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Aurintricarboxylic acid modulates the affinity of hepatitis C virus NS3 helicase for both nucleic acid and ATP.

ABSTRACT: Aurintricarboxylic acid (ATA) is a potent inhibitor of many enzymes needed for cell and virus replication, such as polymerases, helicases, nucleases, and topoisomerases. This study examines how ATA interacts with the helicase encoded by the hepatitis C virus (HCV) and reveals that ATA interferes with both nucleic acid and ATP binding to the enzyme. We show that ATA directly binds HCV helicase to prevent the enzyme from interacting with nucleic acids and to modulate the affinity of HCV helicase for ATP, the fuel for helicase action. Amino acid substitutions in the helicase DNA binding cleft or its ATP binding site alter the ability of ATA to disrupt helicase-DNA interactions. These data, along with molecular modeling results, support the notion that an ATA polymer binds between Arg467 and Glu493 to prevent the helicase from binding either ATP or nucleic acids. We also characterize how ATA affects the kinetics of helicase-catalyzed ATP hydrolysis, and thermodynamic parameters describing the direct interaction between HCV helicase and ATA using microcalorimetry. The thermodynamics of ATA binding to HCV helicase reveal that ATA binding does not mimic nucleic acid binding in that ATA binding is driven by a smaller enthalpy change and an increase in entropy.

SUBMITTER: Shadrick WR

PROVIDER: S-EPMC4272346 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Aurintricarboxylic acid modulates the affinity of hepatitis C virus NS3 helicase for both nucleic acid and ATP.

Shadrick William R WR Mukherjee Sourav S Hanson Alicia M AM Sweeney Noreena L NL Frick David N DN

Biochemistry 20130826 36

Aurintricarboxylic acid (ATA) is a potent inhibitor of many enzymes needed for cell and virus replication, such as polymerases, helicases, nucleases, and topoisomerases. This study examines how ATA interacts with the helicase encoded by the hepatitis C virus (HCV) and reveals that ATA interferes with both nucleic acid and ATP binding to the enzyme. We show that ATA directly binds HCV helicase to prevent the enzyme from interacting with nucleic acids and to modulate the affinity of HCV helicase f ...[more]

PMID: 23947785

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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

Aurintricarboxylic acid modulates the affinity of hepatitis C virus NS3 helicase for both nucleic acid and ATP.

Publications

Aurintricarboxylic acid modulates the affinity of hepatitis C virus NS3 helicase for both nucleic acid and ATP.

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