Norovirus RNA-Dependent RNA Polymerase Is Phosphorylated by an Important Survival Kinase, Akt

Eden, John‐Sebastian; Sharpe, Laura J.; White, Peter A.; Brown, Andrew

doi:10.1128/jvi.05562-11

Cited by 22 publications

(32 citation statements)

References 29 publications

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“…Indeed, growing numbers of virus-encoded phosphoproteins implicated in viral pathogenesis, virion assembly, and genome replication have been identified recently (2)(3)(4)(5)(6)(7). In plus-strand RNA viruses, including hepatitis C virus (HCV), the viral RNA genome is replicated by virus-encoded RNA-dependent RNA polymerases (RdRps) (7), and their phosphorylation has been suggested to be functionally linked to viral genome replication (6)(7)(8)(9)(10)(11)(12)(13).…”

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confidence: 99%

Phosphorylation of Hepatitis C Virus RNA Polymerases Ser29 and Ser42 by Protein Kinase C-Related Kinase 2 Regulates Viral RNA Replication

Han

Kim

et al. 2014

J Virol

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Hepatitis C virus (HCV) nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase (RdRp), is the key enzyme for HCV RNA replication. We previously showed that HCV RdRp is phosphorylated by protein kinase C-related kinase 2 (PRK2). In the present study, we used biochemical and reverse-genetics approaches to demonstrate that HCV NS5B phosphorylation is crucial for viral RNA replication in cell culture. Two-dimensional phosphoamino acid analysis revealed that PRK2 phosphorylates NS5B exclusively at its serine residues in vitro and in vivo. Using in vitro kinase assays and mass spectrometry, we identified two phosphorylation sites, Ser29 and Ser42, in the ⌬1 finger loop region that interacts with the thumb subdomain of NS5B. Colonyforming assays using drug-selectable HCV subgenomic RNA replicons revealed that preventing phosphorylation by Ala substitution at either Ser29 or Ser42 impairs HCV RNA replication. Furthermore, reverse-genetics studies using HCV infectious clones encoding phosphorylation-defective NS5B confirmed the crucial role of these PRK2 phosphorylation sites in viral RNA replication. Molecular-modeling studies predicted that the phosphorylation of NS5B stabilizes the interactions between its ⌬1 loop and thumb subdomain, which are required for the formation of the closed conformation of NS5B known to be important for de novo RNA synthesis. Collectively, our results provide evidence that HCV NS5B phosphorylation has a positive regulatory role in HCV RNA replication. IMPORTANCEWhile the role of RNA-dependent RNA polymerases (RdRps) in viral RNA replication is clear, little is known about their functional regulation by phosphorylation. In this study, we addressed several important questions about the function and structure of phosphorylated hepatitis C virus (HCV) nonstructural protein 5B (NS5B). Reverse-genetics studies with HCV replicons encoding phosphorylation-defective NS5B mutants and analysis of their RdRp activities revealed previously unidentified NS5B protein features related to HCV replication and NS5B phosphorylation. These attributes most likely reflect potential structural changes induced by phosphorylation in the ⌬1 finger loop region of NS5B with two identified phosphate acceptor sites, Ser29 and Ser42, which may transiently affect the closed conformation of NS5B. Elucidating the effects of dynamic changes in NS5B phosphorylation status during viral replication and their impacts on RNA synthesis will improve our understanding of the molecular mechanisms of NS5B phosphorylation-mediated regulation of HCV replication.

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confidence: 99%

Phosphorylation of Hepatitis C Virus RNA Polymerases Ser29 and Ser42 by Protein Kinase C-Related Kinase 2 Regulates Viral RNA Replication

Han

Kim

et al. 2014

J Virol

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“…These multiple effects of Akt/PKB can modulate cap-versus internal ribosome entry site (IRES)-dependent translation as well as infected-cell survival, which may facilitate virus persistence. Thus, not surprisingly, several viral proteins can modulate PI3K/Akt-dependent signaling pathways (9)(10)(11)(12).…”

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confidence: 99%

Hepatitis C Virus RNA-Dependent RNA Polymerase Interacts with the Akt/PKB Kinase and Induces Its Subcellular Relocalization

