predominance of codon 215 mutation in reverse...

1278

Predominance of Codon 215 Mutation in Reverse

Transcriptase-Coding Region of 3'-azido-3'-

deoxythymidine (AZT)-Resistant HIV-1

Isolates after Long-Term

AZT Therapy

Makiko KONDO

Department of Virology, Kanagawa Prefectural Public Health Laboratory,Department of Bacteriology, Yokohama City University, School of Medicine

(Received: June 16, 1995)(Accepted: August 7, 1995)

Key words: AZT, drug resistance, HIV-1, PCR, reverse transcriptase

Abstract

Among human immunodeficiency type 1 viruses (HIV-1) isolated during long-term 3'-azido-

3'-deoxythymidine (AZT) therapy, 4 condons (Asp67, Lys70, Thr215, and Lys219) in the HIV-1 reversetranscriptase (RT)-coding region have been considered to be related to the AZT resistance of HIV-

1. Therefore we determined these mutation patterns in HIV-1 isolates from patients undergoing

long-term AZT treatment. In 41 clones of HIV-1 from 7 patients, the Thr215 mutation was the most

predominant (97.6%), and more frequent than Asp67 (48.8%), Lys70 (31.7%) and Lys219 (9.8%)mutations.

All 22 clones from isolates cultured in the presence of 1 ,u M AZT showed Thr215 mutation; such

a high frequency was not found for the other 3 codon mutations.In a clinical follow-up study, Thr215 mutation appeared in the late stage of AZT treatment in

parallel with the emergence of AZT insusceptibility. It is worthnoting that this Thr215 mutation toTyr or Phe is a 2-nucleotide mutation in contrast to the 1-nucleotide mutations seen in the other 3codons. Isolates with the single amino acid change of Thr215 of the RT-coding region obtained after

long-term AZT treatment grew in the presence of 1 ,u M AZT. This single amino acid mutation in the

Thr215 codon is the most important factor in AZT resistance.

Introduction

For prevention and therapy of acquired immunodeficiency syndrome (AIDS), the hypervariability

of human immunodeficiency type 1 virus (HIV-1) has raised many obstacles. Hypervariability in the

envelope coding region has hindered the development of an HIV vaccine and immunoprophylacticmeasures. Hypervariability in the reverse transcriptase (RT)-coding region has raised concerns

regarding long-term anti-retroviral therapy with 3'-azido-3'-deoxythmidine (AZT). Because the

RT-coding region of HIV-1 is the target of nucleoside analogues such as AZT, multiple mutations in

HIV-1 RT are thought to be correlated with a high level of resistance to AZT. Comparativenucleotide sequence analysis of the RT-coding region from AZT-sensitive and AZT-resistant HIV-1

Correspondence to: Makiko KONDO

Department of Virology, Kanagawa Prefectnral Public Health Laboratory, 52- 2 Nakao-cho Asahi-ku Yokohama,

241, Japan

感染症学雑誌第69巻第11号

Mutation in AZT-resistant HIV-1 1279

isolates identified 4 predicted amino acid substitutions among resistant strains (Asp67•¨Asn, Lys70

→Arg , Thr215→Tyr or Phe, Lys 219→Gln)1)～3).

To determine the mechanism of AZT resistance after long-term AZT treatment in hopes of

improving anti-retroviral therapy, we isolated HIV strains from patients treated with AZT at various

times during therapy and compared the mutation patterns in amino acid residues in the RT region of

those strains with the patterns of the AZT-susceptible wild type HIV-1 strains.

In this paper we report 1) the prevalence of mutation at each of the 4 codons in isolates obtainedafter long-term AZT therapy, 2) amino acid mutation patterns of these HIV-1 isolates grown in the

presence or absence of AZT, 3) comparison of HIV-1 mutations over time, and 4) changes innucleotide mutation patterns in individuals over the course of AZT treatment and the susceptibility

of each isolate to AZT.

