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The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation.

ABSTRACT: Hepatitis C virus (HCV) has been the subject of intensive studies for nearly two decades. Nevertheless, some aspects of the virus life cycle are still a mystery. The HCV nonstructural protein 5A (NS5A) has been shown to be a modulator of cellular processes possibly required for the establishment of viral persistence. NS5A is heavily phosphorylated, and a switch between a basally phosphorylated form of NS5A (p56) and a hyperphosphorylated form of NS5A (p58) seems to play a pivotal role in regulating HCV replication. Using kinase inhibitors that specifically inhibit the formation of NS5A-p58 in cells, we identified the CKI kinase family as a target. NS5A-p58 increased upon overexpression of CKI-alpha, CKI-delta, and CKI-epsilon, whereas the RNA interference of only CKI-alpha reduced NS5A hyperphosphorylation. Rescue of inhibition of NS5A-p58 was achieved by CKI-alpha overexpression, and we demonstrated that the CKI-alpha isoform is targeted by NS5A hyperphosphorylation inhibitors in living cells. Finally, we showed that down-regulation of CKI-alpha attenuates HCV RNA replication.

SUBMITTER: Quintavalle M

PROVIDER: S-EPMC1642135 | biostudies-literature | 2006 Nov

REPOSITORIES: biostudies-literature

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The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation.

Quintavalle Manuela M Sambucini Sonia S Di Pietro Chiara C De Francesco Raffaele R Neddermann Petra P

Journal of virology 20060830 22

Hepatitis C virus (HCV) has been the subject of intensive studies for nearly two decades. Nevertheless, some aspects of the virus life cycle are still a mystery. The HCV nonstructural protein 5A (NS5A) has been shown to be a modulator of cellular processes possibly required for the establishment of viral persistence. NS5A is heavily phosphorylated, and a switch between a basally phosphorylated form of NS5A (p56) and a hyperphosphorylated form of NS5A (p58) seems to play a pivotal role in regulat ...[more]

PMID: 16943283

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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 I?, 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:UnlabelledNonstructural 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.

Project description:The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIII?) is an essential host factor of hepatitis C virus (HCV) replication. PI4KIII? catalyzes the synthesis of phosphatidylinositol 4-phosphate (PI4P) accumulating in HCV replicating cells due to enzyme activation resulting from its interaction with nonstructural protein 5A (NS5A). This study describes the interaction between PI4KIII? and NS5A and its mechanistic role in viral RNA replication. We mapped the NS5A sequence involved in PI4KIII? interaction to the carboxyterminal end of domain 1 and identified a highly conserved PI4KIII? functional interaction site (PFIS) encompassing seven amino acids, which are essential for viral RNA replication. Mutations within this region were also impaired in NS5A-PI4KIII? binding, reduced PI4P levels and altered the morphology of viral replication sites, reminiscent to the phenotype observed by silencing of PI4KIII?. Interestingly, abrogation of RNA replication caused by mutations in the PFIS correlated with increased levels of hyperphosphorylated NS5A (p58), indicating that PI4KIII? affects the phosphorylation status of NS5A. RNAi-mediated knockdown of PI4KIII? or pharmacological ablation of kinase activity led to a relative increase of p58. In contrast, overexpression of enzymatically active PI4KIII? increased relative abundance of basally phosphorylated NS5A (p56). PI4KIII? therefore regulates the phosphorylation status of NS5A and viral RNA replication by favoring p56 or repressing p58 synthesis. Replication deficiencies of PFIS mutants in NS5A could not be rescued by increasing PI4P levels, but by supplying functional NS5A, supporting an essential role of PI4KIII? in HCV replication regulating NS5A phosphorylation, thereby modulating the morphology of viral replication sites. In conclusion, we demonstrate that PI4KIII? activity affects the NS5A phosphorylation status. Our results highlight the importance of PI4KIII? in the morphogenesis of viral replication sites and its regulation by facilitating p56 synthesis.

Dataset Information

The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation.

Publications

The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation.

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