Long terminal repeats of porcine endogenous retrovirusesin Sus scrofa

Abstract

Yun-Ji Kim1 Jae-Won Huh1, Byung-Wook Cho2, Dae-Soo Kim3, Hong-Seok Ha1, Ja-Rang Lee1, Kung Ahn1, and Heui-Soo Kim1,3

1 Division of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea2 Department of Animal Science, College of Life Sciences, Pusan National University,Miryang 627-706, Republic of Korea

3 PBBRC, Interdisciplinary Research Program of Bioinformatics, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea http://www..primate.or.kr

Introduction Materials & Methods

Results & Discussion

Using PCR, sequencing, and bioinformatic approaches with the genomic DNAs of Korean pigs (domestic, wild, and hybrid with Yorkshire), twelve solitary PERV long terminal repeat elements were identified and analyzed. Structure analysis of the LTR elements indicated that they have different repeat sequences in the U3 region. The PERV-A6-KWP1 and –KWP2 elements bear seven and eight 39 bp repeats, respectively. The R region of the PERV LTR elements was highly conserved in pig and mouse genomes, suggesting that they seem to have originated from a common exogenous viral element, and then independently evolved throughout the course of mammalian evolution.

1.Denner J, Specke V, Thiesen U, Karlas A, Kurth R (2003) Genetic alteration of the long terminal repeat of an ecotropic porcine endogenous retrovirus during passage in human cells. Virology 314: 125-133.

2. Akiyoshi DE, Denaro M, Zhu S, Greenstein JL, Banerjee P, Fishman JA (1998) Identification of a full-length cDNA for an endogenous retrovirus of miniature swine. J Virol 72: 4503-4507.

PCR

ElectrophoresisGel purification

Transformation1

Transformation2

Ligation

Inoculation

Plasmid DNAIsolation

Gene Cloning

Genomic DNAs& cDNA

PCR & RT-PCR

Bioinformatics

References

KWP,KDP,KDP×KYP

QIAEX II gel extraction kit(Qiagen)High Pure plasmid isolation kit

(A)

Conserved region

PERV-A5

U3 R U5

Group1

Tandem repeated region

PERV-A(Repbase)

PERV-B(Repbase)

PERV-A6

PERV-A7(KYP, KDP, KWP)

PERV-A8(KYP, KDP, KWP)

LTR-IS(Repbase)

Mouse

S1 S5 S1 S5 S1

S1 S5 S1 S5 S1

S7 S5 S2 S5 S2 S5 S2 S5 S2 S5 S2

S7 S6 S1 S5 S2

S7 S5 S2

S1 S5 S1

1 73 74 109 110 246 343 373 402 484

1 73 74 109 110 246 343 373 402 481

1 52 53 139 140 175 176 312 370 406 429 508

1 52 77 162 163 198 199 335 550 580 609 688

1 52 77 163 214 249 250 385 473 503 532 612

1 52 77 162 163 198 199 335 395 425 454 533

339 468

(KYP, KDP, KWP)PERV-A5

PERV-A6(KWP1)

PERV-A6(KWP2)

Group1a

S1 S5 S1 S5 S1

S7 S5 S2 S5 S2 S5 S2 S5 S2 S5

S7 S5 S3 S5 S3 S5 S3 S5 S3 S5 S2S3 S5 S3 S5 S2 S5

S2 S5 S2 S5 S2

1 52 53 139 140 175 176 312 409 439 468 547

1 52 77 162 163 198 199 335 628 658 687 766

1 52 77 162 163 198 199 335 667 697 726 805

Group2PERV-X9(KYP, KDP, KWP)

PERV-X10

S5 S8 S1 S9

S5 S8 S1 S9

1 52 53 90 91 131 132 215 218 253 254 402 481 501 512 541 620

1 52 53 90 91 131 132 215 239 274 275 423 502 522 533 562 641

Deleted region: GCCAGTAA

S4

S4

: GAGCCCTAACTCCAGCTTCCTAAA: CTCTGTATGAACTAGGTGAAAGGACGTAAAATAGGCCCTTGAATGCGTG: TGAGATAACAGGGAAAAGGGTT

S1 : TATTTTAAAATGATTGGT (Original 18bp repeat)

S5 : CCACGGAGCGCGGGCTCTCGA (Original 21bp repeat)

S7 : TGTAGGAAAAATGATTGGT (Subtype of 18bp repeat)

S3 : TGTTTTAAAACGATTGGT (Subtype of 18bp repeat)S2 : TGTTTTAAAATGATTGGT (Subtype of 18bp repeat)