Materials and Methods

1) Subjects

The viruses used in this study were obtained from 9 patients, 6 with hemophilia, 2 with heterosex-

ual contact and 1 with unknown risk factor. They were in clinical stages II to IV as defined by the

U. S. Centers for Disease Control and Prevention.

2) AZT treatment

All patients were treated with 400 mg of AZT per day for longer than 6 months, except patient

107 who was treated with 800 mg/day.

3) Isolation of HIV-1

Viruses were isolated from patients' peripheral blood mononuclear cells (PBMC) by co-

cultivation with phytohemagglutination (PHA)-stimulated PBMC from healthy individuals4'5)

4) HIV-1 infection marker

During cultivation, HIV-1 infection of the co-cultured cells was confirmed by 3 steps: (1) syncytial

formation by cultured cells; (2) HIV-1-related antigen appearance, detected by the indirect immuno-

fluorescent antibody test using anti-HIV-1 antibody-positive serum; (3) p24 antigen detection in

culture fluid by ELISA (Abbott).

5) AZT resistance test in vitro

Isolates obtained from patients before AZT treatment, and 2 and more than 6 months after

commencing treatment with AZT were cultured in the presence of AZT (0.1 and 1 ƒÊ M) or in its

absence. After 7 days of cultivation, the culture supernatants were transferred to PHA-stimulated

PBMC from healthy donors. This procedure was carried out 4 times and the cultures were examined

for infection markers.

6) Nested PCR for RT region of HIV-1 isolates

To determine the mutation patterns in the RT-coding region gene, DNA was extracted from the

HIV-1 isolate obtained from each patient, and was amplified by the nested PCR6'7. A region en-

compassing codons 67, 70, 215, and 219 was amplified by using oligonucleotide primers that anneal to

conserved sequences flanking this regin. The first PCR was carried out in 50 ƒÊl of reaction mixture

(200 ƒÊmol each of dATP, dGTP, dCTP, dTTP; 50 mM of KC1; 10 mM of tris-HC1 pH 8.8; 1.5 mM

of MgCl2, and Triton X-100, 0.1%), to which were added 2.5 U of Taq DNA polymerase (Wako) and

0.5 ƒÊmol each of KH55 (5'-GACTCAGATTGGTTGCAGTTT-3') and KH52 (5'-

CTTATCTATTCCATCTAGAAATAGT-3')8' as the first PCR primer. The second PCR was conduct-

ed with 5 ƒÊl of the sample amplified by the first PCR and the second PCR primer, 0.5 ƒÊmol each of

SAl (5'-CCATACAATACTGCAGTATTTGC-3') and SA3 (5'-AGTGCTTTGGATCCTCTAAGGAG-

3')7) in the same reaction mixture. Primers SA1 and SA3 were designed to contain Pst 1 and BamHI

平成7年11月20日

1280 Makiko KONDO

restriction enzyme-susceptible regions respectively. Samples were subjected to 30 cycles of denatura-

tion for 1 minute at 92•Ž, primer annealing for 1.5 minutes at 54•Ž and DNA synthesis for 3 minutes

at 71•Ž with a Taitec Thermo processor TR-100. The PCR products of the RT-coding region were

analyzed on 2% agarose gels and a 705-base pair product was identified by ethidium bromide staining.

7) Sequencing of PCR products

Amplified products of the RT-coding region were digested with restriction enzymes Pst 1 and

BamHI (Takara). The fragments were ligated into bacteriophage M13mp18 DNA predigested with

Pst 1 and Barn HI. The ligation solution was used to transform Escherichia coli (strain TG1).

Recombinant M13 clones containing the RT-coding region were screened for expression of an

approximately 700-base pair RT, and single-stranded DNA was prepared from these constructs for

nucleotide sequencing. The dideoxy nucleotide chain termination method') was used for sequencing

(United States Biochemical Co.).

Results

1) Prevalence of amino acid mutants in the RT-coding region of HIV-1 isolates from patients treatedwith AZT for longer than 6 months.

