title: Vinexin β Interacts with Hepatitis C Virus NS5A, Modulating Its Hyperphosphorylation To Regulate Viral Propagation.
abstract: Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is essential for HCV genome replication and virion production and is involved in the regulation of multiple host signaling pathways. As a proline-rich protein, NS5A is capable of interacting with various host proteins containing Src homology 3 (SH3) domains. Previous studies have suggested that vinexin, a member of the sorbin homology (SoHo) adaptor family, might be a potential binding partner of NS5A by yeast two-hybrid screening. However, firm evidence for this interaction is lacking, and the significance of vinexin in the HCV life cycle remains unclear. In this study, we demonstrated that endogenously and exogenously expressed vinexin β coimmunoprecipitated with NS5A derived from different HCV genotypes. Two residues, tryptophan (W307) and tyrosine (Y325), in the third SH3 domain of vinexin β and conserved Pro-X-X-Pro-X-Arg motifs at the C terminus of NS5A were indispensable for the vinexin-NS5A interaction. Furthermore, downregulation of endogenous vinexin β significantly suppressed NS5A hyperphosphorylation and decreased HCV replication, which could be rescued by expressing a vinexin β short hairpin RNA-resistant mutant. We also found that vinexin β modulated the hyperphosphorylation of NS5A in a casein kinase 1α-dependent on manner. Taken together, our findings suggest that vinexin β modulates NS5A phosphorylation via its interaction with NS5A, thereby regulating HCV replication, implicating vinexin β in the viral life cycle.
  
  
  OBJECTIVE
  Hepatitis C virus (HCV) nonstructural protein NS5A is a phosphoprotein, and its phosphorylation states are usually modulated by host kinases and other viral nonstructural elements. Additionally, cellular factors containing Src homology 3 (SH3) domains have been reported to interact with proline-rich regions of NS5A. However, it is unclear whether there are any relationships between NS5A phosphorylation and the NS5A-SH3 interaction, and little is known about the significance of this interaction in the HCV life cycle. In this work, we demonstrate that vinexin β modulates NS5A hyperphosphorylation through the NS5A-vinexin β interaction. Hyperphosphorylated NS5A induced by vinexin β is casein kinase 1α dependent and is also crucial for HCV propagation. Overall, our findings not only elucidate the relationships between NS5A phosphorylation and the NS5A-SH3 interaction but also shed new mechanistic insight on Flaviviridae NS5A (NS5) phosphorylation. We believe that our results may afford the potential to offer an antiviral therapeutic strategy.
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Xiong
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Mingzhen
authors.initials: M
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Hailong
authors.initials: H
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Zhipeng
authors.initials: Z
authors.lastname: Rao
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhong
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Xiu
authors.initials: X
authors.lastname: Xin
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Mo
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Shujuan
authors.initials: S
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China China Center for Type Culture Collection, Wuhan University, Wuhan, China cctcc202@whu.edu.cn shenchao@whu.edu.cn.
authors.forename: Chao
authors.initials: C
authors.lastname: Shen
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Wuhan, China China Center for Type Culture Collection, Wuhan University, Wuhan, China cctcc202@whu.edu.cn shenchao@whu.edu.cn.
authors.forename: Congyi
authors.initials: C
authors.lastname: Zheng
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D002460:Cell Line; D016174:Hepacivirus / Q000502:physiology*; D022781:Hepatocytes / Q000821:virology; D054884:Host-Pathogen Interactions; D006801:Humans; D047468:Immunoprecipitation; D009124:Muscle Proteins; D010766:Phosphorylation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D020798:Two-Hybrid System Techniques; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication; D012324:RNA-Dependent RNA Polymerase
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D009124:Muscle Proteins; D017361:Viral Nonstructural Proteins; C486523:SORBS3 protein, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase