Internal proteolysis of the NS3 protein specified by dengue virus 2

The NS3 protein of flaviviruses is a multifunctional polypeptide required for virus replication. Enzymic activities that have been demonstrated or predicted from the presence of sequence motifs include protease, NTPase, helicase and RNA triphosphatase. Both full-length and truncated forms of NS3 have been identified in infected cells. To examine internal cleavage of the NS3 protein of dengue virus 2 (DEN-2), infected cells or COS cells transfected with cDNA encoding NS2B/3 were radiolabelled and immunoprecipitated with antiserum against NS3 or hyperimmune mouse ascitic fluid, The polypeptides detected were NS2B/3 (M(r) 83000), NS3 (M(r) 69000), NS3' (M(r) 50000) and NS3 '' (M(r) 19000). The latter polypeptide has not been previously identified. For DEN-2, it has been proposed that NS3' results from cleavage at the site ...R(457)R\GR(460)... within an RNA helicase sequence motif of NS3. Our results demonstrated that cleavage occurred at this site, and that prior cleavage between NS2B/NS3 was not necessary.