Hepatitis C virus (HCV) infection is a widespread major human health concern. Significant obstacles in the study of this virus include the absence of a reliable tissue culture system and a small-animal model. Recently, we constructed full-length HCV cDNA clones and successfully initiated HCV infection 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 contained 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 highly 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 into a chimpanzee. No signs of HCV infection were detected in the 8 months following the injection. Inoculation of the same animal with nonmutant RNA transcripts resulted in productive HCV infection, as evidenced by viremia, elevated serum alanine aminotransferase, and HCV-specific seroconversion. These data suggest that these four HCV-encoded enzymatic activities and the conserved 3 terminal RNA element are essential for productive replication in vivo.Prior to the development of specific blood donor screening assays, hepatitis C virus (HCV) was the major cause of transfusion-associated hepatitis (see reference 25 for a review). While transfusion-associated HCV infections are rare, about 30,000 new cases of hepatitis C are estimated to occur in the United States each year. HCV is not easily cleared by the host's immunological defenses; as many as 85% of the people infected with HCV become chronically infected. Many of these persistent infections result in chronic liver disease, including cirrhosis and hepatocellular carcinoma (24). There are an estimated 170 million HCV carriers worldwide, and HCV-associated end-stage liver disease is now the leading cause of liver transplantation. In the United States alone, hepatitis C is responsible for 8,000 to 10,000 deaths annually, and without effective intervention, that number is predicted to triple in the next 10 to 20 years. There is no vaccine to prevent hepatitis C infection. Prolonged treatment of chronically HCV-infected patients with interferon or interferon plus ribavirin is the only currently approved therapy, but it results in a sustained response in fewer than 50% of the cases (37, 46). HCV belongs to the family Flaviviridae, which comprises three genera of small enveloped positive-strand RNA viruses (see reference 47 and references therein). The 9.6-kb genome of HCV consists of a long open reading frame (ORF) flanked by 5Ј and 3Ј nontranslated regions (NTRs). The HCV 5Ј NTR is 341 nucleotides in length and functions as an internal ribosome entry site for cap-independent translation initiation (34). The HC...
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