HIV-1 isolates were obtained from 9 patients treated with AZT for longer than 6 months. From

these, 48 HIV-1 RT-coding clones were obtained and sequenced. Fourty-one of the 48 clones showedmutation in the RT-coding region at 1 or more of codons 67, 70, 215, or 219. These mutants were

isolated from 7 of 9 patients (77.8%).

Among the 41 mutant clones, mutations at codons 67, 70, 215, and 219 were observed in 48.8, 31.7,

97.6 and 9.8 % respectively (Table 1) . It is worthynoting that almost all mutants showed a mutationat codon 215.

2) Amino acid mutation patterns of 22 HIV-1 RT-coding clones from isolates grown in the presence

Table 1 Prevalence of amino acid mutations in the RT-coding region of

HIV-1 isolates from patients with long-term AZT treatment

Table 2 Amino acid mutation patterns of 22 clones from 4 HIV-1 strains grown in the presence of 1,uM AZT for

4 weeks and 17 clones from 4 HIV-1 strains grown without AZT

Underline indicates amino acid mutation



of 1 ƒÊM of AZT for 4 weeks, and 17 HIV-1 RT-coding clones from isolates grown without AZT.

From 4 patients (043-2, 107, 091-1 and 202, treated with AZT for 6, 13, 14, and 31 months

respectively), 22 clones from isolates grown in vitro in the presence of 1ƒÊM AZT and 17 clones from

isolates grown without AZT were obtained.

Sequence analysis of these clones is shown in Table 2. The 22 clones from isolates grown in the

presence of 1ƒÊM AZT all showed mutation in at least one of codons 67, 70, 215, and 219, with

mutation frequencies for each codon of 18.2, 36.4, 100 and 31.8% respectively. It should be noted that

mutation was uniformly observed in codon 215. Fourteen of these 22 clones showed only a single

codon, 215, mutation from Thr to Tyr. For codon 215 in these 22 clones, 18 mutated from Thr (ACC)

to Tyr (TAC) and 4 from Thr (ACC) to Phe (TTC). These are all mutations of 2 nucleotides. Mutation

in Asp67 (GAC) to Ser (AGC); Lys70 (AAA) to Arg (AGA); and Lys 219 (AAA) to Gln (CAA) was

observed in 18.2, 36.4, and 31.8% of these clones respectively. Mutation at these 3 codons was much

less frequent than at codon 215, and the changes all involved 1-nucleotide substitution.

Of the 17 clones obtained from isolates grown in the absence of AZT, mutationat codon 215 was

also observed in 100% of the clones. The frequencies for codons 67, 70, and 219 were 58.8, 52.9 and

52.9%, respectively. All the particular mutation patterns in RT-coding clonesfrom isolates grown in

the presence of 1ƒÊM AZT were observed also in RT-coding clones from isolatesgrown without AZT.

One additional pattern, Ser67, Lys70, Phe215, Glu219 was observed only among the isolates grown

without AZT. Two clones of Asp67, Arg70, Thr215, Lys219 (215 codon not mutated) were obtained

from isolates grown in the presence of 0.1ƒÊM AZT (data not shown) but not 1ƒÊM AZT, and they

are considered to be weakly resistant to AZT.

3) Comparison of HIV-1 mutations over time.

HIV-1 RT-coding clones from isolates obtained at various times during AZT treatment were

sequenced and compared: 12 clones established before treatment, 19, 2 months after starting treat-

ment, 10, 6-7 months after starting, and 12, more than 8 months after starting. Results are shown in

Table 3.

All 12 clones from HIV-1 isolates before AZT treatment were wild type ,and the isolates did not

grow in the presence of 1ƒÊM AZT. After 2 months of AZT treatment, 9 out of 19 clones (47.4%)

showed mutation. Among these, all 9 showed mutation at codon 70 (47.4%), 1 atcodon 215 (0.1%),

and 1 at codon 219 (0.1%). After 6-7 months of AZT treatment, all 10 clones (100%) showed mutation:

4 at codon 67 (40%), 5 at codon 70 (50%) and 9 at codon 215 (90%). After AZT therapy longer than

8 months, all 12 clones were mutants: 1 at codon 67 (8.3%), 1 at codon 70 (8.3%) and all 12 at codon

