Seng Lai Tan

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-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.

Inhibition of Double-Stranded RNA-Dependent Protein Kinase PKR by Vaccinia Virus E3: Role of Complex Formation and the E3 N-Terminal Domain

ABSTRACT The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2α (eIF2α). Vaccinia virus E3Lencodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2α, dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized, not augmented, by overexpressing the catalytically defective PKR-K296R protein containing functional DRBMs. Coimmunoprecipitation experiments showed that a majority of PKR in yeast extracts was in a complex with E3, whose formation was completely dependent on the dsRNA binding activity of E3 and enhanced by the N-terminal half of E3. In yeast two-hybrid assays and in vitro protein binding experiments, segments of E3 and PKR containing their respective DRBMs interacted in a manner requiring E3 residues Lys-167 and Arg-168. We also detected interactions between PKR and the N-terminal half of E3 in the yeast two-hybrid and λ repressor dimerization assays. In the latter case, the N-terminal half of E3 interacted with the kinase domain of PKR, dependent on E3 residue Trp-66. We propose that effective inhibition of PKR in yeast requires formation of an E3-PKR-dsRNA complex, in which the N-terminal half of E3 physically interacts with the protein kinase domain of PKR.

Subversion of Cell Signaling Pathways by Hepatitis C Virus Nonstructural 5A Protein via Interaction with Grb2 and P85 Phosphatidylinositol 3-Kinase

ABSTRACT Hepatitis C virus (HCV) sets up a persistent infection in patients that likely involves a complex virus-host interaction. We previously found that the HCV nonstructural 5A (NS5A) protein interacts with growth factor receptor-binding protein 2 (Grb2) adaptor protein and inhibits the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by epidermal growth factor (EGF). In the present study, we extended this analysis and investigated the specificity of the Grb2-NS5A interaction and whether the subversion of mitogenic signaling involves additional pathways. NS5A containing mutations within the C-terminal proline-rich motif neither bound Grb2 nor inhibited ERK1/2 activation by EGF, demonstrating that NS5A-Grb2 binding and downstream effects were due to direct interactions. Interestingly, NS5A could also form a complex with the Grb2-associated binder 1 (Gab1) protein in an EGF treatment-dependent manner. However, the NS5A-Gab1 association, which appeared indirect, was not mediated by direct NS5A-Grb2 interaction but was likely dependent on direct NS5A interaction with the p85 subunit of phosphatidylinositol 3-kinase (PI3K). The in vivo association of NS5A with p85 PI3K required the N-terminal, but not the C-terminal, region of NS5A. The downstream effects of the NS5A-p85 PI3K interaction included increased tyrosine phosphorylation of p85 PI3K in response to EGF. Consistent with this observation and the antiapoptotic properties of NS5A, we also detected enhanced tyrosine phosphorylation of the downstream AKT protein kinase and increased serine phosphorylation of BAD, a proapoptotic factor and an AKT substrate, in the presence of NS5A. These results collectively suggest a model in which NS5A interacts with Grb2 to inhibit mitogenic signaling while simultaneously promoting the PI3K-AKT cell survival pathway by interaction with p85 PI3K, which may represent a crucial step in HCV persistence and pathogenesis.

The cellular inhibitor of the PKR protein kinase, P58(IPK), is an influenza virus-activated co-chaperone that modulates heat shock protein 70 activity

P58IPK, a member of the tetratricopeptide repeat and J-domain protein families, was first recognized for its ability to inhibit the double-stranded RNA-activated protein kinase, PKR. PKR is part of the interferon-induced host defense against viral infection, and down-regulates translation initiation via phosphorylation of eukaryotic initiation factor 2 on the α-subunit. P58IPK is activated in response to infection by influenza virus, and inhibits PKR through direct protein-protein interaction. Previously, we demonstrated that the molecular chaperone heat shock protein 40 (hsp40) was a negative regulator of P58IPK. We could now report that influenza virus activates the P58IPKpathway by promoting the dissociation of hsp40 from P58IPKduring infection. We also found that the P58IPK-hsp40 association was disrupted during recovery from heat shock, which suggested a regulatory role for P58IPK in the absence of virus infection. The PKR pathway is even more complex as we show in this report that the molecular chaperone, hsp/Hsc70, was a component of a trimeric complex with hsp40 and P58IPK. Moreover, like other J-domain proteins, P58IPK stimulated the ATPase activity of Hsc70. Taken together, our data suggest that P58IPK is a co-chaperone, possibly directing hsp/Hsc70 to refold, and thus inhibit kinase function.

