Characterization of amino acids in the Non‐Structural Protein 2B Hydrophilic domain required for efficient activation of the Flavivirus Non‐Structural 3 Protein Serine Protease

Abstract

West Nile virus (WNV) and dengue virus type 2 (DV2) are pathogens causing widespread disease for which there is no cure. These viruses encode a two-component protease that is essential for polyprotein processing. Protease activation requires interaction between the nonstructural (NS) protein 3 and the NS2B. Previous studies of NS2B identified a conserved minimal hydrophilic domain of 40 amino acids (NS2BH) required for protease activation. To understand the mechanism of NS3 protease (NS3pro) activation by NS2BH, chimeric proteases were constructed in which either a complete substitution of NS2BH from one virus (DV2 and WNV) for that of the other; or just the N- or C-terminus of NS2BH. In vitro protease assays revealed complete replacement or substitution of the C-terminus of NS2BH resulted in loss of activity. Chimeras containing N-terminal substitutions of NS2BH were found to have normal activity. These results indicate the C-terminus of NS2BH is important for virus specific NS3pro activation. DV2 replicon studies with a similar chimera resulted in a replication defective replicon. Additional NS2BH mutants containing specific residue changes should identify replication important substitutions.