Molecular and Cellular Biology, September 1998, p. 5208-5218, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Department of Microbiology,
Received 13 February 1998/Returned for modification 13 April
1998/Accepted 16 June 1998
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-2
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.
*
Corresponding author. Mailing address: Department of
Microbiology, School of Medicine, University of Washington, Box 357242, Seattle, WA 98195. Phone: (206) 543-8837. Fax: (206) 685-0305. E-mail:
honey{at}u.washington.edu.
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