ABSTRACT

INTRODUCTION

REFERENCES

MATERIALS & METHODS

RESULTS & DISCUSSION

Hong-Seok Ha1, Jae-Won Huh1, Dae-Soo Kim2, Yun-Ji Kim1, Ja-Rang Lee1, Kung Ahn1, Byung-Wook Cho3, and Heui-Soo Kim1,2 1 Division of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea

2 PBBRC, Interdisciplinary Research Program of Bioinformatics, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea 3 Department of Animal Science, College of Life Sciences, Pusan National University,

Miryang 627-706, Republic of Korea

HTTP://WWW.PRIMATE.OR.KR

1. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang W, Miller W, Lipman DJ (1997) Gapped Blast and PSI-BLAST: a new generation of protein database search program. Nucleic Acids Res 25: 3389-3402.

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.

3. 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.

Porcine endogenous retroviruses (PERVs) represent a potential infectious risk in the pig-to-human transplantation. Solitary long terminal repeat (LTR) elements of the PERV specifically affect the replication properties of single viruses and adjacent genes. Using PCR, sequencing, and bioinformatic approach, 12 members of solitary PERV LTR elements in various loci were identified and analyzed in various Korean pigs. The promoter activity of solitary LTR elements belonging to the PERV-A and –B families in Korean pigs using a luciferase assay were examined. Potential negative and positive factors for the PERV transcription regulation are identified from the deletion mutant analysis. The solitary PERV LTR element recently integrated into the haploid genome showed less promoter activity than the older in diploid genome of pigs. Methylation analyses of those solitary LTR elements indicated that the ‘young’ solitary LTR element was hypomethylated, whereas the ‘old’ solitary LTR elements were hypermethylated in most tissues examined. Our data suggest that the LTR elements having high promoteractivity are hypermethylated to protect their biological activity in various tissues of pigs.

Identification and characterization of PERV LTR elements in various Korean pig genomes

1. Pig transplant donor with infectious endogenous provirus (PERV)

2. “Donation” of organ to human recopient

3. Production of PERV, infection of nearby host cells

4. Systemic spread Adaptiation of virus to human host

5. Induction of disease

6. Epidemic spread

GAG POL ENV

GAG POL ENV

Identification and characterization of PERV LTR elements in various Korean pig genomes

Cell culture and transient transfection assay

Molecular cloning and sequencing of PERV LTR elements

Methylation analysis

5’-GCGC-3’3’-CGCG-5’

HhaI

X 30

Denature

Anneal

Extension

Template DNA

94℃

Primer

DNA polymerase

55℃

72℃

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

S1S5 S1S5 S1

S1 S5 S1S5 S1

S7 S5 S2S5 S2S5 S2 S5 S2 S5 S2

S7 S6 S1S5 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

S7S5 S2 S5 S2S5 S2 S5 S2S5

S7 S5 S3 S5 S3S5 S3 S5 S3 S5 S2S3 S5 S3S5 S2 S5

S2S5 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

Conserved region

structure

Relative Luciferase Activity

(Fold of pGL-2 control)

KDP

KYP

KDP

0 2 4 6 8 10 12 14 16 18 20 22 24 26

KWP

Vector Only

KDP

KYP

KWP

KWP-2

KWP-1

KYP

KWP

YKP

PERV-A5

PERV-A6

PERV-A7

PERV-A8

PERV-X9

(A) HCT116 (B) Cos7Relative Luciferase Activity

(Fold of pGL-2 control)

KDP

KYP

KDP

0

KWP

Vector Only

KDP

KYP

KWP

KWP-2

KWP-1

KYP

KWP

YKP

PERV-A5

PERV-A6

PERV-A7

PERV-A8

PERV-X9

300 600 900 1200 1500 1800 2100 2400

promoter activity

MEF-2

PPAR

Oct-1

AREB6

AhR

v-ErbA

C-Myb

SMAD-3

E2F

NF-Y

Oct-1

Gfi

HFH-3

Muscle initiator sequence-20

HCT116

Cos7

Relative Luciferase Activity

(Fold of pGL-2 control)

Del 1-157

Del 1-207

Del 1-389

Del 1-480

Vector Only

Luc

Luc

Luc

Luc

Luc

+1

+1

+1

+1

+1

Luc

+1

None

0 2 4 6 8 1012 14 16 18 0 2 4 6 8 10 12 14

1.

1 2 3456 7 8 9 10 1112 13 14

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

Full

KDP deletion mutants

methylation