215. As shown in Fig. 1, the frequency of individual mutant patterns among total clones changed over

the period of AZT treatment. After treatment for 8 months or longer, clones of all isolates were

mutants clones. In the early stage, codon 70 mutants were more frequent but these decreased in later

stages. On the other hand, mutation at codon 215 appeared rarely in the earlystage but later became

Table 3 HIV mutations at 4 codons over time in patients treated with AZT


1282 Makiko KONDO

Fig. 1 Frequency of HIV-1 mutations at 4 codons in patients treated with AZT

over time.*M

/ T=mutant clones (M) per total clones (T) isolated , 67, 70, 215, 219 =codons in RT,

N = number of clones

before therapy (N=12) after 2 monthsof AZT therapy

(N=19)

after 6-7 monthsof AZT therapy

(N=10)

after 8 monthsof AZT therapy

(N=12)

Table 4 Nucleic acid changes in HIV-1 RT-coding region codons and AZT sensitivity of

HIV-1 isolates in the course of AZT treatment of 2 patients

Patient 038

Underline indicates nucleotide mutation.



predominant.

4) Changes in the HIV-1 RT-coding genome obtained from individual patients inthe course of AZT

treatment and AZT sensitivity of HIV-1 isolates.

From 2 patients (038, 005) HIV-1 RT-coding clones were obtained from HIV-1 isolated before

therapy, and 2 months and 8 months or 12 months after AZT treatment. The mutation patterns in

codons 67, 70, 215 and 219 are shown in Table 4. The susceptibility of these isolates to AZT at each

time was tested in vitro. The results are shown in Table 4. Twelve clones from isolates from these

2 patients before AZT treatment were all wild type and did not grow in the presence of 0 .1 ,uM and

1 At A4 AZT.

Among clones from isolates cultured after 2 months of therapy, 3 of 5 from patient 038 and 5 of

10 from patient 005 showed mutation from AAA to AGA at codon 70. Isolates from 005 did not grow

in the presence of 0.1 ,u M or 1 ,uM AZT.

All 7 clones from the isolate from patient 038 after 8 months of AZT therapy showed codon 215

mutation from ACC to TAC. The isolate grew in the presence of 0.1 ,uM and 1 ,uM AZT.

Among the 5 clones of the isolate from patient 005 after 12 months of AZT therapy, 4 clones

showed mutation at codon 215 from ACC to TAC only, and 1 clone also showed mutation at codon

67 (GAC AAC) and codon 70 (AAA•¨AGA). This isolate grew in the presence of 0.1,u M and 1,u M

AZT.

Discussion

We found 41 HIV-1 RT-coding clone mutants among 48 clones obtained from 10 isolates from 9

patients who underwent long-term AZT therapy. As it has been reported that Asn67, Lys70, Thr215,and Lys219 RT mutations are associated with AZT resistance1-3), we determinedthe occurrence ofthese 4 codon mutations among our HIV-1 mutants. Of these 4 mutant amino acid, Thr215 was

observed quite frequently (40 of 41 or 97.6%) compared with other codon mutations. Asp67 occurred

in only 48.8% of the clones, Lys70 in 31.7%, and Lys219 in 9.8%. These results showed the possibility

of predominance of codon 215 mutation in AZT resistance after long-term AZT therapy.

We tried to culture HIV-1 from patients treated with AZT for 6 to 31 months in the presence of1 ,uM AZT. Assuming that the concentration of AZT in the blood 4 hours after a 250 mg dose (given

every 4 hours) is 0.94-1 ,umol/L10), 1 ,uM in vitro represents an appropriateconcentration to

determine AZT resistance in vivo. Twenty-two HIV-1 clones were obtained from these long-termAZT-treated patients by culture with AZT; their amino acid mutation patterns in the RT-coding

region were compared with those of clones obtained from the same patients by culture without AZT.

