Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3 ' nontranslated region are essential for virus replication in vivo

Hepatitis C virus (HCV) infection is a widespread major human health concer n, Significant obstacles in the study of this virus include the absence of a reliable tissue culture system and a small-animal model. Recently, we con structed full-length HCV cDNA clones and successfully initiated HCV infecti on in two chimpanzees by intrahepatic injection of in vitro-transcribed RNA (A, A, Kolykhalov et al., Science 277:570-574, 1997). In order to validate potential targets for development of anti-HCV therapeutics, we constructed six mutant derivatives of this prototype infectious clone. Four clones con tained point mutations ablating the activity of the NS2-3 protease, the NS3 -4A serine protease, the NS3 NTPase/helicase, and the NS5B polymerase. Two additional clones contained deletions encompassing all or part of the highl y conserved 98-base sequence at the 3' terminus of the HCV genome RNA. The RNA transcript from each of the six clones was injected intrahepatically in to a chimpanzee. No signs of HCV infection were detected in the 8 months fo llowing the injection. Inoculation of the same animal with nonmutant RNA tr anscripts resulted in productive HCV infection, as evidenced by viremia, el evated serum alanine aminotransferase, and HCV-specific seroconversion, The se data suggest that these four HCV-encoded enzymatic activities and the co nserved 3' terminal RNA element are essential for productive replication in vivo.