Study On The Promoter Activity In The HCV 5'UTR Sequence

As a Flaviviridace family member,HCV possesses a 9.6kb positive-sense ,ssRNA genosome. The HCV genosome consists of highly conserved 5'-and 3'-nontranslated regions(UTR),and a large open reading frame(ORE): 5'UTR-C-E1-E2/NS1-NS2-NS3-NS4-NS5-3'UTR.Although HCV has been identified since 1989,a cellular model system allowing for the efficient replication of the entire viral genome is not available,and the detailed replication and translation mechanism still remain unknown .HCV 5'UTR is highly conserved and is shown to have an important regulating effect on both the virus replication and translation.In this study,the plasmids carrying HCV 5'UTR cDNA sequence and the reported genes were constructed and HCV 5'UTR cDNA was found to have promoter activity and this promoter activity is available in various cells ;DomainⅢwas the core structure . Objectives : To investigate the promoter activity of the cDNA sequence corresponding to HCV 5'UTR in eukaryotic cells, consequently to reveal the regulation mechanism for HCV replication.Methods:pGL3-5'UTR and pGL3-a-5'UTR were constructed by substitution of SV40 promoter with HCV 5'UTR cDNA or its antisense sequence. In the new plasmids, reporter gene luciferase is driven by HCV 5'UTR cDNA or its antisense sequence, repectively. Then pGL3-5'UTR and pGL3-a-5'UTR were transfected into HepG2 cells and the firefly luciferase activities and mRNA were detected.The 5'UTR sequence was also cloned into pEGFP-Enhancer to make pEGFP-5'UTR and pEGFP-a-5'UTR,and then the GFP expression was confirmed by fluorescence microscopy.Results:pGL3-5'UTR had an obvious luciferase activity whereas pGL3-a-5'UTR had nearly no luciferase activity.The former also had a high level of luciferase mRNA while the latter could not be detected.An intense green fluorescence expression was observed in the cells transfected with the plasmid pEGFP-5'UTR .Conclusions:The cDNA sequence corresponding to HCV 5'UTR possessed a promoter activity in HepG2 cells. Objective:To identify the promoter domain corresponding to the secondary structure of the HCV 5'UTR.Method:Based on the plasmid pGL3-5'UTR, a set of deletion mutants plasmid : pGL3-5'UTR1,with domain II,III,IV;pGL3-5'UTR2,with domain I,II,III;pGl3-5'UTR3,with domain III and IV;pGl3-5'UTR4,with domain I and II,were constructed respectively,and subsequently transfected into hepG2 cell .The firefly luciferase activities expressed by the plasmids and the luciferase mRNA were measured then.Results : The relative luciferase activity associated with the pGL3-5'UTR,pGL3-5'UTR1,pGL3-5'UTR2, pGL3-5'UTR3 and pGL3- 5'UTR4,Were 5.94±0.69, 9.45±2.03, 2.72±0.79, 2.64±0.79, 0.32±0.06, respectively.The mRNA levels of luciferase activity were in according with the luciferase activity .Conclusion: The DNA sequence of stem-loop III was the core structure and the domain I ,II and IV had the regulating effects. Objective: To investigate the promoter activities of HCV 5'UTR cDNA in cell lines derived from various tissues, and to reveal whether these cells can provide efficient accessory factors for this promoter sequence.Methods: pGL3-5'UTR containing full sequence as the promoter for reporter gene luciferase was transfected into human hepatocytes and hepatoma cell line HepG2, human embryonic kidney cell line HEK293, hunan liver cell line L02 and cervical cancer cell line HeLa to express firefly luciferase. Then the firefly luciferase expressed in different cells were measured and to be compared each other.Results: The reporter gene luciferase can be expressed in different degrees in the four kinds of cell lines. And ranked by the firefly luciferase activity from the highest to the lowest, the order is L02, HepG2, HEK293 and HeLa , with the percents 23.42%,20.64%,10.99% and 9.09% , respectively, compared to the firefly luciferase promoted by SV40 promoter.Conclusion: Cells derived from various tissues can provide efficient accessory factors for HCV 5'UTR sequence when it acts as a promoter, and hepatic cells maybe the most suitable for it. This results accord with the clinical feartures of HCV .