Among 22 clones with AZT and 17 clones without AZT, the codon 215 (Thr) mutation was observedin all isolates. By contrast, of the clones cultured with AZT, Asn67, Lys70, and Lys219 mutation was

observed in only 18.2, 36.4 and 31.8% respectively. These 3 amino acid mutation patterns were more

frequently found in isolates grown in the absence of AZT (52.9-58.8%) than inthose grown in its

presence (18.2-36.4%). These results also demonstrate that the codon Thr215 mutation in theRT-coding region plays a more important role in AZT resistance than the other3 amino acid

mutations. Asp67 mutation to Asn, which was observed in isolates cultured without AZT, was not

observed in isolates cultured with AZT. These findings suggest a rather minorrole for Asp67

mutation in AZT resistance.

In 14 of 22 (63.6%) mutant clones grown in the presence of AZT, and in 7 of 17 (41.2%) grownwithout AZT, the single amino acid substitution (Thr215 to Tyr) was the only mutation found. This

finding underlines the predominance of this particular mutation of HIV-1 in relation to AZT resist-

ance in long-term AZT treatment. In contrast to this single amino acid mutation among AZT-


1284 Makiko KONDO

resistant isolates, the other Thr215 mutants (Thr215 to Phe) are all associated with 2 or 3 additionalamino acid mutations in Asp67, Lys70 or Lys219.

The appearance of HIV-1 mutations in these 4 codons was investigated over thecourse of AZT

treatment. Growth of HIV-1 isolates cultured in the presence of AZT was not observed before of AZT

therapy, but the virus appeared after 6-7 months. The low rate of Thr215 mutation in the early stage

of AZT treatment and extremely high rate of Thr215 mutation in later stages, such as after 6 monthsof AZT therapy, demonstrates the predominant role of Thr215 mutation in AZT resistance. This is

in contrast to Lys70 mutation which was frequently observed in the early stage but decreased in later

stages.The nucleotide sequences of 2 patients individually followed up clinically are shown in Table 4.

After 2 months of AZT treatment, the observed mutations had only 1 nucleotideshift, codon 70 from

AAA to AGA or codon 215 from ACC to CCC, and these isolates were not resistant to AZT. However,

the codon 215 mutation from ACC to TAC (Tyr) or TTC (Phe) observed in AZT-resistant isolates

after 8 or 12 months of therapy are 2-nucleotide mutations. This may explain the late appearance ofthe predominant mutation pattern of Thr215 to Phe or especially to Tyr in relation to AZT resist-

ance. Single nucleotide mutations such as codon 70 from AAA to AGA which appear in the early

stages of AZT therapy may not play such a significant role in acquisition of AZT resistance by HIV-1.

Acknowledgments

I am deeply indebted to Professor Kenji Okuda (Department of Bacteriology, Yokohama City

University, School of Medicine), for his guidance in the study. Thanks are also due to Dr. Mitsunobu

Imai and Mr. Takayuki Saito (Department of virology, Kanagawa Prefectural Public Health

Laboratory), Dr. Akira Ito (Division of Clinical Laboratory, Yokohama City University, School ofMedicine), Dr. Shinnichi Oka (Department of Infectious Disease, Institute of Medical Science,

University of Tokyo), and Dr. Kusuya Nishioka (Viral Hepatitis Research Foundation of Japan), for

their advice and encouragement; and Ms. Carson Gleberman for careful reading of the manuscript.This study was supported by a research grant from Kanagawa Prefecture and theMinistry of

Health and Welfare, HIV Epidemiology Project.

References

1) Larder, B. A., Darby, G. & Richman, D. D.: HIV with reduced sensitivity tozidovudine (AZT) isolated duringprolonged therapy. Science, 243: 1731-1734, 1989.

2) Larder, B. A. & Kemp, S. D.: Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance tozidovudin (AZT). Science, 246: 1151-1158, 1989.

3) Boucher, C. A. B., Tersmette, M., Lange, J. M. A., Kellam, P., Goede, R. E. Y., Mulder, J. W., Darby, G., Goudsmit, J.& Larder, B. A.: Zidovudine sensitivity of human immunodeficiency viruses from high-risk symptom-free individ-uals during therapy. Lancet, 336: 585-590, 1990.

