Effects of mutation in hepatitis C virus nonstructural protein 5A on interferon resistance mediated by inhibition of PKR kinase activity in mammalian cells.
The IFN-induced double-stranded RNA (dsRNA)-activated protein kinase PKR is one of the key molecules in the antiviral effects of IFN. To clarify the effects of hepatitis C virus nonstructural protein 5A (NS5A) on antiviral activity of IFN, in particular on PKR kinase activity, in mammalian cells, we established inducible NS5A-expressing cell lines derived from human osteosarcoma (Saos-2). The cells expressing NS5A derived from an IFN-resistant clone (NS5A-lb) that interacted with endogenous PKR in vitro, showed a suppressive effect on IFN function as determined by interference with vesicular stomatitis virus (VSV) infection, whereas NS5A (NS5A-2a) from an IFN-sensitive clone did not block the antiviral effect of IFN. A mutant with deletion of the IFN sensitivity determining region (ISDR) in NS5A-1b (NS5A-AISDR) also interacted with PKR and suppressed its activity in vitro. However, neither NS5A-2a nor the C-terminal truncated mutant of NS5A-1b (NS5A-deltaC) blocked PKR activity. These observations confirmed the previous report that the inhibitory effect of NS5A on IFN activity is mediated at least in part by the repression of PKR. In addition, we showed that IFN sensitivity was determined not only by the ISDR but that the involvement of the C-terminal region of NS5A-1b is important for the suppression of PKR activity.
Noguchi
T
T
Central Pharmaceutical Research Institute, Japan Tobacco Inc., Takatsuki, Osaka.
Satoh
S
S
Noshi
T
T
Hatada
E
E
Fukuda
R
R
Kawai
A
A
Ikeda
S
S
Hijikata
M
M
Shimotohno
K
K
D000595:Amino Acid Sequence; D015153:Blotting, Western; D024882:Drug Resistance, Viral / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D016174:Hepacivirus / Q000235:genetics / Q000276:immunology*; D006526:Hepatitis C / Q000188:drug therapy / Q000276:immunology* / Q000821:virology; D006801:Humans; D016898:Interferon-alpha / Q000276:immunology / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014407:Tumor Cells, Cultured; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000276:immunology*; D019892:eIF-2 Kinase / Q000037:antagonists & inhibitors / Q000276:immunology*; D012324:RNA-Dependent RNA Polymerase
D015852:Eukaryotic Initiation Factor-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D019892:eIF-2 Kinase; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase