Hepatitis C virus replication is directly inhibited by IFN-alpha in a full-length binary expression system.
Hepatitis C virus (HCV) is a leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The absence of culture systems permissive for HCV replication has presented a major bottleneck to antiviral development. We sought to recapitulate the early steps in the life cycle of HCV by means of DNA-based expression of viral genomic sequences. Here we report expression of replicating HCV RNA by using a, to our knowledge, novel binary expression system in which cells were transfected with a T7 polymerase-driven full-length HCV cDNA plasmid containing a cis-acting hepatitis Delta ribozyme to control 3' cleavage, and infected with vaccinia-T7 polymerase. HCV genomic and replicative strand synthesis, in addition to protein synthesis, was detectable and depended on full-length HCV sequences. Moreover, the system was capable of generating HCV RNA quasispecies, consistent with the action of the low-fidelity HCV NS5B RNA polymerase. IFN-alpha, but not ribavirin, directly inhibited the viral replicative cycle in these cells, identifying the virus itself and not solely the immune system as a direct target of IFN action. The availability of a cell-based test for viral replication will facilitate screening of inhibitory compounds, analysis of IFN-resistance mechanisms, and analysis of virus-host cell interactions.
Chung
R T
RT
Gastrointestinal Unit, Cancer Center, and Hospital for Children, Massachusetts General Hospital, Boston, MA 02114, USA. rtchung@partners.org
He
W
W
Saquib
A
A
Contreras
A M
AM
Xavier
R J
RJ
Chawla
A
A
Wang
T C
TC
Schmidt
E V
EV
D000547:Amantadine / Q000494:pharmacology; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D017123:Bacteriophage T7 / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D005813:Genes, Synthetic; D016174:Hepacivirus / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology*; D008113:Liver Neoplasms / Q000473:pathology; D020319:Oligodeoxyribonucleotides, Antisense / Q000138:chemical synthesis / Q000235:genetics; D016133:Polymerase Chain Reaction; D016337:RNA, Catalytic / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012254:Ribavirin / Q000494:pharmacology; D013728:Terminator Regions, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects*
D000998:Antiviral Agents; D018076:DNA, Complementary; D016898:Interferon-alpha; D020319:Oligodeoxyribonucleotides, Antisense; D016337:RNA, Catalytic; D012367:RNA, Viral; D014764:Viral Proteins; D012254:Ribavirin; D000547:Amantadine