Interferon alpha-2b inhibits negative-strand RNA and protein expression from full-length HCV1a infectious clone.
We have established a T7-based model system for hepatitis C virus (HCV) 1a strain, which involves the use of a replication-defective adenovirus that carries the gene for T7 RNA polymerase and a transcription plasmid containing full-length HCV cDNA clone. To facilitate high-level expression of HCV, sub-confluent Huh7 cells were first infected with adenovirus containing the gene for the T7 RNA polymerase and then transfected with the transcription plasmid. As a negative control, part of NS5B gene of this clone was deleted which abolishes the HCV RNA-dependent RNA polymerase and prevents replication of viral RNA. This model produces high levels of structural (core, E1, E2) and nonstructural proteins (NS5), which were detected by Western blot analysis and immunofluorescence assay. Negative-strand HCV RNA was detected only in the wild-type clone in the presence of actinomycin D, and no RNA was detected with the NS5B deleted mutant control. As a practical validation of this model, we showed that IFN alpha-2b selectively inhibits negative-strand RNA synthesis by blocking at the level of protein translation. The inhibitory effect of IFN alpha-2b is not due reduction of transcription by T7 polymerase or due to intracellular degradation of HCV RNA. This in vitro model provides an efficient and reliable means of assaying negative-strand RNA, protein processing, and testing the antiviral properties of interferon.
Prabhu
Ramesh
R
Department of Pathology and Laboratory Medicine, Tulane University Health Science Center, New Orleans, LA 70112, USA.
Joshi
Virendra
V
Garry
Robert F
RF
Bastian
Frank
F
Haque
Salima
S
Regenstein
Fredric
F
Thung
Swan
S
Dash
Srikanta
S
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000494:pharmacology*; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002679:Pan troglodytes; D014176:Protein Biosynthesis / Q000187:drug effects; D012367:RNA, Viral / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D014407:Tumor Cells, Cultured; D014759:Viral Envelope Proteins / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000998:Antiviral Agents; D012321:DNA-Directed RNA Polymerases; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C062210:bacteriophage T7 RNA polymerase; C087036:NS-5 protein, hepatitis C virus