQQ06JWK7-3042 HRQ

HRQ08JWK1-4772 NRQQ

NRQQ11JWK4-8993 NRQQ12JWK1-10125 NRQQ13JWK1-11366 NRQQ

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ +

10JWK4 9159

06 JWK12 5624

91JWK6 6949

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 9

92-13

93-04

93-60

92CWS5-9398 HSE93CWS3-9925 HE93CWS5-6669 EHTE93CWS7-802 KHTE96CWS11-5341 KHTE99CWS10-5338 HPES01CWS2-10532 HPDE03CWS6-684 HPE03CWS10-5773 HPDE04CWS5-3239 HPTE05CWS5-5783 HPTD----05CWS5-5785 HPTD----06CWS2-3071 HPTDE06CWS2-3203 HPTD----06CWS2-3221 HPTD----06CWS2-3222 VHPTD----06CWS2-3223 HPTD----06CWS8-3038 HPTD----06CWS8-3039 HPTD----07CWS4-3820 HPTATD----07CWS4-3821 HPTATD----07CWS4-10174 HPTATD----07CWS4-10175 HPTATD----07CWS4-10176 HPTATD----07CWS4-10177 VHRD----07CWS9-6252 HPE07CWS9-6253 HPTATD----07CWS9-6254 HPTATD----07CWS12-4777 HPTATD----08CWS5-6214 HPTATD----08CWS8-5160 HPTATD----08CWS8-5161 HPTATD----08CWS12-5720 HPTATD----08CWS12-5721 HPTATD----08CWS12-5722 HPTATD----09CWS5-6256 HPTATD----09CWS5-6257 HPTTATD----09CWS5-6258 HPTAD----09CWS12-9108 HPDE09CWS12-9109 HPTATTD----

93KYR2-9472 NSRIRKE94KYR6-10137 NSRRKE94KYR12-9441 NSRIRKER95KYR5-815 NSRIRKE00KYR9-9913 NSRRRKE02KYR7-9911 NSRRRKE03KYR1-817 NSRIRKE06KYR7-5236 QKPHRIRKE06KYR11-5249 QKPHRIRKE07KYR3-5265 NSHRIRKE07KYR3-11387s1 ---------------QNSHRIRRKER07KYR8-4845 QNSRRIRKER07KYR8-4846 NSRRIRKE07KYR8-5166 KNSHRRRIRKER07KYR8-5167 NKHRRIRRKE07KYR8-5168 IQRKPHRRIRKE

94KMH5 HVPR96KMH11-8534 VRT97KMH4-9452 HVQR01KMH11-5526 HVPR03KMH5-687 HVPR03KMH11-5725 HVQRS04KMH5 VQRS05KMH3-5727 HVPR06KMH11-3026 HVPR07KMH4-3281s1 VHVPRV----------------------------07KMH10-5732 HVQPRVRS08KMH5-5165 HIQPRVRS08KMH8-5427s1 ----------------------------HVQPTRVRS09KMH5-11475 HVQPRVRS09KMH5-11475s2 ------------------------------------------------------------------------------------------------10KMH10-9907s1 -----------------------------------------RVRS11KMH4-9917 HVPRVRS12KMH4-10096 HVPRVRRS

00YJN10-9385 ETVSRE01YJN9-4850 ETVSRE03YJN10-840 ETVSRE05YJN2-1948 ETQVSRE06YJN10-3044 ETQVSRE07YJN4-3248 ETQVSREY07YJN8-5627 ETQVSREY08YJN3-5606 ETQVSREY08YJN8-5408 ETQVSREY09YJN2-6041 ETQVSREY09YJN12-9156 ETQVSREY10YJN6-9918 ETQVSRIEY11YJN2-9921 ETQVSREY12YJN7-11382 ETQVSREY13YJN1-11381 ETQVSREYKor consensus QVDPDLADKLIHLHYFDCFSDSAIRHAILGRLVRPKCEYQAGHNKVGSLQYLALTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH

97 120 140 160 180 192

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ +

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

96-51

Figure 2 Sequential alignment of the amino acids of the Vif protein over 20 years All sequences that demonstrated small in-frame deletionsgross deletions and stop codons are depicted together with the baseline and last sequences of each patient Patients 90-18 demonstrated a9 bp deletion (positions 182ndash184) in April 1998 and patient 92-13 demonstrated a 12 bp deletion (positions 185ndash188) in May 2005 Patient90-05 consistently demonstrated a 9 bp insertion (RQTRAR-RAR-NGASRP) at the same position at the terminus of vpr beginning in August2000 (data not shown) The code 92LSH3-6951 (sampling year initials of the patient sampling month and sequence number) denotes thatthe sampling date is March 1992 and the sequence number is 6951 in patient LSH (90-18) The dot (sdot) hyphen (-) and asterisk (lowast) denotesequence identity deletions and premature stop codons respectively compared with the Korean consensus [20] In the right column theplus (+) and minus (minus) signs denote the presence or absence of the corresponding therapy respectively

10 Evidence-Based Complementary and Alternative Medicine

61 69 112 120 208 216 264 272 520 528Patient WT nucleotide TGGAAAAGT TGGGTCTAT TGGGGTCTG TGGAGGAAA TGGAACAAG Hypermut 20

sequence no P value90-05 10LSK4-9168 G A lt005

12LSK4-10131 A A AA A A lt000113LSK5-11470 A A A 015 13LSK5-11472 A AA lt001

90-18 08LSH5-5645 AA 019 08LSH11-5694 A 05710LSH4-9192 AA 013

90-54 07KJin3-5961 A 00693-04 94KYR12-9441 AA AG 009

07KYR8-5166 A A AA A AG lt000107KYR8-4846 A AA G 00507KYR8-5167 A A A AG 00507KYR8-5168 A A AG 02907KYR8-4845 AA G 005

93-60 03KMH11-5725 AA A 015

WT amino acid W K S W V Y W G L W R K W N K21 22 23 38 39 40 70 71 72 89 90 91 174 175 176

Figure 3 Positions of the premature stop codons in the 15 vif genes from five patients All stop codons resulted from GG rarr GA orGG rarr AG changes APOBEC3G exhibits an intrinsic preference for the second cytosine in a 51015840CC dinucleotide motif leading to 51015840GG-to-AG mutations [38]

In total five of our patients demonstrated in-frame deletionsof the vif gene of the 75 KSB-infected patients who weretreated with KRG Also in our present study cohort fivepatients including 90-18 and 92-13 demonstrated deletedvif genes (small in-frame and gross deletions) during KRGintake whereas all of our 10 LTSP patients demonstrateddeletions in the nef gene (119875 lt 005) [14] In additioncompared with the reported deletions of the nef and 51015840LTRgag genes the locations of the deleted vif gene werepositioned within a narrow range at the terminus [15 16 23]

Regarding gross deletions to date only two patientshave demonstrated gross deletions in the vif gene [17 18]and only one patient has demonstrated the insertion of twoamino acids and these patients were part of a nonprogress-ing mother-child pair [28] However in our present studyof the 10 LTSP patients who were assessed four patientsdemonstrated gross deletion in the vif gene during KRGintake prior to receiving HAART (15) This frequency issignificantly lower than the previously reported incidence of10 of 10 patients demonstrating gross deletions in the nef(187) and 51015840 LTRgag genes (376 119875 lt 001) [27 28]The frequency of deletion in the nef gene was reported tosignificantly decrease during KRG plus HAART (119875 lt 001)[16] In contrast to previous studies on the nef and gag genes[16 29] the detection of in-frame deletions in the vif genewas not suppressed in the patients receiving HAART in ourpresent cohort The reasons for this include the following (1)the proportion and frequency (approximately two-thirds) ofdeleted vif genes after the first occurrence were much higherthan in the nef and 51015840 LTRgag genes (lt20 and 30 resp)(2) small deletions in the vif gene occurred as a single bandwhereas gross deletions in the nef and 51015840 LTRgag genes weremainly detected as two bands (wild type and short) per PCRreaction and (3) the number of amplicons indicating PSCdueto G-to-A hypermutation was significantly higher in patients

receiving KRG plus HAART than patients receiving onlyKRG This phenomenon is consistent with our previous datain 51015840 LTRgag gene [29] However the frequency of G-to-Ahypermutationwas significantly lower in vif gene than that in51015840 LTRgag gene [29] Thus for the vif gene the detection ofsmall deletionsmight be less affected by limit-diluting effectsas has been shown for the nef gene [29] Taken together theseadditional defects in the vif genes in the same patients mightbe related to the replicative impairment in vif defective HIV-1 which ultimately results in defects in the synthesis of viralDNA [30]

In our present study it appears that the frequency ofhypermutation was higher in patients who maintained goodcompliance with HAART during HAART plus KRG (90-0590-18 and 93-04) than in the patients who demonstratedpoor compliance (Figure 1) In our extended data analysis124 (60485) 31 (5161) 30 (19617) and 09 (1108)of vif genes during KRG plus HAART HAART aloneKRG alone and control revealed PSC respectively Thesedata indicate the presence of synergistic effect of KRG plusHAART on PSC (119875 lt 001) There was no case that smalldeletions were induced by HAART alone About 6ndash43 ofthe integrated proviral pol gene is hypermutated by HAART[22 31 32] although hypermutated viral genomes are notpresent in plasma [32]

ApoBec3G-induced hypermutation in LTNPs has beenreported previously [33] Indeed it has been reported alsothat LTNP patients harbor PSC-containing vif genes morefrequently than progressors [25] although the reportedfrequencies of defective genes vary among studies [34] Inour current analyses vif gene-containing PSC due to G-to-A hypermutation was found to be significantly higher duringKRG plus HAART than only KRG intake prior to HAARTThis finding is consistent with our previous data obtainedfrom a cohort of the Korean hemophiliacs (21 incidence

Evidence-Based Complementary and Alternative Medicine 11

of G-to-A hypermutation while receiving only KRG versus98 on KRG plus HAART unpublished data) and otherobservations regarding the pol gene [22]

Previous studies report that certain vif mutations such asL81M R132S S130I and the insertion of two amino acids areassociatedwith slow progression or low viral loads [17 18 28]In our present study L81M [35] and R132S were consistentlydetected in patient 96-51 and patients 87-05 and 96-51respectively although both mutations were found in only in2 and 3 of 169 Korean patients respectively Although thevif gene is highly conserved in HIV-1 genomes the recoveryof hypermutants might be severely underestimated in ourpresent analysis because of the primer mismatching andthe difficulties involved in detection following amplification[36 37] Overall our current data suggest that small in-frame deletion and PSCs are associated with KRG intake andHAART respectively although further studies are needed

5 Conclusions

Our data suggest that HAART and KRG intake might inducePSCs and small in-frame deletions respectively

Conflict of Interests

The authors have no conflict of interests to declare

Acknowledgments

This work was supported by a grant from the Korean Societyof Ginseng (2010ndash2012) The authors thank Professor Seung-Chul Yun the Division of Biostatistics University of UlsanCollege of Medicine Asan Medical Center for the statisticalconsultation

References

[1] E Ernst ldquoPanax ginseng an overview of the clinical evidencerdquoJournal of Ginseng Research vol 34 no 4 pp 259ndash263 2010

[2] K T Choi ldquoBotanical characteristics pharmacological effectsand medicinal components of Korean Panax ginseng C AMeyerrdquoActa Pharmacologica Sinica vol 29 no 9 pp 1109ndash11182008

[3] S J Fulder ldquoGinseng and the hypothalamic-pituitary control ofstressrdquo The American Journal of Chinese Medicine vol 9 no 2pp 112ndash118 1981

[4] V K Singh S S Agarwal and B M Gupta ldquoImmunomodula-tory activity of Panax ginseng extractrdquo Planta Medica vol 50no 6 pp 462ndash465 1984

[5] X Song and S Hu ldquoAdjuvant activities of saponins fromtraditional Chinese medicinal herbsrdquo Vaccine vol 27 no 36pp 4883ndash4890 2009

[6] D G Yoo M C Kim M K Park et al ldquoProtective effectof ginseng polysaccharides on influenza viral infectionrdquo PLoSONE vol 7 no 3 Article ID e33678 2012

[7] H X Sun Y P Ye H J Pan and Y J Pan ldquoAdjuvant effectof Panax notoginseng saponins on the immune responses toovalbumin in micerdquo Vaccine vol 22 no 29-30 pp 3882ndash38892004

[8] J Sun S Hu and X Song ldquoAdjuvant effects of protopanaxadioland protopanaxatriol saponins from ginseng roots on theimmune responses to ovalbumin in micerdquo Vaccine vol 25 no6 pp 1114ndash1120 2007

[9] P W Hunt ldquoHIV and inflammation mechanisms and conse-quencesrdquo Current HIVAIDS Reports vol 9 no 2 pp 139ndash1472012

[10] T Yayeh K H Jung H Y Jeong et al ldquoKorean red ginsengsaponin fraction downregulates proinflammatory mediators inLPS stimulated RAW264 7 cells and protects mice againstendotoxic shockrdquo Journal of Ginseng Research vol 36 no 3 pp263ndash269 2012

[11] A T Smolinski and J J Pestka ldquoModulation of lipopolysaccha-ride-induced proinflammatory cytokine production in vitroand in vivo by the herbal constituents apigenin (chamomile)ginsenoside Rb1 (ginseng) and parthenolide (feverfew)rdquo Foodand Chemical Toxicology vol 41 no 10 pp 1381ndash1390 2003

[12] A Rhule S Navarro J R Smith and D M ShepherdldquoPanax notoginseng attenuates LPS-induced pro-inflammatorymediators in RAW2647 cellsrdquo Journal of Ethnopharmacologyvol 106 no 1 pp 121ndash128 2006

[13] J M Brenchley D A Price T W Schacker et al ldquoMicrobialtranslocation is a cause of systemic immune activation inchronic HIV infectionrdquo Nature Medicine vol 12 no 12 pp1365ndash1371 2006

[14] Y K Cho J Y Lim Y S Jung S K Oh H J Lee and H SungldquoHigh frequency of grossly deleted nef genes in HIV-1 infectedlong-term slow progressors treated with Korean red ginsengrdquoCurrent HIV Research vol 4 no 4 pp 447ndash457 2006

[15] Y K Cho and Y S Jung ldquoHigh frequency of gross deletions inthe 51015840 LTR and gag regions in HIV type 1-infected long-termsurvivors treated with Korean red ginsengrdquo AIDS Research andHuman Retroviruses vol 24 no 2 pp 181ndash193 2008

[16] Y K Cho Y S Jung andH S Sung ldquoFrequent gross deletion intheHIV type 1 nef gene in hemophiliacs treatedwith korean redginseng inhibition of detection by highly active antiretroviraltherapyrdquo AIDS Research and Human Retroviruses vol 25 no 4pp 419ndash424 2009

[17] V Sandonıs C Casado T Alvaro et al ldquoA combination ofdefective DNA and protective host factors are found in a setof HIV-1 ancestral LTNPsrdquo Virology vol 391 no 1 pp 73ndash822009

[18] H R Rangel D Garzaro A K Rodrıguez et al ldquoDeletioninsertion and stop codon mutations in vif genes of HIV-1 infecting slow progressor patientsrdquo Journal of Infection inDeveloping Countries vol 3 no 7 pp 531ndash538 2009

[19] Y K Cho J E Kim and B T Foley ldquoPhylogenetic analysisof near full-length HIV-1 genomic sequences in 21 Koreanindividualsrdquo AIDS Research and Human Retroviruses vol 29no 4 pp 738ndash743 2013

[20] Y K Cho Y S Jung J S Lee and B T Foley ldquoMolecu-lar evidence of HIV-1 transmission in 20 Korean individu-als with haemophilia phylogenetic analysis of the vif generdquoHaemophilia vol 18 no 2 pp 291ndash299 2012

[21] P P Rose and B T Korber ldquoDetecting hypermutations inviral sequences with an emphasis on GrarrA hypermutationrdquoBioinformatics vol 16 no 4 pp 400ndash401 2000

[22] S Fourati S Lambert-Niclot C Soulie et al ldquoHIV-1 genomeis often defective in PBMCs and rectal tissues after long-termHAART as a result of APOBEC3 editing and correlates with thesize of reservoirsrdquo Journal of Antimicrobial Chemotherapy vol67 no 10 pp 2323ndash2326 2012

12 Evidence-Based Complementary and Alternative Medicine

[23] U Wieland A Seelhoff A Hofmann et al ldquoDiversity of thevif gene of human immunodeficiency virus type 1 in UgandardquoJournal of General Virology vol 78 no 2 pp 393ndash400 1997

[24] P Sova M van Ranst P Gupta et al ldquoConservation of an intacthuman immunodeficiency virus type 1 vif gene in vitro and invivordquo Journal of Virology vol 69 no 4 pp 2557ndash2564 1995

[25] T Yamada and A Iwamoto ldquoComparison of proviral accessorygenes between long-term nonprogressors and progressors ofhuman immunodeficiency virus type 1 infectionrdquo Archives ofVirology vol 145 no 5 pp 1021ndash1027 2000

[26] K Tominaga S Kato M Negishi and T Takano ldquoA high fre-quency of defective vif genes in peripheral blood mononuclearcells from HIV type 1-infected individualsrdquo AIDS Research andHuman Retroviruses vol 12 no 16 pp 1543ndash1549 1996

[27] Y K Cho H Sung I G Bae et al ldquoFull sequence of HIV type 1Korean subtype B in anAIDS case with atypical seroconversionTAAAA at TATA boxrdquoAIDS Research andHuman Retrovirusesvol 21 no 11 pp 961ndash964 2005

[28] L Alexander M J Aquino-DeJesus M Chan and W AAndiman ldquoInhibition of human immunodeficiency virus type 1(HIV-1) replication by a two-amino-acid insertion in HIV-1 Viffrom a nonprogressing mother and childrdquo Journal of Virologyvol 76 no 20 pp 10533ndash10539 2002

[29] YK Cho Y S JungH S Sung andCH Joo ldquoFrequent geneticdefects in the HIV-1 51015840LTRgag gene in hemophiliacs treatedwith Korean red ginseng decreased detection of genetic defectsby highly active antiretroviral therapyrdquo Journal of GinsengResearch vol 35 no 4 pp 413ndash420 2011

[30] M Nascimbeni M Bouyac F Rey B Spire and F Clavel ldquoThereplicative impairment of Vif-mutants of human immunodefi-ciency virus type 1 correlates with an overall defect in viral DNAsynthesisrdquo Journal of General Virology vol 79 no 8 pp 1945ndash1950 1998

