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We describe an infectious molecular clone of a Japanese genotype 1b strain of hepatitis C virus (HCV-N). The molecularly cloned sequence of HCV-N was compared with alignments of other HCV sequences, leading to the identification of 15 unique, nonconservative amino acid substitutions within the HCV-N open reading frame (ORF). These were repaired to the consensus genotype 1b residue, and the infectivity of RNA transcribed from the repaired clone was assessed by intrahepatic inoculation of a chimpanzee. Viral RNA was first detected in the serum of this chimpanzee 3 weeks following inoculation, and was intermittently present over the next 14 weeks. A strong and persistent anti-HCV serological response developed 13 weeks following inoculation, with seroconversion in the recombinant immunoblot assay (RIBA). A weaker, transient serological response, characterized by seroconversion in a thirdgeneration enzyme-linked immunosorbent assay (ELISA) but not RIBA, occurred between weeks 1 and 5. This may have represented an anamnestic response to HCV antigens translated directly from the intrahepatically inoculated RNA, because the animal previously had undergone 2 unsuccessful attempts at rescue of HCV by intrahepatic RNA inoculation. There was neither biochemical nor histological evidence of liver disease. Although this is within the range of expected outcomes in an HCV-naive chimpanzee, prior immunologic priming may have modified the infection in this animal. The HCV-N clone is the first infectious molecular clone of HCV that is comprised entirely of genotype 1b sequence, and it contains an ORF sequence that is significantly divergent from that of a previously described genotype 1a/1b chimera. (HEPATOLOGY 1999;30:316-324.)Unlike other human hepatitis viruses, the majority of immunocompetent persons who become infected with hepatitis C virus (HCV) are unable to clear the virus and go on to develop persistent infection. 1,2 Such infections are frequently associated with active necroinflammatory liver disease, and eventually result in cirrhosis in 20% to 30% of individuals. 2 These individuals are also at risk for the development of hepatocellular carcinoma. 2 Thus, it is not surprising that HCV is the most important infectious cause of chronic liver disease in the United States and in many other countries. Despite the fact that chronic hepatitis C is increasingly recognized as an important disease, treatment options remain very limited. Even the most efficacious treatment, the combination of interferon with ribavirin, 3 results in a lack of sustained response in at least one half of all persons receiving therapy. There is an urgent need for better therapeutics. However, antiviral discovery efforts are severely restricted by the absence of a cell culture system that supports the efficient replication of HCV, as well as the lack of a small-animal model. Overcoming these limitations to hepatitis C research efforts will likely require a greater understanding of the molecular mechanisms involved in HCV replication. While it is pos...

GB virus B (GBV-B) is a recently discovered hepatotropic flavivirus that is distantly related to hepatitis C virus (HCV).We show here that translation of its polyprotein is initiated by internal entry of ribosomes on GBV-B RNA. We analyzed the translational activity of dicistronic RNA transcripts containing wild-type or mutated 5 nontranslated GBV-B RNA (5NTR) segments, placed between the coding sequences of two reporter proteins, in vitro in rabbit reticulocyte lysate and in vivo in transfected BT7-H cells. We related these results to a previously proposed model of the secondary structure of the GBV-B 5NTR (M. Honda, et al. RNA 2:955-968, 1996). We identified an internal ribosome entry site (IRES) bounded at its 5 end by structural domain II, a location analogous to the 5 limit of the IRES in both the HCV and pestivirus 5NTRs. Mutational analysis confirmed the structure proposed for domain II of GBV-B RNA, and demonstrated that optimal IRES-mediated translation is dependent on each of the putative RNA hairpins in this domain, including two stem-loops not present in the HCV or pestivirus structures. IRES activity was also absolutely dependent on (i) phylogenetically conserved, adenosine-containing bulge loops in domain III and (ii) the primary nucleotide sequence of stem-loop IIIe. IRES-directed translation was inhibited by a series of point mutations predicted to stabilize stem-loop IV, which contains the initiator AUG codon in its loop segment. A reporter gene was translated most efficiently when fused directly to the initiator AUG codon, with no intervening downstream GBV-B sequence. This finding indicates that the 3 limit of the GBV-B IRES is at the initiator AUG and that it does not require downstream polyprotein-coding sequence as suggested for the HCV IRES. These results show that the GBV-B IRES, while sharing a common general structure, differs both structurally and functionally from other flavivirus IRES elements.