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U3. U5. R. Comparative Transcription Promoter Activity of Different PERV LTR Subtypes in Human, Monkey and Pig cell lines Yi-Deun Jung 1 , Jae-Won Huh 1,2 , Dae-Soo Kim 3 , Hong-Seok Ha 1 and Heui-Soo Kim 1

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U3

U5

R

Comparative Transcription Promoter Activity

of Different PERV LTR Subtypes in

Human, 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 Korea

2 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

  • Abstract

Transcription 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.

  • Introduction

  • Materials & Methods

Virus (Virus-like particle)

Virus

11. Budding

1.Receptor

interaction

10. Virus assembly

(Virus –like particles)

9. Translation

Proteins

Cap

(A)n

(A)n

Cap

Viral genomic RNA

RNA

Sample

RT-PCR

cloning

Sequencing

&

analysis

Viral spliced RNA

4. Reverse

Transcription

8. RNA transport

7. RNA processing

6.Transcription

3. Uncoating

U3

U5

R

gag

pol

env

LTR

LTR

Recombination

5. Integration

2. Penetration

Luciferase

assay

Deletion

mapping

Solitary LTR

Subtype

identification

gag : coding for matrix and capsid proteins

pol : reverse transcriptase and integrase proteins

env : product viral envelope and transmembrane structures

3’UTR

5’UTR

  • Results & Discussion

U3

R

U5

U3

R

U5

gag

pol

env

U3

R

U5

  • Luciferase reporter gene assay for 10 deletion mutant

  • Identification of actively expressed PERV LTR elements

PBS

full

full

full

deletion

deletion

MAZR

C-ETS-1

U3-2

  • PERV LTR structure

TATA

IRF

HIF-1

STAT5A

GATA-4

U3

R

U5

606 bp

67 bp

26 bp

  • Transient transfection assay of PERV-LTR in different cell lines

  • HEK293

  • COS7

  • PK15

R

U5

  • Reference

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

  • 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


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