title: The NTPase/helicase domain of hepatitis C virus nonstructural protein 3 inhibits protein kinase C independently of its NTPase activity.
abstract: Helicase motif VI is a short arginine-rich motif within the NTPase/helicase domain of the non-structural protein 3 (NS3) of the hepatitis C virus (HCV). We previously demonstrated that it reduces the catalytic activity and intracellular shuttling of protein kinase C (PKC). Thus, NS3-mediated PKC inhibition may be involved in HCV-associated hepatocellular carcinoma (HCC). In this study, we expand on our earlier results, which were obtained in experiments with short fragments of NS3, to show for the first time that the catalytically active, longer C-terminal NTPase/helicase of NS3 acts as a potent PKC inhibitor in vitro. PKC inhibition assays with the NTPase-inactive mutant NS3h-D1316A revealed a mixed type kinetic inhibition pattern. A broad range of 11 PKC isotypes was tested and all of the PKC isotypes were inhibited with IC₅₀-values in the low micromolar range. These findings were confirmed for the wild-type NTPase/helicase domain in a non-radiometric PKC inhibition assay with ATP regeneration to rule out any effect of ATP hydrolysis caused by its NTPase activity. PKCα was inhibited with a micromolar IC₅₀ in this assay, which compares well with our result for NS3h-D1316A (IC₅₀ = 0.7 μM). In summary, these results confirm that catalytically active NS3 NTPase/helicase can act in an analogous manner to shorter NS3 fragments as a pseudosubstrate inhibitor of PKC.
authors.affiliation: Department of Virology, Bernhard-Nocht-Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany. philip.hartjen@gmail.com
authors.forename: Philip
authors.initials: P
authors.lastname: Hartjen
authors.forename: Bastian
authors.initials: B
authors.lastname: Höchst
authors.forename: Denise
authors.initials: D
authors.lastname: Heim
authors.forename: Henning
authors.initials: H
authors.lastname: von der Kammer
authors.forename: Judith
authors.initials: J
authors.lastname: Lucke
authors.forename: Michael
authors.initials: M
authors.lastname: Reinholz
authors.forename: Andrea
authors.initials: A
authors.lastname: Baier
authors.forename: Ralf
authors.initials: R
authors.lastname: Smeets
authors.forename: Henning
authors.initials: H
authors.lastname: Wege
authors.forename: Peter
authors.initials: P
authors.lastname: Borowski
authors.forename: Julian
authors.initials: J
authors.lastname: Schulze Zur Wiesch
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D004591:Electrophoresis, Polyacrylamide Gel; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D043583:Nucleoside-Triphosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000255:Adenosine Triphosphate; D043583:Nucleoside-Triphosphatase; D011493:Protein Kinase C; D020365:RNA Helicases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
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