Interaction of Hepatitis C Virus Nonstructural Protein 5A with Core Protein Is Critical for the Production of Infectious Virus Particles

Department of Virology II, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
Journal of Virology (Impact Factor: 4.44). 08/2008; 82(16):7964-76. DOI: 10.1128/JVI.00826-08
Source: PubMed


Nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) possesses multiple and diverse functions in RNA replication, interferon resistance, and viral pathogenesis. Recent studies suggest that NS5A is involved in the assembly and maturation of infectious viral particles; however, precisely how NS5A participates in virus production has not been fully elucidated. In the present study, we demonstrate that NS5A is a prerequisite for HCV particle production as a result of its interaction with the viral capsid protein (core protein). The efficiency of virus production correlated well with the levels of interaction between NS5A and the core protein. Alanine substitutions for the C-terminal serine cluster in domain III of NS5A (amino acids 2428, 2430, and 2433) impaired NS5A basal phosphorylation, leading to a marked decrease in NS5A-core interaction, disturbance of the subcellular localization of NS5A, and disruption of virion production. Replacing the same serine cluster with glutamic acid, which mimics the presence of phosphoserines, partially preserved the NS5A-core interaction and virion production, suggesting that phosphorylation of these serine residues is important for virion production. In addition, we found that the alanine substitutions in the serine cluster suppressed the association of the core protein with viral genome RNA, possibly resulting in the inhibition of nucleocapsid assembly. These results suggest that NS5A plays a key role in regulating the early phase of HCV particle formation by interacting with core protein and that its C-terminal serine cluster is a determinant of the NS5A-core interaction.

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    • "NS5A, in addition to its roles in viral RNA replication, is also necessary for viral assembly [8]. HCV assembly requires cellular lipid droplets as a platform [9] and interactions between NS5A and the core protein [10]. Assembled particles bud into the ER and traffic through the secretory pathway. "
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    ABSTRACT: Hepatitis C virus (HCV) infection relies on virus-host interactions with human hepatocytes, a context in which host cell kinases play critical roles in every step of the HCV life cycle. During viral entry, cellular kinases, including EGFR, EphA2 and PKA, regulate the localization of host HCV entry factors and induce receptor complex assembly. Following virion internalization, viral genomes replicate on endoplasmic reticulum-derived membranous webs. The formation of membranous webs depends on interactions between the HCV NS5a protein and PI4KIIIα. The phosphorylation status of NS5a, regulated by PI4KIIIα, CKI and other kinases, also acts as a molecular switch to virion assembly, which takes place on lipid droplets. The formation of lipid droplets is enhanced by HCV activation of IKKα. In view of the multiple crucial steps in the viral life cycle that are mediated by host cell kinases, these enzymes also represent complementary targets for antiviral therapy. This article is part of a Special Issue entitled:Inhibitors of Protein Kinases. Copyright © 2015. Published by Elsevier B.V.
    No preview · Article · Apr 2015 · Biochimica et Biophysica Acta
    • "Antibodies.Thefollowingantibodieswereused:mousemonoclonalantibodiesspecificforHCV coreprotein(ThermoScientific),NS5A(9E10;agiftfromCharlesRice,TheRockefellerUniversity, NewYork,NY),AGO2(MBL,Nagoya,JapanandAbnova,Taipei,Taiwan),PCBP2(Abnova),and hnRNPL(Millipore,Billerica,MA);ratmonoclonalantibodytoAGO2;andrabbitpolyclonal antibodiesagainstNS5A(TB0705#1;agiftfromTakajiWakita,NationalInstituteofInfectious Diseases,Tokyo,Japan)(Masakietal.,2008),Xrn1(BethylLaboratories,Montgomery,TX),AGO2 (CellSignaling,Danvers,MA),greenfluorescentprotein(GFP)(ab290;Abcam,Cambridge,UK),and β-actin(A2066;Sigma-Aldrich). "
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    ABSTRACT: The liver-specific microRNA, miR-122, stabilizes hepatitis C virus (HCV) RNA genomes by recruiting host argonaute 2 (AGO2) to the 5' end and preventing decay mediated by exonuclease Xrn1. However, HCV replication requires miR-122 in Xrn1-depleted cells, indicating additional functions. We show that miR-122 enhances HCV RNA levels by altering the fraction of HCV genomes available for RNA synthesis. Exogenous miR-122 increases viral RNA and protein levels in Xrn1-depleted cells, with enhanced RNA synthesis occurring before heightened protein synthesis. Inhibiting protein translation with puromycin blocks miR-122-mediated increases in RNA synthesis, but independently enhances RNA synthesis by releasing ribosomes from viral genomes. Additionally, miR-122 reduces the fraction of viral genomes engaged in protein translation. Depleting AGO2 or PCBP2, which binds HCV RNA in competition with miR-122 and promotes translation, eliminates miR-122 stimulation of RNA synthesis. Thus, by displacing PCBP2, miR-122 reduces HCV genomes engaged in translation while increasing the fraction available for RNA synthesis. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Feb 2015 · Cell Host & Microbe
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    • "Yet, low levels of hyperphosphorylated NS5A seem to be essential for viral replication since viral replication is inhibited upon its complete loss (Appel et al., 2005a; Fridell et al., 2011). The increase in viral replication caused by reduced levels of the NS5A hyperphosphorylated form inversely correlates with viral production, leading to the notion that the hyperphosphorylated form of NS5A has adverse effects on viral replication but is required for viral assembly (Masaki et al., 2008; Miyanari et al., 2007; Pietschmann et al., 2009; Tellinghuisen et al., 2008a). It was also proposed to regulate the interface between replication and assembly (Tellinghuisen et al., 2008a). "
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    ABSTRACT: Hepatitis C virus (HCV) replicates in membrane associated, highly ordered replication complexes (RCs). These complexes include viral and host proteins necessary for viral RNA genome replication. The interaction network among viral and host proteins underlying the formation of these RCs is yet to be thoroughly characterized. Here, we investigated the association between NS4B and NS5A, two critical RC components. We characterized the interaction between these proteins using fluorescence resonance energy transfer and a mammalian two-hybrid system. Specific tryptophan residues within the C-terminal domain (CTD) of NS4B were shown to mediate this interaction. Domain I of NS5A, was sufficient to mediate its interaction with NS4B. Mutations in the NS4B CTD tryptophan residues abolished viral replication. Moreover, one of these mutations also affected NS5A hyperphosphorylation. These findings provide new insights into the importance of the NS4B-NS5A interaction and serve as a starting point for studying the complex interactions between the replicase subunits. Copyright © 2014 Elsevier Inc. All rights reserved.
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