Control of PKR Protein Kinase by Hepatitis C Virus Nonstructural 5A Protein: Molecular Mechanisms of Kinase Regulation

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 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.

Binding and Nuclear Relocalization of Protein Kinase R by Human Cytomegalovirus TRS1

“…For example, poliovirus infection results in the degradation of PKR (6,7). Several viruses block activation of PKR by sequestering dsRNA or by interfering with PKR dimerization or enzymatic activity (26,27,32,64,65,72). The herpes simplex virus ␥34.5 gene product blocks the effects of PKR by cooperating with cellular protein phosphatase 1␣ to dephosphorylate eIF2␣-phosphate (37).…”

Interferon alpha (IFNα)-induced TRIM22 interrupts HCV replication by ubiquitinating NS5A

“…Furthermore, experimental evidence suggests that the function of NS5A includes abrogation of STAT-1 translocation, 29 suppression of STAT-1 phosphorylation 30 and repression of the protein kinase R protein kinase. [31][32][33] Because of its important function in controlling HCV replication, many molecules have been designed to target NS5A, some of which have exhibited promising treatment outcomes in clinical research. 34,35 Other potential mechanisms of the anti-HCV effects of TRIM22 should be evaluated in future research.…”

Immune responses during acute and chronic infection with hepatitis C virus

“…HCV core protein also inhibits the function of ISGF3 by inducing expression of suppressor of cytokine signaling 3 (SOCS-3) [104; 113; 118; 119] and blocks DNA binding by ISGF3 [120]. NS5A represses activation of PKR [121; 122; 123] through binding to PKR and inhibiting formulation of the PKR dimer [122]. NS5A also blocks the PKR-dependent activation of IRF-1 [123].…”