[31] J P Vartanian M Henry and S Wain-Hobson ldquoSustainedGrarrA hypermutation during reverse transcription of an entirehuman immunodeficiency virus type 1 strain Vau group Ogenomerdquo Journal of General Virology vol 83 no 4 pp 801ndash8052002

[32] T L Kieffer P Kwon R E Nettles Y Han S C Ray andR F Siliciano ldquoGrarrA hypermutation in protease and reversetranscriptase regions of human immunodeficiency virus type 1residing in resting CD4+ T cells in vivordquo Journal of Virology vol79 no 3 pp 1975ndash1980 2005

[33] B Wang M Mikhail W B Dyer J J Zaunders A D Kelleherand N K Saksena ldquoFirst demonstration of a lack of viralsequence evolution in a nonprogressor defining replication-incompetent HIV-1 infectionrdquo Virology vol 312 no 1 pp 135ndash150 2003

[34] G Hassaine I Agostini D Candotti et al ldquoCharacterizationof human immunodeficiency virus type 1 vif gene in long-termasymptomatic individualsrdquoVirology vol 276 no 1 pp 169ndash1802000

[35] F A de Maio C A Rocco P C Aulicino R Bologna AMangano and L Sen ldquoUnusual substitutions in HIV-1 Vif fromchildren infected perinatally without progression to AIDS formore than 8 years without therapyrdquo Journal of Medical Virologyvol 84 no 12 pp 1844ndash1852 2012

[36] M Janini M Rogers D R Birx and F E McCutchan ldquoHumanimmunodeficiency virus type 1 DNA sequences geneticallydamaged by hypermutation are often abundant in patientperipheral blood mononuclear cells and may be generated

during near-simultaneous infection and activation of CD4+ Tcellsrdquo Journal of Virology vol 75 no 17 pp 7973ndash7986 2001

[37] G H Kijak L M Janini S Tovanabutra et al ldquoVariablecontexts and levels of hypermutation inHIV-1 proviral genomesrecovered from primary peripheral blood mononuclear cellsrdquoVirology vol 376 no 1 pp 101ndash111 2008

[38] E W Refsland J F Hultquist and R S Harris ldquoEndogenousorigins of HIV-1 G-to-A hypermutation and restriction in thenonpermissive T cell line CEM2nrdquo PLoS Pathogens vol 8 no7 Article ID e1002800 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 9: Effects of Korean Red Ginseng and HAART on vif Gene in 10 Long

Evidence-Based Complementary and Alternative Medicine 9

92-13

93-04

93-60

92CWS5-9398 HSE93CWS3-9925 HE93CWS5-6669 EHTE93CWS7-802 KHTE96CWS11-5341 KHTE99CWS10-5338 HPES01CWS2-10532 HPDE03CWS6-684 HPE03CWS10-5773 HPDE04CWS5-3239 HPTE05CWS5-5783 HPTD----05CWS5-5785 HPTD----06CWS2-3071 HPTDE06CWS2-3203 HPTD----06CWS2-3221 HPTD----06CWS2-3222 VHPTD----06CWS2-3223 HPTD----06CWS8-3038 HPTD----06CWS8-3039 HPTD----07CWS4-3820 HPTATD----07CWS4-3821 HPTATD----07CWS4-10174 HPTATD----07CWS4-10175 HPTATD----07CWS4-10176 HPTATD----07CWS4-10177 VHRD----07CWS9-6252 HPE07CWS9-6253 HPTATD----07CWS9-6254 HPTATD----07CWS12-4777 HPTATD----08CWS5-6214 HPTATD----08CWS8-5160 HPTATD----08CWS8-5161 HPTATD----08CWS12-5720 HPTATD----08CWS12-5721 HPTATD----08CWS12-5722 HPTATD----09CWS5-6256 HPTATD----09CWS5-6257 HPTTATD----09CWS5-6258 HPTAD----09CWS12-9108 HPDE09CWS12-9109 HPTATTD----

93KYR2-9472 NSRIRKE94KYR6-10137 NSRRKE94KYR12-9441 NSRIRKER95KYR5-815 NSRIRKE00KYR9-9913 NSRRRKE02KYR7-9911 NSRRRKE03KYR1-817 NSRIRKE06KYR7-5236 QKPHRIRKE06KYR11-5249 QKPHRIRKE07KYR3-5265 NSHRIRKE07KYR3-11387s1 ---------------QNSHRIRRKER07KYR8-4845 QNSRRIRKER07KYR8-4846 NSRRIRKE07KYR8-5166 KNSHRRRIRKER07KYR8-5167 NKHRRIRRKE07KYR8-5168 IQRKPHRRIRKE

94KMH5 HVPR96KMH11-8534 VRT97KMH4-9452 HVQR01KMH11-5526 HVPR03KMH5-687 HVPR03KMH11-5725 HVQRS04KMH5 VQRS05KMH3-5727 HVPR06KMH11-3026 HVPR07KMH4-3281s1 VHVPRV----------------------------07KMH10-5732 HVQPRVRS08KMH5-5165 HIQPRVRS08KMH8-5427s1 ----------------------------HVQPTRVRS09KMH5-11475 HVQPRVRS09KMH5-11475s2 ------------------------------------------------------------------------------------------------10KMH10-9907s1 -----------------------------------------RVRS11KMH4-9917 HVPRVRS12KMH4-10096 HVPRVRRS

00YJN10-9385 ETVSRE01YJN9-4850 ETVSRE03YJN10-840 ETVSRE05YJN2-1948 ETQVSRE06YJN10-3044 ETQVSRE07YJN4-3248 ETQVSREY07YJN8-5627 ETQVSREY08YJN3-5606 ETQVSREY08YJN8-5408 ETQVSREY09YJN2-6041 ETQVSREY09YJN12-9156 ETQVSREY10YJN6-9918 ETQVSRIEY11YJN2-9921 ETQVSREY12YJN7-11382 ETQVSREY13YJN1-11381 ETQVSREYKor consensus QVDPDLADKLIHLHYFDCFSDSAIRHAILGRLVRPKCEYQAGHNKVGSLQYLALTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH

97 120 140 160 180 192

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +

minus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ ++ +

minus minusminus minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ ++ ++ +

+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ minus+ ++ ++ ++ +

96-51

Figure 2 Sequential alignment of the amino acids of the Vif protein over 20 years All sequences that demonstrated small in-frame deletionsgross deletions and stop codons are depicted together with the baseline and last sequences of each patient Patients 90-18 demonstrated a9 bp deletion (positions 182ndash184) in April 1998 and patient 92-13 demonstrated a 12 bp deletion (positions 185ndash188) in May 2005 Patient90-05 consistently demonstrated a 9 bp insertion (RQTRAR-RAR-NGASRP) at the same position at the terminus of vpr beginning in August2000 (data not shown) The code 92LSH3-6951 (sampling year initials of the patient sampling month and sequence number) denotes thatthe sampling date is March 1992 and the sequence number is 6951 in patient LSH (90-18) The dot (sdot) hyphen (-) and asterisk (lowast) denotesequence identity deletions and premature stop codons respectively compared with the Korean consensus [20] In the right column theplus (+) and minus (minus) signs denote the presence or absence of the corresponding therapy respectively

10 Evidence-Based Complementary and Alternative Medicine

61 69 112 120 208 216 264 272 520 528Patient WT nucleotide TGGAAAAGT TGGGTCTAT TGGGGTCTG TGGAGGAAA TGGAACAAG Hypermut 20

sequence no P value90-05 10LSK4-9168 G A lt005

12LSK4-10131 A A AA A A lt000113LSK5-11470 A A A 015 13LSK5-11472 A AA lt001

90-18 08LSH5-5645 AA 019 08LSH11-5694 A 05710LSH4-9192 AA 013

90-54 07KJin3-5961 A 00693-04 94KYR12-9441 AA AG 009

07KYR8-5166 A A AA A AG lt000107KYR8-4846 A AA G 00507KYR8-5167 A A A AG 00507KYR8-5168 A A AG 02907KYR8-4845 AA G 005

93-60 03KMH11-5725 AA A 015

WT amino acid W K S W V Y W G L W R K W N K21 22 23 38 39 40 70 71 72 89 90 91 174 175 176

Figure 3 Positions of the premature stop codons in the 15 vif genes from five patients All stop codons resulted from GG rarr GA orGG rarr AG changes APOBEC3G exhibits an intrinsic preference for the second cytosine in a 51015840CC dinucleotide motif leading to 51015840GG-to-AG mutations [38]

In total five of our patients demonstrated in-frame deletionsof the vif gene of the 75 KSB-infected patients who weretreated with KRG Also in our present study cohort fivepatients including 90-18 and 92-13 demonstrated deletedvif genes (small in-frame and gross deletions) during KRGintake whereas all of our 10 LTSP patients demonstrateddeletions in the nef gene (119875 lt 005) [14] In additioncompared with the reported deletions of the nef and 51015840LTRgag genes the locations of the deleted vif gene werepositioned within a narrow range at the terminus [15 16 23]