S6 : CCACGGAGCGCA (Subtype of 21bp repeat)

S8 : AGTTTTGAATTGACTGGTTTGTGA (24bp, C type)S9 : TTGTAAAAGCGCGGGCTTG (19bp, C type)

S4 : TTAAAATTAATTGGT (Subtype of 18bp repeat)

: ATAAAA (TATA signal): cap site

(B)

4677-46985'-CATTTCTACTGAGCCACAACAG-3'PERV-X10-ASCT009542

3331-33521369

5'-CCTGAAAACTGCACTCTCCTCT-3'PERV-X10-S

30524-305445'-AGCAGGTTTAGGTTCACAGCA-3'PERV-X9-ASCT955978

31434-31453930

5'-AGGAGTGGGTTCCAGGTTTC-3'PERV-X9-S

24380-243995'-CTTGCATACTTGGGCTTGTG-3'PERV-A8-ASCT827949

25321-25340961

5'-GGAGGGTAGGACACAGTGGA-3'PERV-A8-S

17097-171165'-TGCTTTCACAAGTATTCATCCA-3'PERV-A7-ASCT797462

17981-18000904

5'-CTCAGTGGGTTGGGGTTCT-3'PERV-A7-S

118450-1184695'-CCCCAAATCACTCACGAGAA-3'PERV-A6-ASAC138167

117570-117589899

5'-CGTATCCAATCACCTGCATC-3'PERV-A6-S

124785-1247665'-TATTTCCATCCCTGAACCCA-3'PERV-A5-ASCT737311

125513-125494748

5'-AAGCCAATCTCCCTTCTTCC-3'PERV-A5-S

　　　　　Acession no.LocationPCR product size (bp)Sequences (5'-3')Primer

　　　　　

Table 1. PCR primers used for amplification of PERV LTR element in Korean pigs

Sus scrofaX10CT009542 (722 bp)PERV-X10-GenBamk

Hybrid (KDPⅹYorkshire)X9AB275600 (701 bp)PERV-X9-KYP

Sus scrofaX9CT955978 (701 bp)PERV-X9-GenBamk

Korean wild pigA8AB275599 (609 bp)PERV-A8-KWP

Korean domestic pigA8AB275598 (609 bp)PERV-A8-KDP

Hybrid (KDPⅹYorkshire)A8AB275597 (609 bp)PERV-A8-KYP

Sus scrofaA8CT827949 (609 bp)PERV-A8-GenBamk

Korean wild pigA7AB275596 (694 bp)PERV-A7-KWP

Korean domestic pigA7AB275595 (694 bp)PERV-A7-KDP

Hybrid (KDPⅹYorkshire)A7AB275594 (694 bp)PERV-A7-KYP

Sus scrofaA7CT797462 (694 bp)PERV-A7-GenBamk

Korean wild pigA6AB275593 (794 bp)PERV-A6-KWP2

Korean wild pigA6AB275592 (833 bp)PERV-A6-KWP1

Sus scrofaA6AC138167 (762 bp)PERV-A6-GenBamk

Korean wild pigA5AB275591 (628 bp)PERV-A5-KWP

Korean domestic pigA5AB275590 (628 bp)PERV-A5-KDP

Hybrid (KDPⅹYorkshire)A5AB275589 (628 bp)PERV-A5-KYP

Sus scrofaA5CT737311 (589 bp)PERV-A5-GenBamk

　　　　SourcesLTR TypesAcession no. (LTR size)Clone

　　　　

Table 2. Clone information of PERV LTR elements in Korean pigs

BLAST,MEGA2

Fig. 1. Schematic representation of various PERV LTR elements (A) and consensus sequences of figured boxes (B). Open boxes indicate the conserved regions, and figured boxes indicate the specific sequences. Closed boxes indicate the different consensus sequences. Different boxes in tandem repeated region indicate the repeat number of tandem repeat sequences. Numbers indicate the specific position in individual LTR sequences. The underlined nucleotide sequences indicate the binding site for the transcription factor, NF-Y. The structures of solitary PERV LTR elements (PERV-A5, -A6, -A7, -A8, -X9, -X10) are derived from Korean pigs, KYP-hybrid of Korean domestic pig × Yorkshire, KDP-Korean domestic pig, and KWP-Korean wild pig (see also Table 2). KWP1 and KWP2 indicate the allele difference in the same individual. Group 2 is PERV-C LTR element and Group 1a is defined by the different copy number of tandem repeated sequences in our sequencing analysis. In the case of the mouse LTR-IS element, only the conserved region (open gray box) has high homology with PERV LTR elements...…………………………………………………………………….