The Emerging Role of the Interferon-Induced PKR Protein Kinase as an Apoptotic Effector: A New Face of Death?

Recent research has thrown a spotlight on the interferon (IFN)-induced PKR protein kinase, implicating it as an important effector of apoptosis induced by several cellular stress conditions, including viral infection, cytokine treatment, and growth factor deprivation. In this review, we summarize the evidence for the role of PKR as a death accomplice and discuss how PKR might promote cell demise in light of current knowledge of the molecular mechanisms of apoptosis. Given its new found role and its established antiviral function, it is no wonder that PKR is a popular target for viral evasion of the host defense. PKR-dependent apoptosis may offer a novel cell-death pathway for specific manipulation in therapeutic strategies against apoptosis-related diseases.

Double-Stranded RNA-Independent Dimerization of Interferon-Induced Protein Kinase PKR and Inhibition of Dimerization by the Cellular P58IPK Inhibitor

ABSTRACT The interferon (IFN)-induced, double-stranded RNA-activated protein kinase (PKR) mediates the antiviral and antiproliferative actions of IFN, in part, via its translational inhibitory properties. Previous studies have demonstrated that PKR forms dimers and that dimerization is likely to be required for activation and/or function. In the present study we used multiple approaches to examine the modulation of PKR dimerization. Deletion analysis with the λ repressor fusion system identified a previously unrecognized site involved in PKR dimerization. This site comprised amino acids (aa) 244 to 296, which span part of the third basic region of PKR and the catalytic subdomains I and II. Using the yeast two-hybrid system and far-Western analysis, we verified the importance of this region for dimerization. Furthermore, coexpression of the 52-aa region alone inhibited the formation of full-length PKR dimers in the λ repressor fusion and two-hybrid systems. Importantly, coexpression of aa 244 to 296 exerted a dominant-negative effect on wild-type kinase activity in a functional assay. Due to its role as a mediator of IFN-induced antiviral resistance, PKR is a target of viral and cellular inhibitors. Curiously, PKR aa 244 to 296 contain the binding site for a select group of specific inhibitors, including the cellular protein P58IPK. We demonstrated, utilizing both the yeast and λ systems, that P58IPK, a member of the tetratricopeptide repeat protein family, can block kinase activity by preventing PKR dimerization. In contrast, a nonfunctional form of P58IPK lacking a TPR motif did not inhibit kinase activity or perturb PKR dimers. These results highlight a potential mechanism of PKR inhibition and define a novel class of PKR inhibitors. Finally, the data document the first known example of inhibition of protein kinase dimerization by a cellular protein inhibitor. On the basis of these results we propose a model for the regulation of PKR dimerization.

Interaction of the interferon-induced PKR protein kinase with inhibitory proteins P58IPK and vaccinia virus K3L is mediated by unique domains: implications for kinase regulation.

