PROCESSING OF DENGUE TYPE-4 AND OTHER FLAVIVIRUS NONSTRUCTURAL PROTEINS

Dengue type 4 (DEN4) and other flaviviruses employ host and viral prot eases for polyprotein processing. Most proteolytic cleavages in the DE N4 nonstructural protein (NS) region are mediated by the viral NS2B-NS 3 protease. The N-terminal third of NS3, containing sequences homologo us to serine protease active sites, is the protease domain. To determi ne required sequences in NS2B, deletions were introduced into DEN4 NS2 B-30%NS3 cDNA and the expressed polyproteins assayed for self-cleavage . A 40 amino acid segment within NS2B was essential. Sequence analysis of NS2B predicts that this segment constitutes a hydrophilic domain s urrounded by hydrophobic regions. Hydophobicity profiles of other flav ivirus NS2Bs show similar patterns. Cleavage of DEN4 NS1-NS2A requires an octapeptide sequence at the NS1 C terminus and downstream NS2A. Co mparison of the analogous octapeptide sequences among flaviviruses ind icates a consensus cleavage sequence of (P8)/Met/Leu-Val-Xaa-Ser-Xaa-V al-Ala(P1) where Xaa are nonconserved amino acids. The effects on clea vage of amino acid substitutions in this consensus sequence were analy zed. Most substitutions of the conserved residues interfered with clea vage, whereas substitutions of non-conserved residues had little or no effect. These findings indicate that the responsible enzyme recognize s well-defined sequences at the cleavage site.