Regarding gross deletions to date only two patientshave demonstrated gross deletions in the vif gene [17 18]and only one patient has demonstrated the insertion of twoamino acids and these patients were part of a nonprogress-ing mother-child pair [28] However in our present studyof the 10 LTSP patients who were assessed four patientsdemonstrated gross deletion in the vif gene during KRGintake prior to receiving HAART (15) This frequency issignificantly lower than the previously reported incidence of10 of 10 patients demonstrating gross deletions in the nef(187) and 51015840 LTRgag genes (376 119875 lt 001) [27 28]The frequency of deletion in the nef gene was reported tosignificantly decrease during KRG plus HAART (119875 lt 001)[16] In contrast to previous studies on the nef and gag genes[16 29] the detection of in-frame deletions in the vif genewas not suppressed in the patients receiving HAART in ourpresent cohort The reasons for this include the following (1)the proportion and frequency (approximately two-thirds) ofdeleted vif genes after the first occurrence were much higherthan in the nef and 51015840 LTRgag genes (lt20 and 30 resp)(2) small deletions in the vif gene occurred as a single bandwhereas gross deletions in the nef and 51015840 LTRgag genes weremainly detected as two bands (wild type and short) per PCRreaction and (3) the number of amplicons indicating PSCdueto G-to-A hypermutation was significantly higher in patients

receiving KRG plus HAART than patients receiving onlyKRG This phenomenon is consistent with our previous datain 51015840 LTRgag gene [29] However the frequency of G-to-Ahypermutationwas significantly lower in vif gene than that in51015840 LTRgag gene [29] Thus for the vif gene the detection ofsmall deletionsmight be less affected by limit-diluting effectsas has been shown for the nef gene [29] Taken together theseadditional defects in the vif genes in the same patients mightbe related to the replicative impairment in vif defective HIV-1 which ultimately results in defects in the synthesis of viralDNA [30]

In our present study it appears that the frequency ofhypermutation was higher in patients who maintained goodcompliance with HAART during HAART plus KRG (90-0590-18 and 93-04) than in the patients who demonstratedpoor compliance (Figure 1) In our extended data analysis124 (60485) 31 (5161) 30 (19617) and 09 (1108)of vif genes during KRG plus HAART HAART aloneKRG alone and control revealed PSC respectively Thesedata indicate the presence of synergistic effect of KRG plusHAART on PSC (119875 lt 001) There was no case that smalldeletions were induced by HAART alone About 6ndash43 ofthe integrated proviral pol gene is hypermutated by HAART[22 31 32] although hypermutated viral genomes are notpresent in plasma [32]

ApoBec3G-induced hypermutation in LTNPs has beenreported previously [33] Indeed it has been reported alsothat LTNP patients harbor PSC-containing vif genes morefrequently than progressors [25] although the reportedfrequencies of defective genes vary among studies [34] Inour current analyses vif gene-containing PSC due to G-to-A hypermutation was found to be significantly higher duringKRG plus HAART than only KRG intake prior to HAARTThis finding is consistent with our previous data obtainedfrom a cohort of the Korean hemophiliacs (21 incidence

Evidence-Based Complementary and Alternative Medicine 11

of G-to-A hypermutation while receiving only KRG versus98 on KRG plus HAART unpublished data) and otherobservations regarding the pol gene [22]

Previous studies report that certain vif mutations such asL81M R132S S130I and the insertion of two amino acids areassociatedwith slow progression or low viral loads [17 18 28]In our present study L81M [35] and R132S were consistentlydetected in patient 96-51 and patients 87-05 and 96-51respectively although both mutations were found in only in2 and 3 of 169 Korean patients respectively Although thevif gene is highly conserved in HIV-1 genomes the recoveryof hypermutants might be severely underestimated in ourpresent analysis because of the primer mismatching andthe difficulties involved in detection following amplification[36 37] Overall our current data suggest that small in-frame deletion and PSCs are associated with KRG intake andHAART respectively although further studies are needed

5 Conclusions

Our data suggest that HAART and KRG intake might inducePSCs and small in-frame deletions respectively

Conflict of Interests

The authors have no conflict of interests to declare

Acknowledgments

This work was supported by a grant from the Korean Societyof Ginseng (2010ndash2012) The authors thank Professor Seung-Chul Yun the Division of Biostatistics University of UlsanCollege of Medicine Asan Medical Center for the statisticalconsultation

References

[1] E Ernst ldquoPanax ginseng an overview of the clinical evidencerdquoJournal of Ginseng Research vol 34 no 4 pp 259ndash263 2010

[2] K T Choi ldquoBotanical characteristics pharmacological effectsand medicinal components of Korean Panax ginseng C AMeyerrdquoActa Pharmacologica Sinica vol 29 no 9 pp 1109ndash11182008

[3] S J Fulder ldquoGinseng and the hypothalamic-pituitary control ofstressrdquo The American Journal of Chinese Medicine vol 9 no 2pp 112ndash118 1981

[4] V K Singh S S Agarwal and B M Gupta ldquoImmunomodula-tory activity of Panax ginseng extractrdquo Planta Medica vol 50no 6 pp 462ndash465 1984

[5] X Song and S Hu ldquoAdjuvant activities of saponins fromtraditional Chinese medicinal herbsrdquo Vaccine vol 27 no 36pp 4883ndash4890 2009

[6] D G Yoo M C Kim M K Park et al ldquoProtective effectof ginseng polysaccharides on influenza viral infectionrdquo PLoSONE vol 7 no 3 Article ID e33678 2012

[7] H X Sun Y P Ye H J Pan and Y J Pan ldquoAdjuvant effectof Panax notoginseng saponins on the immune responses toovalbumin in micerdquo Vaccine vol 22 no 29-30 pp 3882ndash38892004

[8] J Sun S Hu and X Song ldquoAdjuvant effects of protopanaxadioland protopanaxatriol saponins from ginseng roots on theimmune responses to ovalbumin in micerdquo Vaccine vol 25 no6 pp 1114ndash1120 2007

[9] P W Hunt ldquoHIV and inflammation mechanisms and conse-quencesrdquo Current HIVAIDS Reports vol 9 no 2 pp 139ndash1472012

[10] T Yayeh K H Jung H Y Jeong et al ldquoKorean red ginsengsaponin fraction downregulates proinflammatory mediators inLPS stimulated RAW264 7 cells and protects mice againstendotoxic shockrdquo Journal of Ginseng Research vol 36 no 3 pp263ndash269 2012

[11] A T Smolinski and J J Pestka ldquoModulation of lipopolysaccha-ride-induced proinflammatory cytokine production in vitroand in vivo by the herbal constituents apigenin (chamomile)ginsenoside Rb1 (ginseng) and parthenolide (feverfew)rdquo Foodand Chemical Toxicology vol 41 no 10 pp 1381ndash1390 2003

[12] A Rhule S Navarro J R Smith and D M ShepherdldquoPanax notoginseng attenuates LPS-induced pro-inflammatorymediators in RAW2647 cellsrdquo Journal of Ethnopharmacologyvol 106 no 1 pp 121ndash128 2006

[13] J M Brenchley D A Price T W Schacker et al ldquoMicrobialtranslocation is a cause of systemic immune activation inchronic HIV infectionrdquo Nature Medicine vol 12 no 12 pp1365ndash1371 2006

[14] Y K Cho J Y Lim Y S Jung S K Oh H J Lee and H SungldquoHigh frequency of grossly deleted nef genes in HIV-1 infectedlong-term slow progressors treated with Korean red ginsengrdquoCurrent HIV Research vol 4 no 4 pp 447ndash457 2006

[15] Y K Cho and Y S Jung ldquoHigh frequency of gross deletions inthe 51015840 LTR and gag regions in HIV type 1-infected long-termsurvivors treated with Korean red ginsengrdquo AIDS Research andHuman Retroviruses vol 24 no 2 pp 181ndash193 2008

[16] Y K Cho Y S Jung andH S Sung ldquoFrequent gross deletion intheHIV type 1 nef gene in hemophiliacs treatedwith korean redginseng inhibition of detection by highly active antiretroviraltherapyrdquo AIDS Research and Human Retroviruses vol 25 no 4pp 419ndash424 2009

[17] V Sandonıs C Casado T Alvaro et al ldquoA combination ofdefective DNA and protective host factors are found in a setof HIV-1 ancestral LTNPsrdquo Virology vol 391 no 1 pp 73ndash822009

[18] H R Rangel D Garzaro A K Rodrıguez et al ldquoDeletioninsertion and stop codon mutations in vif genes of HIV-1 infecting slow progressor patientsrdquo Journal of Infection inDeveloping Countries vol 3 no 7 pp 531ndash538 2009

[19] Y K Cho J E Kim and B T Foley ldquoPhylogenetic analysisof near full-length HIV-1 genomic sequences in 21 Koreanindividualsrdquo AIDS Research and Human Retroviruses vol 29no 4 pp 738ndash743 2013

[20] Y K Cho Y S Jung J S Lee and B T Foley ldquoMolecu-lar evidence of HIV-1 transmission in 20 Korean individu-als with haemophilia phylogenetic analysis of the vif generdquoHaemophilia vol 18 no 2 pp 291ndash299 2012

[21] P P Rose and B T Korber ldquoDetecting hypermutations inviral sequences with an emphasis on GrarrA hypermutationrdquoBioinformatics vol 16 no 4 pp 400ndash401 2000

