Sequences at the 3'UTR of Hepatitis C virus (HCV) negative-strand (-)RNA play an important role in the initiation of positive-strand (+)RNA synthesis. However, the underlying mechanism in cellular context is still unclear. In this report, we designed several cDNA-based HCV-like minigenomes containing different mutations at the 5'UTR of (+)RNA. These (+)RNAs transcribed from the minigenomes in vitro were transfected into HCV replicon cells for producing (-)RNAs with deletions of different stem loops (SL) at the 3'-end. The results showed that expression of the antisense transgene from minigenome increased, when the minigenome containing deletion of SL-C1+D1+E1 at the 3'-end of (-)RNA was transfected into the HCV replicon cells compared to that of the full minigenome. The expression of the transgene from minigenome decreased using other mutant minigenomes containing deletions SL-A1, SL-A1+B1, and SL-A1+B1+C1 at the 3'-end of (-)RNA. Finally, the transgene from SL-C1+D1+E1 of (-)RNA using CMV promoter-driven minigenome was expressed at higher level than full minigenome in HCV replicon cell lines. These results indicated that the region of (-)RNA interacting with HCV replicase may locate in the SL-C1+D1+E1 region of (-)RNA.
Keywords: Hepatitis C virus; minigenome; RNA dependent RNA polymerase; replication.