Destabilization of PDK1 by Hsp90 inactivation suppresses hepatitis C virus replication through inhibition of PRK2-mediated viral RNA polymerase phosphorylation

Kim, Mi‐Gyeong; Moon, Jae Su; Kim, Eun Jung; Lee, Jun Young; Oh, Jisun

doi:10.1016/j.bbrc.2012.03.126

Cited by 24 publications

(20 citation statements)

References 24 publications

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“…Wild-type and mutant subgenomic HCV RNAs prepared by in vitro transcription using the Megascript T7 kit (Ambion, Austin, TX, USA) were transfected into Huh7 cells by electroporation as previously described (26). At 24 h posttransfection, selection of G418-resistant colonies was initiated by adding 0.5 mg/ml G418.…”

Section: Two-dimensional Phosphoamino Acid Analysismentioning

confidence: 99%

“…The plasmid JFH1 and its derivatives with mutations at the NS5B phosphorylation site were used to prepare viral-RNA transcripts as previously reported (26). The viral RNAs were then electroporated into Huh7 cells as previously described (26) or using Lipofectamine 2000 (Invitrogen), and total RNA harvested from the transfected cells was analyzed by Northern blotting to detect HCV genomic RNA (34).…”

Section: Two-dimensional Phosphoamino Acid Analysismentioning

confidence: 99%

“…Total RNA was extracted with TRIzol reagent (Invitrogen) and purified according to the manufacturer's instructions. HCV RNA levels were quantified by real-time quantitative reverse transcription (qRT)-PCR using a TaqMan probe targeting a region within the HCV 5= UTR, as previously described (26).…”

Section: Two-dimensional Phosphoamino Acid Analysismentioning

confidence: 99%

“…With regard to HCV RNA replication, we previously demonstrated that protein kinase C-related kinase 2 (PRK2), a Ser/Thr kinase in the AGC kinase subfamily (25), binds to and phosphorylates HCV RdRp at its N-terminal finger subdomain (amino acids 1 to 187) (7). Silencing of PRK2 expression by small interfering RNA (siRNA), inhibiting PRK2 activity with its pharmacological inhibitors HA1077 and Y27632, or destabilizing the PRK2 upstream kinase phosphoinositide-dependent kinase 1 reduced HCV replication (7,8,26), demonstrating PRK2's regulatory role in the HCV life cycle.…”

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confidence: 99%

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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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Section: Two-dimensional Phosphoamino Acid Analysismentioning

confidence: 99%

Section: Two-dimensional Phosphoamino Acid Analysismentioning

confidence: 99%

Section: Two-dimensional Phosphoamino Acid Analysismentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…Numerous antiviral activity of Hsp90 inhibitors have been proved against tissue culture of ill health conditions such as picornaviruses poliovirus, coxsackievirus, rhinovirus, influenza virus, paramyxoviruses, hepatitis C virus (HCV), Ebola virus, vesicular stomatitis virus, severe acquired respiratory syndrome (SARS), feline viral rhinotracheitis (FHV), HIV, vaccinia virus, and herpes viruses [14,16,32,33,44,57,60,61,64]. Gryase B inhibitors (novobiocin, coumermycin A1 (C-A1) and daunorubicin) have been reported to impair HIV-1 replication the most, by targeting Hsp90 [60].…”

Section: Introductionmentioning

confidence: 99%

Mechanism of Inhibition of Hsp90 Dimerization by Gyrase B Inhibitor Coumermycin A1 (C–A1) Revealed by Molecular Dynamics Simulations and Thermodynamic Calculations

Cele

Kumalo

Soliman

2016

Cell Biochem Biophys

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Heat shock protein (Hsp) 90 an emerging and attracting target in the anti-HIV drug discovery process due to the key role it plays in the pathogenicity of HIV-1 virus. In this research study, long-range all-atom molecular dynamics simulations were engaged for the bound and the unbound proteins to enhance the understanding of the molecular mechanisms of the Hsp90 dimerization and inhibition. Results evidently showed that coumermycin A1 (C-A1), a recently discovered Hsp90 inhibitor, binds at the dimer's active site of the Hsp90 protein and leads to a substantial parting between dimeric opposed residues, which include Arg591.B, Lys594.A, Ser663.A, Thr653.B, Ala665.A, Thr649.B, Leu646.B and Asn669.A. Significant differences in magnitudes were observed in radius of gyration, root-mean-square deviation and root-mean-square fluctuation, which confirms a reasonably more flexible state in the apo conformation associated with it dimerization. In contrast, the bound conformer of Hsp90 showed less flexibility. This visibly highpoints the inhibition process resulting from the binding of the ligand. These findings were further validated by principal component analysis. We believe that the detailed dynamic analyses of Hsp90 presented in this study, would give an imperative insight and better understanding to the function and mechanisms of inhibition. Furthermore, information obtained from the binding mode of the inhibitor would be of great assistance in the design of more potent inhibitors against the HIV target Hsp90.

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Galactosylated Lipidoid Nanoparticles for Delivery of Small Interfering RNA to Inhibit Hepatitis C Viral Replication In Vivo

Park

Jeon

Lee

et al. 2016

Adv Healthcare Materials

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Small interfering RNA (siRNA) delivery can provide an effective therapy for treating viral diseases by silencing genes involved in viral replication. In this study, a liver-targeting formulation of lipidoid nanoparticle for delivery of siRNA that targets protein kinase C-related kinase 2 (PRK2) to inhibit hepatitis C virus (HCV) replication is reported. The most effective, minimally cytotoxic lipidoid for siRNA delivery to hepatic cells is identified from a small library of alkyl epoxide-polyamine conjugates. In vitro transfection of PRK2 siRNA (siPRK2) using this lipidoid induces significant silencing of PRK2 (≈80%), suppressing HCV replication in human hepatic cells transfected with the HCV subgenomic replicon. Systemic administration of siPRK2 using the lipidoid nanoparticles results in significant reduction of host PRK2 in the mouse liver (≈60%). This treatment significantly suppresses HCV replication in an HCV-xenograft mouse model. siRNA delivery to the liver is further improved via galactosylation of the lipidoid. Compared with the unmodified lipidoid formulation, galactosylated lipidoids induce greater silencing of host PRK2 in mouse livers (≈80%) and more rapid suppression of HCV replication in an HCV-xenograft mouse. This study suggests that galactosylated lipidoid nanoparticles could provide a treatment for hepatitis C by mediating delivery of anti-viral RNA interference therapeutics to the liver.

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Destabilization of PDK1 by Hsp90 inactivation suppresses hepatitis C virus replication through inhibition of PRK2-mediated viral RNA polymerase phosphorylation

Cited by 24 publications

References 24 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

Mechanism of Inhibition of Hsp90 Dimerization by Gyrase B Inhibitor Coumermycin A1 (C–A1) Revealed by Molecular Dynamics Simulations and Thermodynamic Calculations

Galactosylated Lipidoid Nanoparticles for Delivery of Small Interfering RNA to Inhibit Hepatitis C Viral Replication In Vivo

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