The inhibition of cAMP-dependent protein kinase by full-length hepatitis Cvirus NS3/4A complex is due to ATP hydrolysis

Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. T he HCV non-structural protein NS3 complexes with NS4A and has three enzymat ic activities: a proteinase and a helicase/NTPase, Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent pro tein kinase (PKA C-subunit). Here we demonstrate that full-length, catalyti cally active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunit in vitro. This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved he licase motif II, both of which also abolished NTPase activity. As PKA C-sub unit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase ac tivity), we hypothesized that PKA C-subunit inhibition could be due to NS3/ 4A-mediated ATP hydrolysis, This was confirmed by experiments in which a co nstant ATP concentration was maintained by addition of an ATP regeneration system-under these conditions PKA C-subunit inhibition was not observed, In terestingly, the mutations also abrogated the ability of wild-type NS3/4A t o inhibit the PKA-regulated transcription factor CREB in transiently transf ected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit, However, in vivo effects of NS3/4A suggest that ATPase activity may play a role in viral pathology i n the infected liver.