Yellow Fever Virus NS2B–NS3 Protease: Charged-to-Alanine Mutagenesis and Deletion Analysis Define Regions Important for Protease Complex Formation and Function

Charged-to-alanine substitutions and deletions within the yellow fever virus NS2B–NS3181 protease were analyzed for effects on protease function. During cell-free translation of NS2B–3181 polyproteins, mutations at three charge clusters markedly impaired cis cleavage activity: a single N-terminal cluster in the conserved domain of NS2B (residues ELKK52–55) and two in NS3 (ED21–22, and residue H47). These mutations inhibited other protease-dependent cleavages of a transiently expressed nonstructural polyprotein, although differential effects occurred. NS2B and NS3181 proteins harboring these mutations were impaired in their ability to associate for trans cleavage activity. N-terminal deletions in NS3 also implicated residues ED21–22 in the association with NS2B. Deletions within NS2B revealed that the conserved domain alone provided minimal cofactor activity, with optimal function requiring both flanking hydrophobic regions. NS2B–3181- and NS3181–green fluorescent protein fusion proteins were used to determine the intracellular distribution of the protease complex. The former localized in membrane-based vesicular structures, whereas the latter localized poorly. The data suggest that NS2B–NS3 complex formation requires charge interactions involving the N-terminus of the conserved domain of NS2B and 22 N-terminal residues of NS3. A role for the putative transmembrane regions of NS2B in targeting of NS3 to intracellular membranes is also suggested.