Dataset Information

Sequential Phosphorylation of the Hepatitis C Virus NS5A Protein Depends on NS3-Mediated Autocleavage between NS3 and NS4A.

ABSTRACT: Replication of the genotype 2 hepatitis C virus (HCV) requires hyperphosphorylation of the nonstructural protein NS5A. It has been known that NS5A hyperphosphorylation results from the phosphorylation of a cluster of highly conserved serine residues (S2201, S2208, S2211, and S2214) in a sequential manner. It has also been known that NS5A hyperphosphorylation requires an NS3 protease encoded on one single NS3-5A polyprotein. It was unknown whether NS3 protease participates in this sequential phosphorylation process. Using an inventory of antibodies specific to S2201, S2208, S2211, and S2214 phosphorylation, we found that protease-dead S1169A mutation abrogated NS5A hyperphosphorylation and phosphorylation at all serine residues measured, consistent with the role of NS3 in NS5A sequential phosphorylation. These effects were not rescued by a wild-type NS3 protease provided in trans by another molecule. Mutations (T1661R, T1661Y, or T1661D) that prohibited proper cleavage at the NS3-4A junction also abolished NS5A hyperphosphorylation and phosphorylation at all serine residues, whereas mutations at the other cleavage sites, NS4A-4B (C1715S) or NS4B-5A (C1976F), did not. In fact, any combinatory mutations that prohibited NS3-4A cleavage (T1661Y/C1715S or T1661Y/C1976F) abrogated NS5A hyperphosphorylation and phosphorylation at all serine residues. In the C1715S/C1976F double mutant, which resulted in an NS4A-NS4B-NS5A fusion polyprotein, a hyperphosphorylated band was observed and was phosphorylated at all serine residues. We conclude that NS3-mediated autocleavage at the NS3-4A junction is critical to NS5A hyperphosphorylation at S2201, S2208, S2211, and S2214 and that NS5A hyperphosphorylation could occur in an NS4A-NS4B-NS5A polyprotein.IMPORTANCE For ca. 20 years, the HCV protease NS3 has been implicated in NS5A hyperphosphorylation. We now show that it is the NS3-mediated cis cleavage at the NS3-4A junction that permits NS5A phosphorylation at serines 2201, 2208, 2211, and 2214, leading to hyperphosphorylation, which is a necessary condition for genotype 2 HCV replication. We further show that NS5A may already be phosphorylated at these serine residues right after NS3-4A cleavage and before NS5A is released from the NS4A-5A polyprotein. Our data suggest that the dual-functional NS3, a protease and an ATP-binding RNA helicase, could have a direct or indirect role in NS5A hyperphosphorylation.

SUBMITTER: Chiang CH

PROVIDER: S-EPMC7495366 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Sequential Phosphorylation of the Hepatitis C Virus NS5A Protein Depends on NS3-Mediated Autocleavage between NS3 and NS4A.

Chiang Cho-Han CH Lai Yen-Ling YL Huang Yu-Ning YN Yu Chun-Chiao CC Lu Christine C CC Yu Guann-Yi GY Yu Ming-Jiun MJ

Journal of virology 20200915 19

Replication of the genotype 2 hepatitis C virus (HCV) requires hyperphosphorylation of the nonstructural protein NS5A. It has been known that NS5A hyperphosphorylation results from the phosphorylation of a cluster of highly conserved serine residues (S2201, S2208, S2211, and S2214) in a sequential manner. It has also been known that NS5A hyperphosphorylation requires an NS3 protease encoded on one single NS3-5A polyprotein. It was unknown whether NS3 protease participates in this sequential phos ...[more]

PMID: 32699091

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Project description:Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a multifunctional protein implicated in both HCV RNA replication and virus particle assembly. NS2-encoded cysteine protease is responsible for autoprocessing of NS2-NS3 precursor, an essential step in HCV RNA replication. NS2 also promotes HCV particle assembly by recruiting envelope protein 2 (E2) to the virus assembly sites located at the detergent-resistant membranes (DRM). However, the fundamental mechanism regulating multiple functions of NS2 remains unclear. In this study, we discovered that NS2 is palmitoylated at the position 113 cysteine residue (NS2/C113) when expressed by itself in cells and during infectious-HCV replication. Blocking NS2 palmitoylation by introducing an NS2/C113S mutation reduced NS2-NS3 autoprocessing and impaired HCV RNA replication. Replication of the NS2/C113S mutant was restored by inserting an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between NS2 and NS3 to separate the two proteins independently of NS2-mediated autoprocessing. These results suggest that NS2 palmitoylation is critical for HCV RNA replication by promoting NS2-NS3 autoprocessing. The NS2/C113S mutation also impaired infectious-HCV assembly, DRM localization of NS2 and E2, and colocalization of NS2 with Core and endoplasmic reticulum lipid raft-associated protein 2 (Erlin-2). In conclusion, our study revealed that two major functions of NS2 involved in HCV RNA replication and virus assembly, i.e., NS2-NS3 autoprocessing and E2 recruitment to the DRM, are regulated by palmitoylation at NS2/C113. Since S-palmitoylation is reversible, NS2 palmitoylation likely allows NS2 to fine tune both HCV RNA replication and infectious-particle assembly.IMPORTANCE Chronic infection with hepatitis C virus (HCV) is a major cause of severe liver diseases responsible for nearly 400,000 deaths per year. HCV NS2 protein is a multifunctional regulator of HCV replication involved in both viral-genome replication and infectious-virus assembly. However, the underlying mechanism that enables the protein to participate in multiple steps of HCV replication remains unknown. In this study, we discovered that NS2 palmitoylation is the master regulator of its multiple functions, including NS2-mediated self-cleavage and HCV envelope protein recruitment to the virus assembly sites, which in turn promote HCV RNA replication and infectious-particle assembly, respectively. This newly revealed information suggests that NS2 palmitoylation could serve as a promising target to inhibit both HCV RNA replication and virus assembly, representing a new avenue for host-targeting strategies against HCV infection.

Project description:Replication of hepatitis C virus (HCV) is dependent on virus-encoded proteins and numerous cellular factors. DDX3 is a well-known host cofactor of HCV replication. In this study, we investigated the role of a DDX3-interacting protein, Y-box binding protein 1 (YB-1), in the HCV life cycle. Both YB-1 and DDX3 interacted with the viral nonstructural protein NS5A. During HCV infection, YB-1 partially colocalized with NS5A and the HCV replication intermediate double-stranded RNA (dsRNA) in HCV-infected Huh-7.5.1 cells. Despite sharing the same interacting partners, YB-1 participated in HCV RNA replication but was dispensable in steady-state HCV RNA replication, different from the action of DDX3. Moreover, knockdown of YB-1 in HCV-infected cells prevented infectious virus production and reduced the ratio of hyperphosphorylated (p58) to hypophosphorylated (p56) forms of NS5A, whereas DDX3 silencing did not affect the ratio of the p58 and p56 phosphoforms of NS5A. Interestingly, silencing of YB-1 severely reduced NS5A protein stability in NS5A-ectopically expressing, replicon-containing, and HCV-infected cells. Furthermore, mutations of serine 102 of YB-1 affected both YB-1-NS5A interaction and NS5A-stabilizing activity of YB-1, indicating that this Akt phosphorylation site of YB-1 plays an important role in stabilizing NS5A. Collectively, our results support a model in which the event of YB-1 phosphorylation-mediated interaction with NS5A results in stabilizing NS5A to sustain HCV RNA replication and infectious HCV production. Overall, our study may reveal a new aspect for the development of novel anti-HCV drugs.Chronic hepatitis C virus (HCV) infection induces liver cirrhosis and hepatocellular carcinoma. The viral nonstructural protein NS5A co-opting various cellular signaling pathways and cofactors to support viral genome replication and virion assembly is a new strategy for anti-HCV drug development. NS5A phosphorylation is believed to modulate switches between different stages of the HCV life cycle. In this study, we identified the cellular protein YB-1 as a novel NS5A-interacting protein. YB-1 is a multifunctional protein participating in oncogenesis and is an oncomarker of hepatocellular carcinoma (HCC). We found that YB-1 protects NS5A from degradation and likely regulates NS5A phosphorylation through its phosphorylation-dependent interaction with NS5A, which might be controlled by HCV-induced signaling pathways. Our observations suggest a model in which HCV modulates NS5A level and the ratio of the p58 and p56 phosphoforms for efficient viral propagation via regulation of cellular signaling inducing YB-1 phosphorylation. Our finding may provide new aspects for developing novel anti-HCV drugs.

Project description:BACKGROUND: Hepatitis C virus (HCV) is a common and leading cause for liver cirrhosis and hepatocellular carcinoma. Current therapies to treat HCV infection are shown to be partially effective and poorly tolerated. Therefore, ample efforts are underway to rationally design therapies targeting the HCV non-structural proteins. Most of the work carried out in this direction has been focusing mainly on HCV genotype 1. Two direct-acting antiviral agents (DAAs) Telaprevir and Boceprevir are being used against genotype 1a infection in combination therapy with interferon and ribavirin. Unfortunately these DAAs are not effective against genotype 3a. Considering the wide spread infection by HCV genotype 3a in developing countries especially South Asia, we have focused on the recombinant production of antiviral drug targets NS3 and NS5A from HCV genotype 3a. These protein targets are to be used for screening of inhibitors. RESULTS: High-level expression of NS3 and NS5A was achieved at 25°C, using ~1 and 0.5 mM Isopropyl ?-D-1-thiogalactopyranoside (IPTG), respectively. Yields of the purified NS3 and NS5A were 4 and 1 mg per liter culture volume, respectively. Although similar amounts of purified NS3 were obtained at 25 and 14°C, specificity constant (Kcat/Km) was somewhat higher at expression temperature of 25°C. Circular dichroism (CD) and Fourier-transform infrared (FT-IR) spectroscopy revealed that both NS3 and NS5A contain a mixture of alpha-helix and beta-sheet secondary structures. For NS3 protein, percentages of secondary structures were similar to the values predicted from homology modeling. CONCLUSIONS: NS3 and NS5A were over-expressed and using Nickel-affinity method both proteins were purified to?~?95% purity. Yield of the purified NS3 obtained is four fold higher than previous reports. CD spectroscopy revealed that difference in activity of NS3 expressed at various temperatures is not related to changes in global structural features of the protein. Moreover, CD and FT-IR analysis showed that NS3 and NS5A contain both alpha-helical and beta-sheet structures and for NS5A, the proportion is almost equal. The production of NS3 and NS5A in milligram quantities will allow their characterization by biophysical and biochemical means that will help in designing new strategies to fight against HCV infection.

Dataset Information

Sequential Phosphorylation of the Hepatitis C Virus NS5A Protein Depends on NS3-Mediated Autocleavage between NS3 and NS4A.

Publications

Sequential Phosphorylation of the Hepatitis C Virus NS5A Protein Depends on NS3-Mediated Autocleavage between NS3 and NS4A.

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