1. 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 , Republic of Korea
2 National Primate Research Center, KRIBB, Ochang, Chungbuk , Republic of Korea
3 Korea Bioinformation Center, KRIBB, Daejeon , Republic of Korea
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
(A)n
Cap
Viral genomic RNA
Viral spliced RNA
Proteins
Virus
Virus (Virus-like particle)
1.Receptor
interaction
2. Penetration
4. Reverse
Transcription
3. Uncoating
5. Integration
8. RNA transport
7. RNA processing
6.Transcription
9. Translation
10. Virus assembly
(Virus –like particles)
11. Budding U3 R U5
gag
pol
env
LTR
Solitary LTR
Recombination
RNA
Sample
RT-PCR
cloning
Sequencing
&
analysis
Luciferase
assay
Deletion
mapping
Subtype
identification
gag : coding for matrix and capsid proteins
pol : reverse transcriptase and integrase proteins
env : product viral envelope and transmembrane structures U5
gag
pol
env
PBS R U3
5’UTR
3’UTR
Results & Discussion
Identification of actively expressed PERV LTR elements
Luciferase reporter gene assay for 10 deletion mutant
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:
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: