Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: Molecular mechanisms of kinase regulation

Michael Gale, Collin M. Blakely, Bart Kwieciszewski, Seng Lai Tan, Michelle Dossett, Norina M. Tang, Marcus J. Korth, Stephen J. Polyak, David R. Gretch, Michael G. Katze

Research output: Contribution to journalArticlepeer-review

Abstract

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 NSSA-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-2α 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.

Original languageEnglish (US)
Pages (from-to)5208-5218
Number of pages11
JournalMolecular and Cellular Biology
Volume18
Issue number9
DOIs
StatePublished - 1998
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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