Expression of the double-stranded RNA-activated protein kinase (PKR) is induced by interferons, with PKR activity playing a pivotal role in establishing the interferon-induced antiviral and antiproliferative states. PKR is directly regulated by physical association with the specific inhibitor, P58IPK, a cellular protein of the tetratricopeptide repeat (TPR) family, and K3L, the product of the corresponding vaccinia virus gene. P58IPK and K3L repress PKR activation and activity. To investigate the mechanism of P58IPK- and K3L-mediated PKR inhibition, we have used a combination of in vitro and in vivo binding assays to identify the interactive regions of these proteins. The P58IPK-interacting site of PKR was mapped to a 52-amino-acid aa segment (aa 244 to 296) spanning the ATP-binding region of the protein kinase catalytic domain. The interaction with PKR did not require the C-terminal DNA-J homology region of P58IPK but was dependent on the presence of the eukaryotic initiation factor 2-alpha homology region, mapping to the 34 aa within the sixth P58IPK TPR motif. Consistent with other TPR proteins, P58IPK formed multimers in vivo: the N-terminal 166 aa were both necessary and sufficient for complex formation. A parallel in vivo analysis to map the K3L-binding region of PKR revealed that like P58IPK , K3L interacted exclusively with the PKR protein kinase catalytic domain. In contrast, however, the K3L-binding region of PKR was localized to within aa 367 to 551, demonstrating that each inhibitor bound PKR in unique, nonoverlapping domains. These data, taken together, suggest that P58IPK and K3L may mediate PKR inhibition by distinct mechanisms. Finally, we will propose a model of PKR inhibition in which P58IPK or a P58IPK complex binds PKR and interferes with nucleotide binding and autoregulation, while formation of a PKR-K3L complex interferes with active-site function and/or substrate association.

Regulation of mRNA Translation and Cellular Signaling by Hepatitis C Virus Nonstructural Protein NS5A

ABSTRACT The NS5A nonstructural protein of hepatitis C virus (HCV) has been shown to inhibit the cellular interferon (IFN)-induced protein kinase R (PKR). PKR mediates the host IFN-induced antiviral response at least in part by inhibiting mRNA translation initiation through phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). We thus examined the effect of NS5A inhibition of PKR on mRNA translation within the context of virus infection by using a recombinant vaccinia virus (VV)-based assay. The VV E3L protein is a potent inhibitor of PKR. Accordingly, infection of IFN-pretreated HeLa S3 cells with an E3L-deficient VV (VVΔE3L) resulted in increased phosphorylation levels of both PKR and eIF2α. IFN-pretreated cells infected with VV in which the E3L locus was replaced with theNS5A gene (VVNS5A) displayed diminished phosphorylation of PKR and eIF2α in a transient manner. We also observed an increase in activation of p38 mitogen-activated protein kinase in IFN-pretreated cells infected with VVΔE3L, consistent with reports that p38 lies downstream of the PKR pathway. Furthermore, these cells exhibited increased phosphorylation of the cap-binding initiation factor 4E (eIF4E), which is downstream of the p38 pathway. Importantly, these effects were reduced in cells infected with VVNS5A. NS5A was also found to inhibit activation of the p38-eIF4E pathway in epidermal growth factor-treated cells stably expressing NS5A. NS5A-induced inhibition of eIF2α and eIF4E phosphorylation may exert counteracting effects on mRNA translation. Indeed, IFN-pretreated cells infected with VVNS5A exhibited a partial and transient restoration of cellular and viral mRNA translation compared with IFN-pretreated cells infected with VVΔE3L. Taken together, these results support the role of NS5A as a PKR inhibitor and suggest a potential mechanism by which HCV might maintain global mRNA translation rate during early virus infection while favoring cap-independent translation of HCV mRNA during late infection.

P58IPK, a novel cochaperone containing tetratricopeptide repeats and a J-domain with oncogenic potential

Abstract. Tetratricopeptide repeats (TPRs) are loosely conserved 34-amino acid sequence motifs that have been shown to function as scaffolding structures to mediate protein-protein interactions. TPRs have been identified in a number of proteins with diverse functions and cellular locations. Recent studies suggest that individual TPR motifs can confer specificity in promoting homotypic and/or heterotypic interactions, often in a mutually exclusive manner. These features are best exemplified by the P58IPK protein, an influenza virus-activated cellular inhibitor of the PKR protein kinase, whose different TPR motifs mediate interactions with distinct proteins. P58IPK, which possesses cochaperone and oncogenic properties, represents a unique class of TPR proteins containing a J-domain. Here we review recent progress on the structural and functional characterization of P58IPK, and discuss the possible mechanisms by which P58IPK modulates PKR and induces tumorigenesis in view of present knowledge of TPR proteins and molecular chaperones.