A Hepatitis C Virus NS5A Phosphorylation Site That Regulates RNA Replication

112

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-␣ (CKI-␣) 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-␣-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-␣ depletion identified peptide fragments that corresponded to the region located within the low-complexity sequence I, which is important for CKI-␣-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. IMPORTANCEMechanisms 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-␣ as an NS5A-associated kinase involved in NS5A hyperphosphorylation and the production of infectious virus. Our results suggest that the impact of CKI-␣ in the HCV life cycle is more profound on virion assembly than viral replication via mediation of NS5A hyperphosphorylation. CKI-␣-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.

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confidence: 99%

Involvement of Hepatitis C Virus NS5A Hyperphosphorylation Mediated by Casein Kinase I-α in Infectious Virus Production

Masaki

Matsunaga

Takahashi

et al. 2014

112

“…The nonstructural 5A (NS5A) protein is a phosphoprotein consisting of 447 amino acid residues. NS5A exists in two forms of polypeptide, p56 and p58, which are phosphorylated at serine residues by cellular kinase (3), and phosphorylation regulates the HCV life cycle (4). NS5A protein forms a part of the HCV RNA replication complex (5) and is involved in liver pathogenesis (6).…”

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confidence: 99%

Nonstructural 5A Protein of Hepatitis C Virus Regulates Soluble Resistance-Related Calcium-Binding Protein Activity for Viral Propagation

Tran

Luong

Park

et al. 2016

Hepatitis C virus (HCV) is a major cause of chronic liver disease and is highly dependent on cellular proteins for virus propagation. To identify the cellular factors involved in HCV propagation, we recently performed protein microarray assays using the HCV nonstructural 5A (NS5A) protein as a probe. Of 90 cellular protein candidates, we selected the soluble resistance-related calcium-binding protein (sorcin) for further characterization. Sorcin is a calcium-binding protein and is highly expressed in certain cancer cells. We verified that NS5A interacted with sorcin through domain I of NS5A, and phosphorylation of the threonine residue 155 of sorcin played a crucial role in protein interaction. Small interfering RNA (siRNA)-mediated knockdown of sorcin impaired HCV propagation. Silencing of sorcin expression resulted in a decrease of HCV assembly without affecting HCV RNA and protein levels. We further demonstrated that polo-like kinase 1 (PLK1)-mediated phosphorylation of sorcin was increased by NS5A. We showed that both phosphorylation and calcium-binding activity of sorcin were required for HCV propagation. These data indicate that HCV modulates sorcin activity via NS5A protein for its own propagation. IMPORTANCESorcin is a calcium-binding protein and regulates intracellular calcium homeostasis. HCV NS5A interacts with sorcin, and phosphorylation of sorcin is required for protein interaction. Gene silencing of sorcin impaired HCV propagation at the assembly step of the HCV life cycle. Sorcin is phosphorylated by PLK1 via protein interaction. We showed that sorcin interacted with both NS5A and PLK1, and PLK1-mediated phosphorylation of sorcin was increased by NS5A. Moreover, calcium-binding activity of sorcin played a crucial role in HCV propagation. These data provide evidence that HCV regulates host calcium metabolism for virus propagation, and thus manipulation of sorcin activity may represent a novel therapeutic target for HCV. Hepatitis C virus (HCV) is a major causative agent of non-A, non-B hepatitis. HCV infection often leads to chronic hepatitis, liver cirrhosis, and ultimately hepatocellular carcinoma (HCC) (1). HCV belongs to the member of the Hepacivirus genus within the Flaviviridae family. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome of ϳ9.6 kb. Its genome encodes a single polyprotein precursor of more than 3,000 amino acids, which is cleaved by both host and viral proteases at the endoplasmic reticulum (ER), yielding structural (core, E1, and E2) and nonstructural (p7 and NS2 to NS5B) proteins (2). The nonstructural 5A (NS5A) protein is a phosphoprotein consisting of 447 amino acid residues. NS5A exists in two forms of polypeptide, p56 and p58, which are phosphorylated at serine residues by cellular kinase (3), and phosphorylation regulates the HCV life cycle (4). NS5A protein forms a part of the HCV RNA replication complex (5) and is involved in liver pathogenesis (6). NS5A is a multifunctional protein that regulates RNA replication, interferon (IFN) resis...

“…On the other hand, phosphorylated NS5A has also been shown to play a critical role in the HCV life cycle, especially in maintenance of the balance between RNA replication and viral assembly (9). Thus, investigating the detailed molecular mechanisms by which cellular factors, including some kinases that interact with NS5A and modulate the phosphorylation of NS5A, would be instrumental in the development of antiviral therapy (64,65). Previous studies have identified various host kinases involved in the regulation of the phosphorylation status of NS5A either directly or indirectly, such as CK1␣, CK2␣, Plk1, lipid kinase phosphatidylinositol-4 kinase III alpha, glycogen synthase kinase 3, and MAPK (45,59,61,63,66).…”

Section: Discussionmentioning

confidence: 99%

Vinexin β Interacts with Hepatitis C Virus NS5A, Modulating Its Hyperphosphorylation To Regulate Viral Propagation

Xiong

Yang

Wang

et al. 2015

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...