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 ␤ coimmunoprecipitated with NS5A derived from different HCV genotypes. Two residues, tryptophan (W307) and tyrosine (Y325), in the third SH3 domain of vinexin ␤ 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 ␤ significantly suppressed NS5A hyperphosphorylation and decreased HCV replication, which could be rescued by expressing a vinexin ␤ short hairpin RNA-resistant mutant. We also found that vinexin ␤ modulated the hyperphosphorylation of NS5A in a casein kinase 1␣-dependent on manner. Taken together, our findings suggest that vinexin ␤ modulates NS5A phosphorylation via its interaction with NS5A, thereby regulating HCV replication, implicating vinexin ␤ in the viral life cycle. IMPORTANCEHepatitis 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 ␤ modulates NS5A hyperphosphorylation through the NS5A-vinexin ␤ interaction. Hyperphosphorylated NS5A induced by vinexin ␤ is casein kinase 1␣ 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. H epatitis C virus (HCV) infection is a global health disease and is a major cause of chronic liver disease leading to hepatic fibrosis, liver cirrhosis, and hepatic carcinoma. No protective vaccine is available. Some directly acting antiviral agents combining pegylated interferon and ribavirin show therapeutic promise for chronic hepatitis C. However, the mechanisms of drug action, the issues of interferon-free therapy, drug resistance, and broad treatment of all HCV genotypes...

Susceptibilities of Genotype 1a, 1b, and 3 Hepatitis C Virus Variants to the NS5A Inhibitor Elbasvir

“…While the replicon assay yields the number of cells that support HCV replication in the laboratory, viral load measurements give the actual number of circulating viruses in the patient. The impact of a substitution within NS5A may to some degree depend on the surrounding context, including the degree of phosphorylation (19). Adaptive amino acid changes elsewhere in and/or outside NS5A in the virus not incorporated into the replicon could enhance replication of certain RAVs in vivo.…”

ASPP2 binds to hepatitis C virus NS5A protein via an SH3 domain/PxxP motif-mediated interaction and potentiates infection

“…ASPP1 and ASPP2 are known to bind an SH3 class II binding motif, PxφPx+ via their C-terminal domains () [34–40]. The PRR between the low complexity sequence 2 (LCSII) and Domain 3 of NS5A contains a PxφPx+ motif and mediates the interaction with several SH3 domain-containing host proteins that have been reported to regulate NS5A phosphorylation and viral replication [30, 41–46] (). We hypothesized that the SH3 domain of ASPP2 binds an NS5A PRR and evaluated this hypothesis using both computational and biochemical analyses.…”

“…1b) [34][35][36][37][38][39][40]. The PRR between the low complexity sequence 2 (LCSII) and Domain 3 of NS5A contains a PxφPx+ motif and mediates the interaction with several SH3 domain-containing host proteins that have been reported to regulate NS5A phosphorylation and viral replication [30,[41][42][43][44][45][46] (Fig. 1c).…”

SPSB2 inhibits hepatitis C virus replication by targeting NS5A for ubiquitination and degradation

“…Infectious HCV (genotype 2a, strain JFH-1) was provided by Takaji Wakita (National Institute of Infectious Diseases, Japan); the virus amplification, titer measure, and storage were carried out as described previously [27]. HCV infection was performed as described previously [28].…”