Abstract: Objective To investigate the roles of Domain I and Domain II of hepatitis C virus (HCV) 5’ untranslated region
(UTR) in the translation initiation activity of HCV 5’UTR in different host cell lines. Methods The eukaryotic expression
plasmid pCMVNCRLuc (pCN1), in which full-length HCV 5’UTR regulates firefly luciferase expression, was modified by
deleting Domain I and the downstream single-stranded sequence (43 bp in total) from the UTR (pCNl-d2), Domain I with the
downstream single-stranded sequence and Domain II (118 bp in total) from the UTR (pCNl-d3), or the total UTR (pCNl-d5).
The modified plasmids were transfected via liposome into different cell lines with pRL-TK plasmid co-transfected as the
normalization control. At 36 h after the transfection, the total cellular RNA was harvested for semi-quantitative RT-PCR, and
the relative expression activities of luciferase were assayed with a dual luciferase reporter gene assay system. The translation
initiation activities of the truncated HCV 5’UTRs in different translation systems were analyzed. Results Deletion of Domain I
and the downstream single-stranded sequence caused no significant changes of the translational activity of HCV 5’UTR in
Hela or C6 cells, but decreased the translational activity by 46% in L-02 cells and increased the translational activity by 46% in
293T cells. Deletion of both Domain I and Domain II resulted in decreased translational activity of HCV 5’UTR by 51% in HeLa
cells, but increased the translational activity by 40% in L-02 cells, 60% in C6 cells and 135% in 293T cells. Conclusions Domain
I and Domain II of HCV 5’UTR perform cell type-specific roles in HCV IRES-driven translation initiation.