Determinants of hepatitis C translational initiation in vitro, in cultured cells and mice

Hepatitis C virus (HCV) is an RNA virus infecting 1 in every 40 people worldwide. Development of new therapeutics for treating HCV has been hampered by the lack of small-animal models. We have adapted existing hydrodynamic transfection methods to optimize the delivery of RNAs to the cytoplasm of mouse liver cells in vivo. Transfected HCV genomic RNA failed to replicate in mouse liver, suggesting a post-entry block to viral replication. Real-time imaging of HCV internal ribosome entry site (IRES) firefly luciferase reporter mRNA translation in living mice demonstrated that the HCV IRES was functional in mouse liver. We then used this system as a model for studying HCV RNA translation in mice. We compared translation by several mutant HCV IRES variants in cell lysates, cultured cells, and mouse liver. We measured the contribution to translation of a cap, HCV 3'-untranslated region (UTR), poly(A) tail, domains II, IIIb, IIIabc, IIIabcd, IIId, and the initiator codon. Efficient translation required a 3'-UTR in mice and HeLa cells, but not in rabbit reticulocyte lysates. Translational regulation of transfected RNAs was stringent in mice. The method we describe could be useful for studies in mice of antisense or ribozyme inhibitors targeting the IRES as well as other RNA biochemical studies in vivo.