MULTIPLE ENZYMATIC-ACTIVITIES ASSOCIATED WITH RECOMBINANT NS3 PROTEINOF HEPATITIS-C VIRUS

The hepatitis C virus (HCV) nonstructural 3 protein (NS3) contains at least two domains associated with multiple enzymatic activities; a ser ine protease activity resides in the N-terminal one-third of the prote in, whereas RNA helicase activity and RNA-stimulated nucleoside tripho sphatase activity are associated with the C-terminal portion. To study the possible mutual influence of these enzymatic activities, a full l ength NS3 polypeptide of 67 kDa was expressed as a nonfusion protein i n Escherichia coli, purified to homogeneity, and shown to retain all t hree enzymatic activities. The protease activity of the full-length NS 3 was strongly dependent on the activation by a synthetic peptide span ning the central hydrophobic core of the NS4A cofactor. Once complexed with the NS4A-derived peptide, the full-length NS3 protein and the is olated N-terminal protease domain cleaved synthetic peptide substrates with comparable efficiency. We show that, as in the case of the isola ted protease domain, the protease activity of full-length NS3 undergoe s inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B and NS5A-NS5B. We have also characteri zed and quantified the NS3 ATPase, RNA helicase? and RNA-binding activ ities under optimized reaction conditions. Compared with the isolated N-terminal and C-terminal domains, recombinant full-length NS3 did not show significant differences in the three enzymatic activities analyz ed in independent in vitro assays. We have further explored the possib le interdependence of the NS3 N-terminal and e-terminal domains by ana lyzing the effect of polynucleotides on the modulation of all NS3 enzy matic functions. Our results demonstrated that the observed inhibition of the NS3 proteolytic activity by single-stranded RNA is mediated by direct interaction with the protease domain rather than with the heli case RNA-binding domain.