Antiviral effects of antisense RNA on hepatitis C virus RNA translation and expression

We developed approaches using antisense RNA to inhibit hepatitis C virus (H CV) RNA translation and HCV core protein expression. An HCV genotype 1b cDN A comprising nt 1-1321 or a fusion construct consisting of HCV (nt 1-584) a nd luciferase cDNAs were inserted downstream of T7 and CMV promoter sequenc es and used to generate HCV RNA target molecules. Such constructs will prod uce HCV core or HCV core-luciferase fusion proteins in vitro or within tran sfected cells. Seven different antisense RNA constructs were designed to ta rget the highly conserved 5' region of HCV RNA at nt positions 1-402. For i n vitro experiments, synthesized HCV RNA target sequences and antisense RNA s were mixed at various molar ratios and subsequently translated in a rabbi t reticulocyte lysate system. In cell culture studies, the HCV core-lucifer ase fusion cDNA was co-transfected with antisense RNA-producing constructs into human hepatocellular carcinoma (HCC) cells. Luciferase activity in cel l lysates was measured to determine quantitatively antiviral effects within the cell. It was found that translation of HCV RNAs was efficiently inhibi ted by antisense RNA in vitro. The specificity of this inhibition was confi rmed using control target RNA sequences or nonrelevant antisense RNA constr ucts. Co-transfection studies demonstrated that antisense RNA inhibited HCV core-luciferase fusion protein expression by 41-57% in HuH-7 HCC cells. Th ese studies indicate that antisense RNA will find viral target RNA sequence s in HuH-7 cells and inhibit HCV RNA translation. More important, these stu dies have defined critical viral RNA target sequences susceptible to antise nse inhibitory effects within the cell. J. Med. Virol. 57:217-222, 1999. (C ) 1999 Wiley-Liss, Inc.