Valero

Sabariegos

Cimas

et al. 2016

Antimicrob Agents Chemother

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i Hepatitis C virus (HCV) interacts with cellular components and modulates their activities for its own benefit. These interactions have been postulated as a target for antiviral treatment, and some candidate molecules are currently in clinical trials. The multifunctional cellular kinase Akt/protein kinase B (PKB) must be activated to increase the efficacy of HCV entry but is rapidly inactivated as the viral replication cycle progresses. Viral components have been postulated to be responsible for Akt/PKB inactivation, but the underlying mechanism remained elusive. In this study, we show that HCV polymerase NS5B interacts with Akt/ PKB. In the presence of transiently expressed NS5B or in replicon-or virus-infected cells, NS5B changes the cellular localization of Akt/PKB from the cytoplasm to the perinuclear region. Sequestration of Akt/PKB by NS5B could explain its exclusion from its participation in early Akt/PKB inactivation. The NS5B-Akt/PKB interaction represents a new regulatory step in the HCV infection cycle, opening possibilities for new therapeutic options. As an obligate parasite, HCV usurps cellular functions and pathways to complete its replication cycle, and some of these routes are related to cell cycle control (2). The Akt/protein kinase B (PKB) family of serine/threonine protein kinases are involved in several signaling pathways that can impact the outcome of viral infections. Akt/PKB resides in the cytosol in an inactive conformation. Initial stimulation of the cell causes activation of a cell surface receptor and subsequently phosphorylation of phosphatidylinositol 3-kinase (PI3K). Activated PI3K is responsible of the formation of phosphatidylinositol(3,4,5)-trisphosphate second messenger. Akt interacts with these phosphoinositides and is recruited to the membrane, where it can be fully activated by PDK1 and other kinases. The Akt/PKB pathway is involved in processes as disparate as nutrient metabolism, regulation of protein synthesis, autophagy, and the balance of apoptosis versus cell survival and proliferation, the latter being critical for the outcome of viral infections (3-6). Akt/PKB can influence the virus-host interaction by several mechanisms. The translational repressors 4E-BPs are eukaryotic translation initiation factor 4E (eIF4E)-binding proteins that prevent cap-dependent translation of cellular mRNAs. Akt/PKB is needed for the inactivation of 4E-BPs, which occurs through a phosphorylation reaction that requires FRAP/mammalian target of rapamycin (mTOR) and results in activation of cellular translation (7). Akt/PKB is the downstream target of the lipid kinase PI3K, and both are involved in cell proliferation and apoptosis. In infections of neuronal cells by dengue virus serotype 2 (dengue-2) and Japanese encephalitis virus, PI3K played an antiapoptotic role, and the blocking of PI3K activation enhanced virus-induced cytopathology with no effect on virus production (8). These multiple effects of Akt/PKB can modulate cap-versus internal ribosome entry site (IRES)-dependent translatio...

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“…Recent work has suggested that Akt may phosphorylate the phosphoprotein of several negative-stranded RNA viruses 60 and that it may also control calicivirus polymerase function. 133 Future discoveries will likely greatly expand the number of viral substrates and more clearly point out the viral dependencies on this signaling axis.…”

Section: Discussionmentioning

confidence: 99%

HijAkt

Dunn

Connor

2012

Progress in Molecular Biology and Translational Science

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As obligate parasites of cellular processes, viruses must take over cellular macromolecular machinery. It is also becoming clear that viruses routinely control intracellular signaling pathways through the direct or indirect control of kinases and phosphatases. This control of cellular phosphoproteins is important to promote a variety of viral processes, from control of entry to nuclear function to the stimulation of viral protein synthesis. This review focuses on the takeover of the cellular phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway by a variety of retroviruses, DNA viruses, and RNA viruses, highlighting the functions ascribed to virus activation of PI3K and Akt activity. This review also describes the role that the PI3K/Akt pathway plays in the host response, noting that it that can trigger anti- as well as proviral functions.

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Norovirus RNA-Dependent RNA Polymerase Is Phosphorylated by an Important Survival Kinase, Akt

Cited by 22 publications

References 29 publications

Phosphorylation of Hepatitis C Virus RNA Polymerases Ser29 and Ser42 by Protein Kinase C-Related Kinase 2 Regulates Viral RNA Replication

Phosphorylation of Hepatitis C Virus RNA Polymerases Ser29 and Ser42 by Protein Kinase C-Related Kinase 2 Regulates Viral RNA Replication

Hepatitis C Virus RNA-Dependent RNA Polymerase Interacts with the Akt/PKB Kinase and Induces Its Subcellular Relocalization

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