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Domain 3 of NS5A protein from the hepatitis C virus has intrinsic alpha-helical propensity and is a substrate of cyclophilin A.

ABSTRACT: Nonstructural protein 5A (NS5A) is essential for hepatitis C virus (HCV) replication and constitutes an attractive target for antiviral drug development. Although structural data for its in-plane membrane anchor and domain D1 are available, the structure of domains 2 (D2) and 3 (D3) remain poorly defined. We report here a comparative molecular characterization of the NS5A-D3 domains of the HCV JFH-1 (genotype 2a) and Con1 (genotype 1b) strains. Combining gel filtration, CD, and NMR spectroscopy analyses, we show that NS5A-D3 is natively unfolded. However, NS5A-D3 domains from both JFH-1 and Con1 strains exhibit a propensity to partially fold into an ?-helix. NMR analysis identifies two putative ?-helices, for which a molecular model could be obtained. The amphipathic nature of the first helix and its conservation in all genotypes suggest that it might correspond to a molecular recognition element and, as such, promote the interaction with relevant biological partner(s). Because mutations conferring resistance to cyclophilin inhibitors have been mapped into NS5A-D3, we also investigated the functional interaction between NS5A-D3 and cyclophilin A (CypA). CypA indeed interacts with NS5A-D3, and this interaction is completely abolished by cyclosporin A. NMR heteronuclear exchange experiments demonstrate that CypA has in vitro peptidyl-prolyl cis/trans-isomerase activity toward some, but not all, of the peptidyl-prolyl bonds in NS5A-D3. These studies lead to novel insights into the structural features of NS5A-D3 and its relationships with CypA.

SUBMITTER: Verdegem D

PROVIDER: S-EPMC3121464 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Domain 3 of NS5A protein from the hepatitis C virus has intrinsic alpha-helical propensity and is a substrate of cyclophilin A.

Verdegem Dries D Badillo Aurélie A Wieruszeski Jean-Michel JM Landrieu Isabelle I Leroy Arnaud A Bartenschlager Ralf R Penin François F Lippens Guy G Hanoulle Xavier X

The Journal of biological chemistry 20110413 23

Nonstructural protein 5A (NS5A) is essential for hepatitis C virus (HCV) replication and constitutes an attractive target for antiviral drug development. Although structural data for its in-plane membrane anchor and domain D1 are available, the structure of domains 2 (D2) and 3 (D3) remain poorly defined. We report here a comparative molecular characterization of the NS5A-D3 domains of the HCV JFH-1 (genotype 2a) and Con1 (genotype 1b) strains. Combining gel filtration, CD, and NMR spectroscopy ...[more]

PMID: 21489988

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

Dataset Information

Domain 3 of NS5A protein from the hepatitis C virus has intrinsic alpha-helical propensity and is a substrate of cyclophilin A.

Publications

Domain 3 of NS5A protein from the hepatitis C virus has intrinsic alpha-helical propensity and is a substrate of cyclophilin A.

Similar Datasets

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