4) Barre-Sinoussi, F., Ghermann, J. C., Ray, F., Nugeyre, M. T., chamaret, S., Gruest, J., Dauguet, C. & Axler-Blin, C.:Isolation of T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS).Science, 220: 868-871, 1983.

5) Saito, T., Kondo, M., Ito, A., Nagao, T. & Imai, M.: Investigation of AZT sensitivity of HIV-1 isolated from

patients treated with AZT. JJA Inf D, 67: 311-314, 1993.6) Kondo, M, Saito, T., Ito, A., Oka, S., Hamamoto, K. & Imai, M.: Reverse transcriptase gene analysis of HIV-1

mutants cultured in the Presence of AZT. JJA Inf D, 67: 992-997, 1993.7) Kondo, M., Saito, T., Ito, A., Oka, S,,Takata, N. & Imai, M.: Mutation of HIV-1 RT gene isolated from patients

treated with AZT. JJA Inf D, 67: 185-189, 1993.8) St. Clair, M. H., Martin, J. L., Tudor-Williams, G., Bach, M. C., Vavro, C. L., King, D. M., Kellam, P., kemp, S. D. &

Larder, B. A.: Resistance to ddI and sensitivity to AZT induced by a mutationin HIV-1 reverse transcriptase.Science, 253: 1557-1559, 1991.



9) Sanger, F., Nicklen, S. & Coulson, A.R.: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad SciUSA, 74: 5463-5467, 1977.

10) Yarchoan, R., Berg, G., Brouwers, P., Fischel, M. A., Spitzer, A. R., Wichman, A., Grafman, J., Thomas, R. V., Safai,B., Brunetti, A., Perno, C. F., Schmidt, P.J., Larson, S. M., Myer, C. E. & Broders, S.: Response of humanimmunodeficiency-virus-associated neurological disease to 3'-azido-3'-deoxythymidine. Lancet. 1: 132-5, 1987.

AZT長期投与後出現したAZT耐性HIV-1分離株に見られる

逆転写酵素遺伝子の変異に関する研究

神奈川県衛生研究所・ウイルス部,横浜市立大学医学部細菌学講座

近藤真規子

要旨

6カ月以上3'-azido-3'-deoxythymidine(AZT)

を投与した患者9名からhuman immuno-

de丘ciencytype-1virus(HIV-1)を分離iし,AZT

耐性に関与する逆転写酵素(RT)遺伝子の4カ所

のアミノ酸(Asp67,Lys70,Thr215,Lys219)にい

て解析した.その結果,9名中7名にこれら4カ

所のアミノ酸いずれかが変異しているクローンが

検出された.これら7名から得られた合計41ク

ローンの内40クローン(97.6%)に215番アミノ酸

のTyrあるいはPheへの変異が認められ,この

変異は他の3カ所のアミノ酸の変異に比べ最も高

率であった.

また,AZT1μMの存在下で培養できたAZT

耐性株から得られた合計22個のクローンも4カ所

のアミノ酸いずれかが必ず変異しており,特に215

番アミノ酸の変異は全てのクローンに認められ

た.

次に一人の患者のAZT投与前から投与後にか

けて経時的にHIV-1を分離し,RT遺伝子を解析

した.AZT投与前に得られた分離株には4カ所の

アミノ酸いずれにも変異は認められなかったが,

投与2カ月後には70番アミノ酸がArgに変異し

たクローンが優勢となった.AZT投与6カ月以上

を経過するとほとんどのクローンで215番アミノ

酸がTyrに変異しており,これら分離株はAZT

1μM存在下で培養が可能であった.

215番アミノ酸の変異は,他の3カ所のアミノ酸

の変異が1コドン内1塩基置換であるのに対し2

塩基置換であり,比較的確率の低い変異にもかか

わらず,全てのAZT耐性株に認められることか

ら,このアミノ酸の変異はAZT耐性に最も重要

であることが確認された.


predominance of codon 215 mutation in reverse...

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