[22] S Fourati S Lambert-Niclot C Soulie et al ldquoHIV-1 genomeis often defective in PBMCs and rectal tissues after long-termHAART as a result of APOBEC3 editing and correlates with thesize of reservoirsrdquo Journal of Antimicrobial Chemotherapy vol67 no 10 pp 2323ndash2326 2012

12 Evidence-Based Complementary and Alternative Medicine

[23] U Wieland A Seelhoff A Hofmann et al ldquoDiversity of thevif gene of human immunodeficiency virus type 1 in UgandardquoJournal of General Virology vol 78 no 2 pp 393ndash400 1997

[24] P Sova M van Ranst P Gupta et al ldquoConservation of an intacthuman immunodeficiency virus type 1 vif gene in vitro and invivordquo Journal of Virology vol 69 no 4 pp 2557ndash2564 1995

[25] T Yamada and A Iwamoto ldquoComparison of proviral accessorygenes between long-term nonprogressors and progressors ofhuman immunodeficiency virus type 1 infectionrdquo Archives ofVirology vol 145 no 5 pp 1021ndash1027 2000

[26] K Tominaga S Kato M Negishi and T Takano ldquoA high fre-quency of defective vif genes in peripheral blood mononuclearcells from HIV type 1-infected individualsrdquo AIDS Research andHuman Retroviruses vol 12 no 16 pp 1543ndash1549 1996

[27] Y K Cho H Sung I G Bae et al ldquoFull sequence of HIV type 1Korean subtype B in anAIDS case with atypical seroconversionTAAAA at TATA boxrdquoAIDS Research andHuman Retrovirusesvol 21 no 11 pp 961ndash964 2005

[28] L Alexander M J Aquino-DeJesus M Chan and W AAndiman ldquoInhibition of human immunodeficiency virus type 1(HIV-1) replication by a two-amino-acid insertion in HIV-1 Viffrom a nonprogressing mother and childrdquo Journal of Virologyvol 76 no 20 pp 10533ndash10539 2002

[29] YK Cho Y S JungH S Sung andCH Joo ldquoFrequent geneticdefects in the HIV-1 51015840LTRgag gene in hemophiliacs treatedwith Korean red ginseng decreased detection of genetic defectsby highly active antiretroviral therapyrdquo Journal of GinsengResearch vol 35 no 4 pp 413ndash420 2011

[30] M Nascimbeni M Bouyac F Rey B Spire and F Clavel ldquoThereplicative impairment of Vif-mutants of human immunodefi-ciency virus type 1 correlates with an overall defect in viral DNAsynthesisrdquo Journal of General Virology vol 79 no 8 pp 1945ndash1950 1998

[31] J P Vartanian M Henry and S Wain-Hobson ldquoSustainedGrarrA hypermutation during reverse transcription of an entirehuman immunodeficiency virus type 1 strain Vau group Ogenomerdquo Journal of General Virology vol 83 no 4 pp 801ndash8052002

[32] T L Kieffer P Kwon R E Nettles Y Han S C Ray andR F Siliciano ldquoGrarrA hypermutation in protease and reversetranscriptase regions of human immunodeficiency virus type 1residing in resting CD4+ T cells in vivordquo Journal of Virology vol79 no 3 pp 1975ndash1980 2005

[33] B Wang M Mikhail W B Dyer J J Zaunders A D Kelleherand N K Saksena ldquoFirst demonstration of a lack of viralsequence evolution in a nonprogressor defining replication-incompetent HIV-1 infectionrdquo Virology vol 312 no 1 pp 135ndash150 2003

[34] G Hassaine I Agostini D Candotti et al ldquoCharacterizationof human immunodeficiency virus type 1 vif gene in long-termasymptomatic individualsrdquoVirology vol 276 no 1 pp 169ndash1802000

[35] F A de Maio C A Rocco P C Aulicino R Bologna AMangano and L Sen ldquoUnusual substitutions in HIV-1 Vif fromchildren infected perinatally without progression to AIDS formore than 8 years without therapyrdquo Journal of Medical Virologyvol 84 no 12 pp 1844ndash1852 2012

[36] M Janini M Rogers D R Birx and F E McCutchan ldquoHumanimmunodeficiency virus type 1 DNA sequences geneticallydamaged by hypermutation are often abundant in patientperipheral blood mononuclear cells and may be generated

during near-simultaneous infection and activation of CD4+ Tcellsrdquo Journal of Virology vol 75 no 17 pp 7973ndash7986 2001

[37] G H Kijak L M Janini S Tovanabutra et al ldquoVariablecontexts and levels of hypermutation inHIV-1 proviral genomesrecovered from primary peripheral blood mononuclear cellsrdquoVirology vol 376 no 1 pp 101ndash111 2008

[38] E W Refsland J F Hultquist and R S Harris ldquoEndogenousorigins of HIV-1 G-to-A hypermutation and restriction in thenonpermissive T cell line CEM2nrdquo PLoS Pathogens vol 8 no7 Article ID e1002800 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 10: Effects of Korean Red Ginseng and HAART on vif Gene in 10 Long

10 Evidence-Based Complementary and Alternative Medicine

61 69 112 120 208 216 264 272 520 528Patient WT nucleotide TGGAAAAGT TGGGTCTAT TGGGGTCTG TGGAGGAAA TGGAACAAG Hypermut 20

sequence no P value90-05 10LSK4-9168 G A lt005

12LSK4-10131 A A AA A A lt000113LSK5-11470 A A A 015 13LSK5-11472 A AA lt001

90-18 08LSH5-5645 AA 019 08LSH11-5694 A 05710LSH4-9192 AA 013

90-54 07KJin3-5961 A 00693-04 94KYR12-9441 AA AG 009

07KYR8-5166 A A AA A AG lt000107KYR8-4846 A AA G 00507KYR8-5167 A A A AG 00507KYR8-5168 A A AG 02907KYR8-4845 AA G 005

93-60 03KMH11-5725 AA A 015

WT amino acid W K S W V Y W G L W R K W N K21 22 23 38 39 40 70 71 72 89 90 91 174 175 176

Figure 3 Positions of the premature stop codons in the 15 vif genes from five patients All stop codons resulted from GG rarr GA orGG rarr AG changes APOBEC3G exhibits an intrinsic preference for the second cytosine in a 51015840CC dinucleotide motif leading to 51015840GG-to-AG mutations [38]

In total five of our patients demonstrated in-frame deletionsof the vif gene of the 75 KSB-infected patients who weretreated with KRG Also in our present study cohort fivepatients including 90-18 and 92-13 demonstrated deletedvif genes (small in-frame and gross deletions) during KRGintake whereas all of our 10 LTSP patients demonstrateddeletions in the nef gene (119875 lt 005) [14] In additioncompared with the reported deletions of the nef and 51015840LTRgag genes the locations of the deleted vif gene werepositioned within a narrow range at the terminus [15 16 23]

Regarding gross deletions to date only two patientshave demonstrated gross deletions in the vif gene [17 18]and only one patient has demonstrated the insertion of twoamino acids and these patients were part of a nonprogress-ing mother-child pair [28] However in our present studyof the 10 LTSP patients who were assessed four patientsdemonstrated gross deletion in the vif gene during KRGintake prior to receiving HAART (15) This frequency issignificantly lower than the previously reported incidence of10 of 10 patients demonstrating gross deletions in the nef(187) and 51015840 LTRgag genes (376 119875 lt 001) [27 28]The frequency of deletion in the nef gene was reported tosignificantly decrease during KRG plus HAART (119875 lt 001)[16] In contrast to previous studies on the nef and gag genes[16 29] the detection of in-frame deletions in the vif genewas not suppressed in the patients receiving HAART in ourpresent cohort The reasons for this include the following (1)the proportion and frequency (approximately two-thirds) ofdeleted vif genes after the first occurrence were much higherthan in the nef and 51015840 LTRgag genes (lt20 and 30 resp)(2) small deletions in the vif gene occurred as a single bandwhereas gross deletions in the nef and 51015840 LTRgag genes weremainly detected as two bands (wild type and short) per PCRreaction and (3) the number of amplicons indicating PSCdueto G-to-A hypermutation was significantly higher in patients

receiving KRG plus HAART than patients receiving onlyKRG This phenomenon is consistent with our previous datain 51015840 LTRgag gene [29] However the frequency of G-to-Ahypermutationwas significantly lower in vif gene than that in51015840 LTRgag gene [29] Thus for the vif gene the detection ofsmall deletionsmight be less affected by limit-diluting effectsas has been shown for the nef gene [29] Taken together theseadditional defects in the vif genes in the same patients mightbe related to the replicative impairment in vif defective HIV-1 which ultimately results in defects in the synthesis of viralDNA [30]

In our present study it appears that the frequency ofhypermutation was higher in patients who maintained goodcompliance with HAART during HAART plus KRG (90-0590-18 and 93-04) than in the patients who demonstratedpoor compliance (Figure 1) In our extended data analysis124 (60485) 31 (5161) 30 (19617) and 09 (1108)of vif genes during KRG plus HAART HAART aloneKRG alone and control revealed PSC respectively Thesedata indicate the presence of synergistic effect of KRG plusHAART on PSC (119875 lt 001) There was no case that smalldeletions were induced by HAART alone About 6ndash43 ofthe integrated proviral pol gene is hypermutated by HAART[22 31 32] although hypermutated viral genomes are notpresent in plasma [32]

