Comparative Transcription Analysis of

1. U3 U5 R c-Ets-1 ccAGGAAgttaataa CDP ccataCTTCTTGCTG HIF-1 agtACGTGcctc STAT5A ctctaggcacgaaaCTTCTtggaa STAT5A aacttcttGGAAAct GATA-4 AGATAacaggga IRF gaaagAGAAAa AREB6 Comparative Transcription Analysis 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 Porcine endogenous retroviruses (PERV) in the pig genome have the potential to act as harmful factors in xenotransplantation (pig-to-human). Long terminal repeats (LTRs) of the porcine endogenous retroviruse (PERV) elements show promoter activity that can affect human functional genes. 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 line. 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 RNA Sample RT-PCR cloning Sequencing & analysis Cap (A)n (A)n Cap Viral genomic RNA 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 Subtype identification Solitary LTR gag : coding for matrix and capsid proteins pol : reverse transcriptase and integrase proteins env : product viral envelope and transmembrane structures U3 R U5 U3 R U5 gag pol env U3 R U5 PBS 3’UTR 5’UTR • Results & Discussion • Deletion mutant structure • Transient transfection assay of PERV-LTR in different cell lines 1 2 3 4 5 6 7 8 9 10 • Luciferase reporter gene assay for 10 deletion mutant 1) Separated by structure 2) Separated by transcription factor (U3 region) • PERV LTR structure TATA U3 R U5 3) Separated by transcription factor ( deletion mutant 3 ) 606 bp 67 bp 26 bp • 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