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 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.
Kolykhalov
A A
AA
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093, USA.
Mihalik
K
K
Feinstone
S M
SM
Rice
C M
CM
D020413:3' Untranslated Regions; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary; D004279:DNA, Viral; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D006801:Humans; D016296:Mutagenesis; D012367:RNA, Viral / Q000502:physiology*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D020413:3' Untranslated Regions; D003546:Cysteine Endopeptidases; D018076:DNA, Complementary; D004279:DNA, Viral; D004792:Enzyme Precursors; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus; C087036:NS-5 protein, hepatitis C virus; C107824:NS2-3 protease