Internal processing of hepatitis C virus NS3 protein

Hepatitis C virus (HCV) NS3 protein contains at least three enzymatic activ ities: NS2-3 protease, NS3 serine protease, and NTPase/RNA helicase. It has been shown that NS2/3 cleavage is mediated by NS2-3 protease, whereas NS3 serine protease is responsible for the other four cleavage sites of the non structural (NS) region. In this study, we showed that the internal cleavage of NS3 protein produced two products of 49 kDa (NS3a) and 23 kDa (NS3b) wh en the entire NS3 region (aa 1027-1657) or the whole open reading frame (aa 1-3010) was expressed in mammalian and insect cells. By means of site-dire cted mutagenesis, we demonstrated that NS3a/NS3b cleavage occurs within the RNA helicase sequence motif that is highly conserved in the Flaviviridae f amily and that neither NS2-3 protease nor NS3 serine protease was responsib le for this cleavage. The NS3 protease of flaviviruses, dengue virus type 2 , for example, has been shown to mediate the internal cleavage of NS3. The NS3 proteins of HCV and dengue virus may thus be cleaved internally at the same sequence by different mechanisms of proteolysis. Also discussed is a p ossible role for the internal processing of HCV NS3 in the viral life cycle and its pathogenesis. (C) 1999 Academic Press.