Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: molecular mechanisms of kinase regulation.
The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2alpha phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.
Gale
M
M
Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195, USA.
Blakely
C M
CM
Kwieciszewski
B
B
Tan
S L
SL
Dossett
M
M
Tang
N M
NM
Korth
M J
MJ
Polyak
S J
SJ
Gretch
D R
DR
Katze
M G
MG
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D003001:Cloning, Molecular; D017931:DNA Primers; D019281:Dimerization; D004926:Escherichia coli; D015971:Gene Expression Regulation, Enzymologic; D016174:Hepacivirus / Q000187:drug effects / Q000472:pathogenicity / Q000502:physiology*; D007372:Interferons / Q000494:pharmacology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D014779:Virus Replication; D019892:eIF-2 Kinase / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D007372:Interferons; D019892:eIF-2 Kinase; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase