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Serine phosphorylation of the hepatitis C virus NS5A protein controls the establishment of replication complexes.

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The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is highly phosphorylated and involved in both virus genome replication and virion assembly. We and others have identified serine 225 in NS5A to be a phosphorylation site, but the function of this posttranslational modification in the virus life cycle remains obscure. Here we describe the phenotype of mutants with mutations at serine 225; this residue was mutated to either alanine (S225A; phosphoablatant) or aspartic acid (S225D; phosphomimetic) in the context of both the JFH-1 cell culture infectious virus and a corresponding subgenomic replicon. The S225A mutant exhibited a 10-fold reduction in genome replication, whereas the S225D mutant replicated like the wild type. By confocal microscopy, we show that, in the case of the S225A mutant, the replication phenotype correlated with an altered subcellular distribution of NS5A. This phenotype was shared by viruses with other mutations in the low-complexity sequence I (LCS I), namely, S229D, S232A, and S235D, but not by viruses with mutations that caused a comparable replication defect that mapped to domain II of NS5A (P315A, L321A). Together with other components of the genome replication complex (NS3, double-stranded RNA, and cellular lipids, including phosphatidylinositol 4-phosphate), the mutation in NS5A was restricted to a perinuclear region. This phenotype was not due to cell confluence or another environmental factor and could be partially transcomplemented by wild-type NS5A. We propose that serine phosphorylation within LCS I may regulate the assembly of an active genome replication complex.

Importance

The mechanisms by which hepatitis C virus replicates its RNA genome remain poorly characterized. We show here that phosphorylation of the viral nonstructural protein NS5A at serine residues is important for the efficient assembly of a complex that is able to replicate the viral genome. This research implicates cellular protein kinases in the control of virus replication and highlights the need to further understand the interplay between the virus and the host cell in order to develop potential avenues for future antiviral therapy.

SUBMITTER: Ross-Thriepland D

PROVIDER: S-EPMC4337517 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Serine phosphorylation of the hepatitis C virus NS5A protein controls the establishment of replication complexes.

Ross-Thriepland Douglas D Mankouri Jamel J Harris Mark M

Journal of virology 20141231 6

<h4>Unlabelled</h4>The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is highly phosphorylated and involved in both virus genome replication and virion assembly. We and others have identified serine 225 in NS5A to be a phosphorylation site, but the function of this posttranslational modification in the virus life cycle remains obscure. Here we describe the phenotype of mutants with mutations at serine 225; this residue was mutated to either alanine (S225A; phosphoablatant) or aspartic a ...[more]

PMID: 25552726

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Project description:Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a phosphoprotein that plays key, yet poorly defined, roles in both virus genome replication and virion assembly/release. It has been proposed that differential phosphorylation could act as a switch to regulate the various functions of NS5A; however, the mechanistic details of the role of this posttranslational modification in the virus life cycle remain obscure. We previously reported (D. Ross-Thriepland, J. Mankouri, and M. Harris, J Virol 89:3123-3135, 2015, doi:10.1128/JVI.02995-14) a role for phosphorylation at serine 225 (S225) of NS5A in the regulation of JFH-1 (genotype 2a) genome replication. A phosphoablatant (S225A) mutation resulted in a 10-fold reduction in replication and a perinuclear restricted distribution of NS5A, whereas the corresponding phosphomimetic mutation (S225D) had no phenotype. To determine the molecular mechanisms underpinning this phenotype we conducted a label-free proteomics approach to identify cellular NS5A interaction partners. This analysis revealed that the S225A mutation disrupted the interactions of NS5A with a number of cellular proteins, in particular the nucleosome assembly protein 1-like protein 1 (NAP1L1), bridging integrator 1 (Bin1, also known as amphiphysin II), and vesicle-associated membrane protein-associated protein A (VAP-A). These interactions were validated by immunoprecipitation/Western blotting, immunofluorescence, and proximity ligation assay. Importantly, small interfering RNA (siRNA)-mediated knockdown of NAP1L1, Bin1 or VAP-A impaired viral genome replication and recapitulated the perinuclear redistribution of NS5A seen in the S225A mutant. These results demonstrate that S225 phosphorylation regulates the interactions of NS5A with a defined subset of cellular proteins. Furthermore, these interactions regulate both HCV genome replication and the subcellular localization of replication complexes.IMPORTANCE Hepatitis C virus is an important human pathogen. The viral nonstructural 5A protein (NS5A) is the target for new antiviral drugs. NS5A has multiple functions during the virus life cycle, but the biochemical details of these roles remain obscure. NS5A is known to be phosphorylated by cellular protein kinases, and in this study, we set out to determine whether this modification is required for the binding of NS5A to other cellular proteins. We identified 3 such proteins and show that they interacted only with NS5A that was phosphorylated on a specific residue. Furthermore, these proteins were required for efficient virus replication and the ability of NS5A to spread throughout the cytoplasm of the cell. Our results help to define the function of NS5A and may contribute to an understanding of the mode of action of the highly potent antiviral drugs that are targeted to NS5A.

Project description:The nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) is a phosphoprotein with two phosphorylation states: hypo- and hyperphosphorylation. Genetic mutation studies have demonstrated a cluster of serine residues responsible for NS5A hyperphosphorylation and functions in viral replication and assembly; however, the phosphorylation levels and potential interactions among the serine residues are unclear. We used three specific antibodies to measure NS5A phosphorylation at S222, S235, and S238 that were identified in our previous proteomics study. In the HCV (J6/JFH-1)-infected Huh7.5.1 cells, S222 phosphorylation was barely detected, whereas S235 phosphorylation and S238 phosphorylation were always detected in parallel in time and intracellular spaces. S235A mutation eliminated S238 phosphorylation whereas S238A mutation did not affect S235 phosphorylation, indicating that S235 phosphorylation occurs independently of S238 phosphorylation while S238 phosphorylation depends on S235 phosphorylation. In line with this, immunoprecipitation coupled with immunoblotting showed that S235 phosphorylation existed alone without S238 phosphorylation, whereas S238 phosphorylation existed only when S235 was phosphorylated on the same NS5A molecule. S235-phosphorylated NS5A constituted the primary hyperphosphorylated NS5A species. S235A mutation blunted viral replication, whereas S238A mutation did not affect replication. We concluded that S235 is the primary NS5A hyperphosphorylation site required for HCV replication. S238 is likely phosphorylated by casein kinase Iα, which requires a priming phosphorylation at S235.IMPORTANCE It has been known for years that the hepatitis C virus nonstructural protein 5A (NS5A) undergoes transition between two phosphorylation states: hypo- and hyperphosphorylation. It is also known that a cluster of serine residues is responsible for NS5A hyperphosphorylation and functions; however, the primary serine residue responsible for NS5A hyperphosphorylation is not clear. Here, we show for the first time that serine 235-phosphorylated NS5A constitutes the primary hyperphosphorylated NS5A species required for viral replication. We also show that NS5A phosphorylation among the serine residues is interdependent and occurs in a directional manner, i.e., phosphorylation at serine 235 leads to phosphorylation at serine 238. Our data provide the first proof-of-principle evidence that NS5A undergoes a sequential phosphorylation cascade.

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:The propagation of the hepatitis C virus (HCV) is regulated in part by the phosphorylation of its nonstructural protein NS5A that undergoes sequential phosphorylation on several highly conserved serine residues and switches from a hypo- to a hyperphosphorylated state. Previous studies have shown that NS5A sequential phosphorylation requires NS3 encoded on the same NS3-NS4A-NS4B-NS5A polyprotein. Subtle mutations in NS3 without affecting its protease activity could affect NS5A phosphorylation. Given the ATPase domain in the NS3 COOH terminus, we tested whether NS3 participates in NS5A phosphorylation similarly to the nucleoside diphosphate kinase-like activity of the rotavirus NSP2 nucleoside triphosphatase (NTPase). Mutations in the NS3 ATP-binding motifs blunted NS5A hyperphosphorylation and phosphorylation at serines 225, 232, and 235, whereas a mutation in the RNA-binding domain did not. The phosphorylation events were not rescued with wild-type NS3 provided in trans. When provided with an NS3 ATPase-compatible ATP analog, N6-benzyl-ATP-γ-S, thiophosphorylated NS5A was detected in the cells expressing the wild-type NS3-NS5B polyprotein. The thiophosphorylation level was lower in the cells expressing NS3-NS5B with a mutation in the NS3 ATP-binding domain. In vitro assays with a synthetic peptide and purified wild-type NS3 followed by dot blotting and mass spectrometry found weak NS5A phosphorylation at serines 222 and 225 that was sensitive to an inhibitor of casein kinase Iα but not helicase. When casein kinase Iα was included in the assay, much stronger phosphorylation was observed at serines 225, 232, and 235. We concluded that NS5A sequential phosphorylation requires the ATP-binding domain of the NS3 helicase and that casein kinase Iα is a potent NS5A kinase. IMPORTANCE For more than 20 years, NS3 was known to participate in NS5A sequential phosphorylation. In the present study, we show for the first time that the ATP-binding domain of NS3 is involved in NS5A phosphorylation. In vitro assays showed that casein kinase Iα is a very potent kinase responsible for NS5A phosphorylation at serines 225, 232, and 235. Our data suggest that ATP binding by NS3 probably results in conformational changes that recruit casein kinase Iα to phosphorylate NS5A, initially at S225 and subsequently at S232 and S235. Our discovery reveals intricate requirements of the structural integrity of NS3 for NS5A hyperphosphorylation and HCV replication.

Dataset Information

Serine phosphorylation of the hepatitis C virus NS5A protein controls the establishment of replication complexes.

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Importance

Publications

Serine phosphorylation of the hepatitis C virus NS5A protein controls the establishment of replication complexes.

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