Gale M, Blakely C M, Kwieciszewski B, Tan S L, Dossett M, Tang N M, Korth M J, Polyak S J, Gretch D R, Katze M G
Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195, USA.
Mol Cell Biol. 1998 Sep;18(9):5208-18. doi: 10.1128/MCB.18.9.5208.
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.
PKR蛋白激酶是干扰素诱导的细胞抗病毒和抗增殖反应的关键组成部分。最近的证据表明，丙型肝炎病毒（HCV）的非结构5A（NS5A）蛋白可在体内抑制PKR功能，这可能使HCV逃避干扰素的抗病毒作用。NS5A提供了一个独特的工具，可用于研究PKR调控的分子机制，因为NS5A中一个称为干扰素敏感性决定区（ISDR）的区域内的突变与HCV对干扰素抗病毒作用的敏感性相关。在本研究中，我们研究了NS5A介导的PKR调控机制以及ISDR突变对这一调控过程的影响。我们观察到，NS5A的ISDR虽然是必需的，但不足以与PKR相互作用；我们发现，在ISDR羧基端还需要另外26个氨基酸（aa）才能形成NS5A-PKR复合物。相反，我们将NS5A的结合定位在PKR的244至296位氨基酸内，该区域最近被认为是PKR二聚化结构域。与这一观察结果一致，我们发现来自干扰素抗性HCV 1b基因型的NS5A在体内破坏了激酶二聚化。NS5A介导的PKR二聚化破坏导致PKR功能受到抑制，并抑制了PKR介导的eIF-2α磷酸化。引入多个ISDR突变消除了NS5A在哺乳动物细胞中与PKR结合以及在酵母功能试验中抑制PKR的能力。这些结果表明，NS5A的PKR结合区域内的突变，包括ISDR内的突变，可破坏NS5A-PKR相互作用，这可能使HCV对干扰素的抗病毒作用敏感。我们提出了一个NS5A对PKR调控的模型，这可能对HCV的治疗策略具有重要意义。
