Involvement of hepatitis C virus NS5A hyperphosphorylation mediated by casein kinase I-α in infectious virus production.
Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) possesses multiple functions in the viral life cycle. NS5A is a phosphoprotein that exists in hyperphosphorylated and basally phosphorylated forms. Although the phosphorylation status of NS5A is considered to have a significant impact on its function, the mechanistic details regulating NS5A phosphorylation, as well as its exact roles in the HCV life cycle, are still poorly understood. In this study, we screened 404 human protein kinases via in vitro binding and phosphorylation assays, followed by RNA interference-mediated gene silencing in an HCV cell culture system. Casein kinase I-α (CKI-α) was identified as an NS5A-associated kinase involved in NS5A hyperphosphorylation and infectious virus production. Subcellular fractionation and immunofluorescence confocal microscopy analyses showed that CKI-α-mediated hyperphosphorylation of NS5A contributes to the recruitment of NS5A to low-density membrane structures around lipid droplets (LDs) and facilitates its interaction with core protein and the viral assembly. Phospho-proteomic analysis of NS5A with or without CKI-α depletion identified peptide fragments that corresponded to the region located within the low-complexity sequence I, which is important for CKI-α-mediated NS5A hyperphosphorylation. This region contains eight serine residues that are highly conserved among HCV isolates, and subsequent mutagenesis analysis demonstrated that serine residues at amino acids 225 and 232 in NS5A (genotype 2a) may be involved in NS5A hyperphosphorylation and hyperphosphorylation-dependent regulation of virion production. These findings provide insight concerning the functional role of NS5A phosphorylation as a regulatory switch that modulates its multiple functions in the HCV life cycle.


OBJECTIVE
Mechanisms regulating NS5A phosphorylation and its exact function in the HCV life cycle have not been clearly defined. By using a high-throughput screening system targeting host protein kinases, we identified CKI-α as an NS5A-associated kinase involved in NS5A hyperphosphorylation and the production of infectious virus. Our results suggest that the impact of CKI-α in the HCV life cycle is more profound on virion assembly than viral replication via mediation of NS5A hyperphosphorylation. CKI-α-dependent hyperphosphorylation of NS5A plays a role in recruiting NS5A to low-density membrane structures around LDs and facilitating its interaction with the core for new virus particle formation. By using proteomic approach, we identified the region within the low-complexity sequence I of NS5A that is involved in NS5A hyperphosphorylation and hyperphosphorylation-dependent regulation of infectious virus production. These findings will provide novel mechanistic insights into the roles of NS5A-associated kinases and NS5A phosphorylation in the HCV life cycle.
Department of Virology II, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo, Japan Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Takahiro
T
Masaki
Proteo-Science Center, Ehime University, Matsuyama, Ehime, Japan.
Satoko
S
Matsunaga
Proteo-Science Center, Ehime University, Matsuyama, Ehime, Japan.
Hirotaka
H
Takahashi
Department of Infectious Diseases, Hamamatsu University School of Medicine, Handayama, Higashi-ku, Hamamatsu, Japan.
Kenji
K
Nakashima
Graduate School of Medical Life Science and Advanced Medical Research Center, Yokohama City University, Fukuura, Kanazawa-ku, Yokohama, Japan.
Yayoi
Y
Kimura
Department of Infectious Diseases, Hamamatsu University School of Medicine, Handayama, Higashi-ku, Hamamatsu, Japan.
Masahiko
M
Ito
Department of Virology II, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo, Japan.
Mami
M
Matsuda
Department of Virology II, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo, Japan.
Asako
A
Murayama
Department of Virology II, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo, Japan.
Takanobu
T
Kato
Graduate School of Medical Life Science and Advanced Medical Research Center, Yokohama City University, Fukuura, Kanazawa-ku, Yokohama, Japan.
Hisashi
H
Hirano
Proteo-Science Center, Ehime University, Matsuyama, Ehime, Japan.
Yaeta
Y
Endo
Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Division of Infectious Diseases, Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Stanley M
SM
Lemon
Department of Virology II, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo, Japan.
Takaji
T
Wakita
Proteo-Science Center, Ehime University, Matsuyama, Ehime, Japan.
Tatsuya
T
Sawasaki
Department of Infectious Diseases, Hamamatsu University School of Medicine, Handayama, Higashi-ku, Hamamatsu, Japan tesuzuki@hama-med.ac.jp.
Tetsuro
T
Suzuki
D000595:Amino Acid Sequence; D015153:Blotting, Western; D048128:Casein Kinase Ialpha / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D016174:Hepacivirus / Q000502:physiology*; D006526:Hepatitis C / Q000378:metabolism / Q000821:virology*; D006801:Humans; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000502:physiology*; D012324:RNA-Dependent RNA Polymerase
D048128:Casein Kinase Ialpha; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase