Isolation and gene analysis of interferon alpha-resistant cell clones of the hepatitis C virus subgenome.
Hepatitis C virus (HCV) proteins appear to play an important role in IFN-resistance, but the molecular mechanism remains unclear. To clarify the mechanism in HCV replicon RNA harboring Huh-7 cells (Huh-9-13), we isolated cellular clones with impaired IFNalpha-sensitivity. Huh-9-13 was cultured for approximately 2 months in the presence of IFNalpha, and 4 IFNalpha-resistant cell clones showing significant resistances were obtained. When total RNA from clones was introduced into Huh-7 cells, the transfected cells also exhibited IFNalpha-resistance. Although no common mutations were present, mutations in NS3 and NS5A regions were accumulated. Transactivation of IFNalpha and IFNalpha-stimulated Stat-1 phosphorylation were reduced, and the elimination of HCV replicon RNA from the clones restored the IFNalpha signaling. These results suggest that the mutations in the HCV replicon RNA, at least in part, cause an inhibition of IFN signaling and are important for acquisition of IFNalpha resistance in Huh-9-13.
Noguchi
Tohru
T
Central Pharmaceutical Research Institute, Japan Tobacco Inc, Takatsuki, Osaka, Japan. toru.noguchi@ims.jti.co.jp
Otsubaki
Tomoko
T
Ando
Izuru
I
Ogura
Naoki
N
Ikeda
Satoru
S
Shimotohno
Kunitada
K
D045744:Cell Line, Tumor; D002999:Clone Cells; D024882:Drug Resistance, Viral; D005814:Genes, Viral / Q000235:genetics*; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology*; D009154:Mutation; D010766:Phosphorylation; D012367:RNA, Viral / Q000235:genetics; D012093:Replicon / Q000235:genetics; D050794:STAT1 Transcription Factor / Q000378:metabolism; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
D016898:Interferon-alpha; D012367:RNA, Viral; D050794:STAT1 Transcription Factor; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C494079:STAT1 protein, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase