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.
Highlights
Chronic hepatitis C virus (HCV) infection is a major risk factor for the development of hepatocellular carcinoma (HCC) [1]
A protein kinase C (PKC) activity assay containing an adenosine triphosphate (ATP)-regenerating system was employed to confirm these results for wild-type non-structural protein 3 (NS3)-NTPase/helicase and rule out the influence of the D1316A point mutation on the accessibility of the arginine-rich stretch
Together with our new finding that NS3h-D1316A acts as a mixed-type inhibitor of rat brain PKC, these novel results strongly suggest that NS3-NTPase/helicase-mediated inhibition of PKC is at least in part due to pseudosubstrate inhibition and follows a complex mechanism similar to the PKC inhibition by the shorter fragments analyzed in our earlier studies conducted [5, 20]
Summary
Chronic hepatitis C virus (HCV) infection is a major risk factor for the development of hepatocellular carcinoma (HCC) [1]. We showed that within its NTPase/helicase domain, NS3 contains an arginine-rich sequence motif consisting of residues 1487 to 1500 of HCV polyprotein, known as helicase motif VI [2]. This motif strongly resembles the pseudosubstrate sequence within the auto-regulatory domain of protein kinase C (PKC) [3, 4] and it interacts with the active site of PKC, thereby inhibiting the catalytic activity and function of this enzyme [5, 6]. Our previous studies on the interaction between HCV-NS3 and PKC were only conducted with smaller, catalytically inactive fragments of NS3. We show that all PKC isotypes are inhibited by NS3 with IC50 values in the low micromolar range independently of the NS3 NTPase activity
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