ApoBec3G-induced hypermutation in LTNPs has beenreported previously [33] Indeed it has been reported alsothat LTNP patients harbor PSC-containing vif genes morefrequently than progressors [25] although the reportedfrequencies of defective genes vary among studies [34] Inour current analyses vif gene-containing PSC due to G-to-A hypermutation was found to be significantly higher duringKRG plus HAART than only KRG intake prior to HAARTThis finding is consistent with our previous data obtainedfrom a cohort of the Korean hemophiliacs (21 incidence

Evidence-Based Complementary and Alternative Medicine 11

of G-to-A hypermutation while receiving only KRG versus98 on KRG plus HAART unpublished data) and otherobservations regarding the pol gene [22]

Previous studies report that certain vif mutations such asL81M R132S S130I and the insertion of two amino acids areassociatedwith slow progression or low viral loads [17 18 28]In our present study L81M [35] and R132S were consistentlydetected in patient 96-51 and patients 87-05 and 96-51respectively although both mutations were found in only in2 and 3 of 169 Korean patients respectively Although thevif gene is highly conserved in HIV-1 genomes the recoveryof hypermutants might be severely underestimated in ourpresent analysis because of the primer mismatching andthe difficulties involved in detection following amplification[36 37] Overall our current data suggest that small in-frame deletion and PSCs are associated with KRG intake andHAART respectively although further studies are needed

5 Conclusions

Our data suggest that HAART and KRG intake might inducePSCs and small in-frame deletions respectively

Conflict of Interests

The authors have no conflict of interests to declare

Acknowledgments

This work was supported by a grant from the Korean Societyof Ginseng (2010ndash2012) The authors thank Professor Seung-Chul Yun the Division of Biostatistics University of UlsanCollege of Medicine Asan Medical Center for the statisticalconsultation

References

[1] E Ernst ldquoPanax ginseng an overview of the clinical evidencerdquoJournal of Ginseng Research vol 34 no 4 pp 259ndash263 2010

[2] K T Choi ldquoBotanical characteristics pharmacological effectsand medicinal components of Korean Panax ginseng C AMeyerrdquoActa Pharmacologica Sinica vol 29 no 9 pp 1109ndash11182008

[3] S J Fulder ldquoGinseng and the hypothalamic-pituitary control ofstressrdquo The American Journal of Chinese Medicine vol 9 no 2pp 112ndash118 1981

[4] V K Singh S S Agarwal and B M Gupta ldquoImmunomodula-tory activity of Panax ginseng extractrdquo Planta Medica vol 50no 6 pp 462ndash465 1984

[5] X Song and S Hu ldquoAdjuvant activities of saponins fromtraditional Chinese medicinal herbsrdquo Vaccine vol 27 no 36pp 4883ndash4890 2009

[6] D G Yoo M C Kim M K Park et al ldquoProtective effectof ginseng polysaccharides on influenza viral infectionrdquo PLoSONE vol 7 no 3 Article ID e33678 2012

[7] H X Sun Y P Ye H J Pan and Y J Pan ldquoAdjuvant effectof Panax notoginseng saponins on the immune responses toovalbumin in micerdquo Vaccine vol 22 no 29-30 pp 3882ndash38892004

[8] J Sun S Hu and X Song ldquoAdjuvant effects of protopanaxadioland protopanaxatriol saponins from ginseng roots on theimmune responses to ovalbumin in micerdquo Vaccine vol 25 no6 pp 1114ndash1120 2007

[9] P W Hunt ldquoHIV and inflammation mechanisms and conse-quencesrdquo Current HIVAIDS Reports vol 9 no 2 pp 139ndash1472012

[10] T Yayeh K H Jung H Y Jeong et al ldquoKorean red ginsengsaponin fraction downregulates proinflammatory mediators inLPS stimulated RAW264 7 cells and protects mice againstendotoxic shockrdquo Journal of Ginseng Research vol 36 no 3 pp263ndash269 2012

[11] A T Smolinski and J J Pestka ldquoModulation of lipopolysaccha-ride-induced proinflammatory cytokine production in vitroand in vivo by the herbal constituents apigenin (chamomile)ginsenoside Rb1 (ginseng) and parthenolide (feverfew)rdquo Foodand Chemical Toxicology vol 41 no 10 pp 1381ndash1390 2003

[12] A Rhule S Navarro J R Smith and D M ShepherdldquoPanax notoginseng attenuates LPS-induced pro-inflammatorymediators in RAW2647 cellsrdquo Journal of Ethnopharmacologyvol 106 no 1 pp 121ndash128 2006

[13] J M Brenchley D A Price T W Schacker et al ldquoMicrobialtranslocation is a cause of systemic immune activation inchronic HIV infectionrdquo Nature Medicine vol 12 no 12 pp1365ndash1371 2006

[14] Y K Cho J Y Lim Y S Jung S K Oh H J Lee and H SungldquoHigh frequency of grossly deleted nef genes in HIV-1 infectedlong-term slow progressors treated with Korean red ginsengrdquoCurrent HIV Research vol 4 no 4 pp 447ndash457 2006

[15] Y K Cho and Y S Jung ldquoHigh frequency of gross deletions inthe 51015840 LTR and gag regions in HIV type 1-infected long-termsurvivors treated with Korean red ginsengrdquo AIDS Research andHuman Retroviruses vol 24 no 2 pp 181ndash193 2008

[16] Y K Cho Y S Jung andH S Sung ldquoFrequent gross deletion intheHIV type 1 nef gene in hemophiliacs treatedwith korean redginseng inhibition of detection by highly active antiretroviraltherapyrdquo AIDS Research and Human Retroviruses vol 25 no 4pp 419ndash424 2009

[17] V Sandonıs C Casado T Alvaro et al ldquoA combination ofdefective DNA and protective host factors are found in a setof HIV-1 ancestral LTNPsrdquo Virology vol 391 no 1 pp 73ndash822009

[18] H R Rangel D Garzaro A K Rodrıguez et al ldquoDeletioninsertion and stop codon mutations in vif genes of HIV-1 infecting slow progressor patientsrdquo Journal of Infection inDeveloping Countries vol 3 no 7 pp 531ndash538 2009

[19] Y K Cho J E Kim and B T Foley ldquoPhylogenetic analysisof near full-length HIV-1 genomic sequences in 21 Koreanindividualsrdquo AIDS Research and Human Retroviruses vol 29no 4 pp 738ndash743 2013

[20] Y K Cho Y S Jung J S Lee and B T Foley ldquoMolecu-lar evidence of HIV-1 transmission in 20 Korean individu-als with haemophilia phylogenetic analysis of the vif generdquoHaemophilia vol 18 no 2 pp 291ndash299 2012

[21] P P Rose and B T Korber ldquoDetecting hypermutations inviral sequences with an emphasis on GrarrA hypermutationrdquoBioinformatics vol 16 no 4 pp 400ndash401 2000

[22] S Fourati S Lambert-Niclot C Soulie et al ldquoHIV-1 genomeis often defective in PBMCs and rectal tissues after long-termHAART as a result of APOBEC3 editing and correlates with thesize of reservoirsrdquo Journal of Antimicrobial Chemotherapy vol67 no 10 pp 2323ndash2326 2012

12 Evidence-Based Complementary and Alternative Medicine

[23] U Wieland A Seelhoff A Hofmann et al ldquoDiversity of thevif gene of human immunodeficiency virus type 1 in UgandardquoJournal of General Virology vol 78 no 2 pp 393ndash400 1997

[24] P Sova M van Ranst P Gupta et al ldquoConservation of an intacthuman immunodeficiency virus type 1 vif gene in vitro and invivordquo Journal of Virology vol 69 no 4 pp 2557ndash2564 1995

[25] T Yamada and A Iwamoto ldquoComparison of proviral accessorygenes between long-term nonprogressors and progressors ofhuman immunodeficiency virus type 1 infectionrdquo Archives ofVirology vol 145 no 5 pp 1021ndash1027 2000

[26] K Tominaga S Kato M Negishi and T Takano ldquoA high fre-quency of defective vif genes in peripheral blood mononuclearcells from HIV type 1-infected individualsrdquo AIDS Research andHuman Retroviruses vol 12 no 16 pp 1543ndash1549 1996

[27] Y K Cho H Sung I G Bae et al ldquoFull sequence of HIV type 1Korean subtype B in anAIDS case with atypical seroconversionTAAAA at TATA boxrdquoAIDS Research andHuman Retrovirusesvol 21 no 11 pp 961ndash964 2005

[28] L Alexander M J Aquino-DeJesus M Chan and W AAndiman ldquoInhibition of human immunodeficiency virus type 1(HIV-1) replication by a two-amino-acid insertion in HIV-1 Viffrom a nonprogressing mother and childrdquo Journal of Virologyvol 76 no 20 pp 10533ndash10539 2002

[29] YK Cho Y S JungH S Sung andCH Joo ldquoFrequent geneticdefects in the HIV-1 51015840LTRgag gene in hemophiliacs treatedwith Korean red ginseng decreased detection of genetic defectsby highly active antiretroviral therapyrdquo Journal of GinsengResearch vol 35 no 4 pp 413ndash420 2011

