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The hepatitis C virus NS3 protein: a model RNA helicase and potential drug target.

ABSTRACT: The C-terminal portion of hepatitis C virus (HCV) nonstructural protein 3 (NS3) forms a three domain polypeptide that possesses the ability to travel along RNA or single-stranded DNA (ssDNA) in a 3' to 5' direction. Fueled byATP hydrolysis, this movement allows the protein to displace complementary strands of DNA or RNA and proteins bound to the nucleic acid. HCV helicase shares two domains common to other motor proteins, one of which appears to rotate upon ATP binding. Several models have been proposed to explain how this conformational change leads to protein movement and RNA unwinding, but no model presently explains all existing experimental data. Compounds recently reported to inhibit HCV helicase, which include numerous small molecules, RNA aptamers and antibodies, will be useful for elucidating the role of a helicase in positive-sense single-stranded RNA virus replication and might serve as templates for the design of novel antiviral drugs.

SUBMITTER: Frick DN

PROVIDER: S-EPMC3571657 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

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The hepatitis C virus NS3 protein: a model RNA helicase and potential drug target.

Frick David N DN

Current issues in molecular biology 20070101 1

The C-terminal portion of hepatitis C virus (HCV) nonstructural protein 3 (NS3) forms a three domain polypeptide that possesses the ability to travel along RNA or single-stranded DNA (ssDNA) in a 3' to 5' direction. Fueled byATP hydrolysis, this movement allows the protein to displace complementary strands of DNA or RNA and proteins bound to the nucleic acid. HCV helicase shares two domains common to other motor proteins, one of which appears to rotate upon ATP binding. Several models have been ...[more]

PMID: 17263143

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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

The hepatitis C virus NS3 protein: a model RNA helicase and potential drug target.

Publications

The hepatitis C virus NS3 protein: a model RNA helicase and potential drug target.

Similar Datasets

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