Comparative Transcription Promoter Activity Comparative Transcription Promoter Activity of Different PERV LTR Subtypes in of Different PERV LTR Subtypes in Human, Monkey and Pig cell linesHuman, Monkey and Pig cell lines

Yi-Deun Jung1, Jae-Won Huh1,2, Dae-Soo Kim3, Hong-Seok Ha1 and Heui-Soo Kim1

1 Division of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea2 National Primate Research Center, KRIBB, Ochang, Chungbuk 363-883, Republic of Korea

3 Korea Bioinformation Center, KRIBB, Daejeon 305-806, Republic of Korea

http://www.primate.or.kr

AbstractTranscription activity of 11 different long terminal repeat (LTR) subtypes of porcine endogenous retroviruse (PERV) are analyzed using human (HEK293), monkey (Cos7) and pig (PK15) kidney cell lines. They show the varied transcription regulation activity in different cell lines. However, E4 type (H2-2) shows the distinct promoter activity in the human kidney HEK293 cell line. We thus constructed the series of deletion mutants based on the boundary of U3, R and U5 region. Transient transfection assay of 10 deletion mutants show that U3 is crucial region for transcription regulation. By the computational approaches, 7 potential transcription factors are identified in U3 region. MAZR, HIF-1, STAT5A, and GATA-4 seem to be positive factors and c-ETS-1, CDP, and IRF should be acted by negative elements in human cell lines.

1. Patience C, Takeuchi Y, Weiss RA (1997) Infection of human cells by an endogenous retrovirus of pigs. Nat Med 3: 276-282.

2. Scheef G, Fischer N, Krach U, Tonjes RR (2001) The number of a U3 repeat box acting as an enhancer in long terminal repeats of polytropic replication-competent porcine endogenous retroviruses dynamically fluctuates during serial virus passages in human cells. J Virol 75: 6933-6940

Introduction

U5 gag pol env

PBS

RU3 U5RU3

5’UTR 3’UTR

gag : coding for matrix and capsid proteins

pol : reverse transcriptase and integrase proteins

env : product viral envelope and transmembrane structures

(A)nCap

Viral genomic RNA

(A)nCap

Viral spliced RNA

Proteins

Virus

Virus (Virus-like particle)

1.Receptorinteraction

2. Penetration

4. ReverseTranscription

3. Uncoating

5. Integration

8. RNA transport7. RNA processing6.Transcription

9. Translation

10. Virus assembly

(Virus –like particles)

11. Budding

U3 R U5gag pol env

LTR LTR

Solitary LTR

Recombination

U3 R U5

U3 R U5

Materials & Methods

RNASample

RT-PCR cloning

Sequencing&

analysis

Results & Discussion

U3 R U5TATA

67 bp 26 bp606 bp

PERV LTR structure

Identification of actively expressed PERV LTR elements

Transient transfection assay of PERV-LTR in different cell lines

Reference

Luciferase reporter gene assay for 10 deletion mutant

deletiondeletion

full full full

U3-2

R

U5

MAZR

HIF-1 STAT5A GATA-4

C-ETS-1

IRF

HEK293 COS7 PK15