[30] M Nascimbeni M Bouyac F Rey B Spire and F Clavel ldquoThereplicative impairment of Vif-mutants of human immunodefi-ciency virus type 1 correlates with an overall defect in viral DNAsynthesisrdquo Journal of General Virology vol 79 no 8 pp 1945ndash1950 1998

[31] J P Vartanian M Henry and S Wain-Hobson ldquoSustainedGrarrA hypermutation during reverse transcription of an entirehuman immunodeficiency virus type 1 strain Vau group Ogenomerdquo Journal of General Virology vol 83 no 4 pp 801ndash8052002

[32] T L Kieffer P Kwon R E Nettles Y Han S C Ray andR F Siliciano ldquoGrarrA hypermutation in protease and reversetranscriptase regions of human immunodeficiency virus type 1residing in resting CD4+ T cells in vivordquo Journal of Virology vol79 no 3 pp 1975ndash1980 2005

[33] B Wang M Mikhail W B Dyer J J Zaunders A D Kelleherand N K Saksena ldquoFirst demonstration of a lack of viralsequence evolution in a nonprogressor defining replication-incompetent HIV-1 infectionrdquo Virology vol 312 no 1 pp 135ndash150 2003

[34] G Hassaine I Agostini D Candotti et al ldquoCharacterizationof human immunodeficiency virus type 1 vif gene in long-termasymptomatic individualsrdquoVirology vol 276 no 1 pp 169ndash1802000

[35] F A de Maio C A Rocco P C Aulicino R Bologna AMangano and L Sen ldquoUnusual substitutions in HIV-1 Vif fromchildren infected perinatally without progression to AIDS formore than 8 years without therapyrdquo Journal of Medical Virologyvol 84 no 12 pp 1844ndash1852 2012

[36] M Janini M Rogers D R Birx and F E McCutchan ldquoHumanimmunodeficiency virus type 1 DNA sequences geneticallydamaged by hypermutation are often abundant in patientperipheral blood mononuclear cells and may be generated

during near-simultaneous infection and activation of CD4+ Tcellsrdquo Journal of Virology vol 75 no 17 pp 7973ndash7986 2001

[37] G H Kijak L M Janini S Tovanabutra et al ldquoVariablecontexts and levels of hypermutation inHIV-1 proviral genomesrecovered from primary peripheral blood mononuclear cellsrdquoVirology vol 376 no 1 pp 101ndash111 2008

[38] E W Refsland J F Hultquist and R S Harris ldquoEndogenousorigins of HIV-1 G-to-A hypermutation and restriction in thenonpermissive T cell line CEM2nrdquo PLoS Pathogens vol 8 no7 Article ID e1002800 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 11: Effects of Korean Red Ginseng and HAART on vif Gene in 10 Long

Evidence-Based Complementary and Alternative Medicine 11

of G-to-A hypermutation while receiving only KRG versus98 on KRG plus HAART unpublished data) and otherobservations regarding the pol gene [22]

Previous studies report that certain vif mutations such asL81M R132S S130I and the insertion of two amino acids areassociatedwith slow progression or low viral loads [17 18 28]In our present study L81M [35] and R132S were consistentlydetected in patient 96-51 and patients 87-05 and 96-51respectively although both mutations were found in only in2 and 3 of 169 Korean patients respectively Although thevif gene is highly conserved in HIV-1 genomes the recoveryof hypermutants might be severely underestimated in ourpresent analysis because of the primer mismatching andthe difficulties involved in detection following amplification[36 37] Overall our current data suggest that small in-frame deletion and PSCs are associated with KRG intake andHAART respectively although further studies are needed

5 Conclusions

Our data suggest that HAART and KRG intake might inducePSCs and small in-frame deletions respectively

Conflict of Interests

The authors have no conflict of interests to declare

Acknowledgments

This work was supported by a grant from the Korean Societyof Ginseng (2010ndash2012) The authors thank Professor Seung-Chul Yun the Division of Biostatistics University of UlsanCollege of Medicine Asan Medical Center for the statisticalconsultation

References

[1] E Ernst ldquoPanax ginseng an overview of the clinical evidencerdquoJournal of Ginseng Research vol 34 no 4 pp 259ndash263 2010

[2] K T Choi ldquoBotanical characteristics pharmacological effectsand medicinal components of Korean Panax ginseng C AMeyerrdquoActa Pharmacologica Sinica vol 29 no 9 pp 1109ndash11182008

[3] S J Fulder ldquoGinseng and the hypothalamic-pituitary control ofstressrdquo The American Journal of Chinese Medicine vol 9 no 2pp 112ndash118 1981

[4] V K Singh S S Agarwal and B M Gupta ldquoImmunomodula-tory activity of Panax ginseng extractrdquo Planta Medica vol 50no 6 pp 462ndash465 1984

[5] X Song and S Hu ldquoAdjuvant activities of saponins fromtraditional Chinese medicinal herbsrdquo Vaccine vol 27 no 36pp 4883ndash4890 2009

[6] D G Yoo M C Kim M K Park et al ldquoProtective effectof ginseng polysaccharides on influenza viral infectionrdquo PLoSONE vol 7 no 3 Article ID e33678 2012

[7] H X Sun Y P Ye H J Pan and Y J Pan ldquoAdjuvant effectof Panax notoginseng saponins on the immune responses toovalbumin in micerdquo Vaccine vol 22 no 29-30 pp 3882ndash38892004

[8] J Sun S Hu and X Song ldquoAdjuvant effects of protopanaxadioland protopanaxatriol saponins from ginseng roots on theimmune responses to ovalbumin in micerdquo Vaccine vol 25 no6 pp 1114ndash1120 2007

[9] P W Hunt ldquoHIV and inflammation mechanisms and conse-quencesrdquo Current HIVAIDS Reports vol 9 no 2 pp 139ndash1472012

[10] T Yayeh K H Jung H Y Jeong et al ldquoKorean red ginsengsaponin fraction downregulates proinflammatory mediators inLPS stimulated RAW264 7 cells and protects mice againstendotoxic shockrdquo Journal of Ginseng Research vol 36 no 3 pp263ndash269 2012

[11] A T Smolinski and J J Pestka ldquoModulation of lipopolysaccha-ride-induced proinflammatory cytokine production in vitroand in vivo by the herbal constituents apigenin (chamomile)ginsenoside Rb1 (ginseng) and parthenolide (feverfew)rdquo Foodand Chemical Toxicology vol 41 no 10 pp 1381ndash1390 2003

[12] A Rhule S Navarro J R Smith and D M ShepherdldquoPanax notoginseng attenuates LPS-induced pro-inflammatorymediators in RAW2647 cellsrdquo Journal of Ethnopharmacologyvol 106 no 1 pp 121ndash128 2006

[13] J M Brenchley D A Price T W Schacker et al ldquoMicrobialtranslocation is a cause of systemic immune activation inchronic HIV infectionrdquo Nature Medicine vol 12 no 12 pp1365ndash1371 2006

[14] Y K Cho J Y Lim Y S Jung S K Oh H J Lee and H SungldquoHigh frequency of grossly deleted nef genes in HIV-1 infectedlong-term slow progressors treated with Korean red ginsengrdquoCurrent HIV Research vol 4 no 4 pp 447ndash457 2006

[15] Y K Cho and Y S Jung ldquoHigh frequency of gross deletions inthe 51015840 LTR and gag regions in HIV type 1-infected long-termsurvivors treated with Korean red ginsengrdquo AIDS Research andHuman Retroviruses vol 24 no 2 pp 181ndash193 2008

[16] Y K Cho Y S Jung andH S Sung ldquoFrequent gross deletion intheHIV type 1 nef gene in hemophiliacs treatedwith korean redginseng inhibition of detection by highly active antiretroviraltherapyrdquo AIDS Research and Human Retroviruses vol 25 no 4pp 419ndash424 2009

[17] V Sandonıs C Casado T Alvaro et al ldquoA combination ofdefective DNA and protective host factors are found in a setof HIV-1 ancestral LTNPsrdquo Virology vol 391 no 1 pp 73ndash822009

[18] H R Rangel D Garzaro A K Rodrıguez et al ldquoDeletioninsertion and stop codon mutations in vif genes of HIV-1 infecting slow progressor patientsrdquo Journal of Infection inDeveloping Countries vol 3 no 7 pp 531ndash538 2009

[19] Y K Cho J E Kim and B T Foley ldquoPhylogenetic analysisof near full-length HIV-1 genomic sequences in 21 Koreanindividualsrdquo AIDS Research and Human Retroviruses vol 29no 4 pp 738ndash743 2013

[20] Y K Cho Y S Jung J S Lee and B T Foley ldquoMolecu-lar evidence of HIV-1 transmission in 20 Korean individu-als with haemophilia phylogenetic analysis of the vif generdquoHaemophilia vol 18 no 2 pp 291ndash299 2012

[21] P P Rose and B T Korber ldquoDetecting hypermutations inviral sequences with an emphasis on GrarrA hypermutationrdquoBioinformatics vol 16 no 4 pp 400ndash401 2000

[22] S Fourati S Lambert-Niclot C Soulie et al ldquoHIV-1 genomeis often defective in PBMCs and rectal tissues after long-termHAART as a result of APOBEC3 editing and correlates with thesize of reservoirsrdquo Journal of Antimicrobial Chemotherapy vol67 no 10 pp 2323ndash2326 2012

12 Evidence-Based Complementary and Alternative Medicine

[23] U Wieland A Seelhoff A Hofmann et al ldquoDiversity of thevif gene of human immunodeficiency virus type 1 in UgandardquoJournal of General Virology vol 78 no 2 pp 393ndash400 1997

[24] P Sova M van Ranst P Gupta et al ldquoConservation of an intacthuman immunodeficiency virus type 1 vif gene in vitro and invivordquo Journal of Virology vol 69 no 4 pp 2557ndash2564 1995

[25] T Yamada and A Iwamoto ldquoComparison of proviral accessorygenes between long-term nonprogressors and progressors ofhuman immunodeficiency virus type 1 infectionrdquo Archives ofVirology vol 145 no 5 pp 1021ndash1027 2000

[26] K Tominaga S Kato M Negishi and T Takano ldquoA high fre-quency of defective vif genes in peripheral blood mononuclearcells from HIV type 1-infected individualsrdquo AIDS Research andHuman Retroviruses vol 12 no 16 pp 1543ndash1549 1996

[27] Y K Cho H Sung I G Bae et al ldquoFull sequence of HIV type 1Korean subtype B in anAIDS case with atypical seroconversionTAAAA at TATA boxrdquoAIDS Research andHuman Retrovirusesvol 21 no 11 pp 961ndash964 2005

[28] L Alexander M J Aquino-DeJesus M Chan and W AAndiman ldquoInhibition of human immunodeficiency virus type 1(HIV-1) replication by a two-amino-acid insertion in HIV-1 Viffrom a nonprogressing mother and childrdquo Journal of Virologyvol 76 no 20 pp 10533ndash10539 2002

[29] YK Cho Y S JungH S Sung andCH Joo ldquoFrequent geneticdefects in the HIV-1 51015840LTRgag gene in hemophiliacs treatedwith Korean red ginseng decreased detection of genetic defectsby highly active antiretroviral therapyrdquo Journal of GinsengResearch vol 35 no 4 pp 413ndash420 2011

[30] M Nascimbeni M Bouyac F Rey B Spire and F Clavel ldquoThereplicative impairment of Vif-mutants of human immunodefi-ciency virus type 1 correlates with an overall defect in viral DNAsynthesisrdquo Journal of General Virology vol 79 no 8 pp 1945ndash1950 1998

[31] J P Vartanian M Henry and S Wain-Hobson ldquoSustainedGrarrA hypermutation during reverse transcription of an entirehuman immunodeficiency virus type 1 strain Vau group Ogenomerdquo Journal of General Virology vol 83 no 4 pp 801ndash8052002

[32] T L Kieffer P Kwon R E Nettles Y Han S C Ray andR F Siliciano ldquoGrarrA hypermutation in protease and reversetranscriptase regions of human immunodeficiency virus type 1residing in resting CD4+ T cells in vivordquo Journal of Virology vol79 no 3 pp 1975ndash1980 2005

[33] B Wang M Mikhail W B Dyer J J Zaunders A D Kelleherand N K Saksena ldquoFirst demonstration of a lack of viralsequence evolution in a nonprogressor defining replication-incompetent HIV-1 infectionrdquo Virology vol 312 no 1 pp 135ndash150 2003

[34] G Hassaine I Agostini D Candotti et al ldquoCharacterizationof human immunodeficiency virus type 1 vif gene in long-termasymptomatic individualsrdquoVirology vol 276 no 1 pp 169ndash1802000

[35] F A de Maio C A Rocco P C Aulicino R Bologna AMangano and L Sen ldquoUnusual substitutions in HIV-1 Vif fromchildren infected perinatally without progression to AIDS formore than 8 years without therapyrdquo Journal of Medical Virologyvol 84 no 12 pp 1844ndash1852 2012

[36] M Janini M Rogers D R Birx and F E McCutchan ldquoHumanimmunodeficiency virus type 1 DNA sequences geneticallydamaged by hypermutation are often abundant in patientperipheral blood mononuclear cells and may be generated

during near-simultaneous infection and activation of CD4+ Tcellsrdquo Journal of Virology vol 75 no 17 pp 7973ndash7986 2001

[37] G H Kijak L M Janini S Tovanabutra et al ldquoVariablecontexts and levels of hypermutation inHIV-1 proviral genomesrecovered from primary peripheral blood mononuclear cellsrdquoVirology vol 376 no 1 pp 101ndash111 2008

[38] E W Refsland J F Hultquist and R S Harris ldquoEndogenousorigins of HIV-1 G-to-A hypermutation and restriction in thenonpermissive T cell line CEM2nrdquo PLoS Pathogens vol 8 no7 Article ID e1002800 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 12: Effects of Korean Red Ginseng and HAART on vif Gene in 10 Long

12 Evidence-Based Complementary and Alternative Medicine

[23] U Wieland A Seelhoff A Hofmann et al ldquoDiversity of thevif gene of human immunodeficiency virus type 1 in UgandardquoJournal of General Virology vol 78 no 2 pp 393ndash400 1997

[24] P Sova M van Ranst P Gupta et al ldquoConservation of an intacthuman immunodeficiency virus type 1 vif gene in vitro and invivordquo Journal of Virology vol 69 no 4 pp 2557ndash2564 1995

[25] T Yamada and A Iwamoto ldquoComparison of proviral accessorygenes between long-term nonprogressors and progressors ofhuman immunodeficiency virus type 1 infectionrdquo Archives ofVirology vol 145 no 5 pp 1021ndash1027 2000

[26] K Tominaga S Kato M Negishi and T Takano ldquoA high fre-quency of defective vif genes in peripheral blood mononuclearcells from HIV type 1-infected individualsrdquo AIDS Research andHuman Retroviruses vol 12 no 16 pp 1543ndash1549 1996

[27] Y K Cho H Sung I G Bae et al ldquoFull sequence of HIV type 1Korean subtype B in anAIDS case with atypical seroconversionTAAAA at TATA boxrdquoAIDS Research andHuman Retrovirusesvol 21 no 11 pp 961ndash964 2005

[28] L Alexander M J Aquino-DeJesus M Chan and W AAndiman ldquoInhibition of human immunodeficiency virus type 1(HIV-1) replication by a two-amino-acid insertion in HIV-1 Viffrom a nonprogressing mother and childrdquo Journal of Virologyvol 76 no 20 pp 10533ndash10539 2002

[29] YK Cho Y S JungH S Sung andCH Joo ldquoFrequent geneticdefects in the HIV-1 51015840LTRgag gene in hemophiliacs treatedwith Korean red ginseng decreased detection of genetic defectsby highly active antiretroviral therapyrdquo Journal of GinsengResearch vol 35 no 4 pp 413ndash420 2011

[30] M Nascimbeni M Bouyac F Rey B Spire and F Clavel ldquoThereplicative impairment of Vif-mutants of human immunodefi-ciency virus type 1 correlates with an overall defect in viral DNAsynthesisrdquo Journal of General Virology vol 79 no 8 pp 1945ndash1950 1998

[31] J P Vartanian M Henry and S Wain-Hobson ldquoSustainedGrarrA hypermutation during reverse transcription of an entirehuman immunodeficiency virus type 1 strain Vau group Ogenomerdquo Journal of General Virology vol 83 no 4 pp 801ndash8052002

[32] T L Kieffer P Kwon R E Nettles Y Han S C Ray andR F Siliciano ldquoGrarrA hypermutation in protease and reversetranscriptase regions of human immunodeficiency virus type 1residing in resting CD4+ T cells in vivordquo Journal of Virology vol79 no 3 pp 1975ndash1980 2005

[33] B Wang M Mikhail W B Dyer J J Zaunders A D Kelleherand N K Saksena ldquoFirst demonstration of a lack of viralsequence evolution in a nonprogressor defining replication-incompetent HIV-1 infectionrdquo Virology vol 312 no 1 pp 135ndash150 2003

[34] G Hassaine I Agostini D Candotti et al ldquoCharacterizationof human immunodeficiency virus type 1 vif gene in long-termasymptomatic individualsrdquoVirology vol 276 no 1 pp 169ndash1802000

[35] F A de Maio C A Rocco P C Aulicino R Bologna AMangano and L Sen ldquoUnusual substitutions in HIV-1 Vif fromchildren infected perinatally without progression to AIDS formore than 8 years without therapyrdquo Journal of Medical Virologyvol 84 no 12 pp 1844ndash1852 2012

[36] M Janini M Rogers D R Birx and F E McCutchan ldquoHumanimmunodeficiency virus type 1 DNA sequences geneticallydamaged by hypermutation are often abundant in patientperipheral blood mononuclear cells and may be generated

during near-simultaneous infection and activation of CD4+ Tcellsrdquo Journal of Virology vol 75 no 17 pp 7973ndash7986 2001

[37] G H Kijak L M Janini S Tovanabutra et al ldquoVariablecontexts and levels of hypermutation inHIV-1 proviral genomesrecovered from primary peripheral blood mononuclear cellsrdquoVirology vol 376 no 1 pp 101ndash111 2008

[38] E W Refsland J F Hultquist and R S Harris ldquoEndogenousorigins of HIV-1 G-to-A hypermutation and restriction in thenonpermissive T cell line CEM2nrdquo PLoS Pathogens vol 8 no7 Article ID e1002800 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 13: Effects of Korean Red Ginseng and HAART on vif Gene in 10 Long

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom