Using constructs that encode the individual West Nile virus (WNV) NS3helicase (NS3hel) and NS3hel linked to the hydrophilic, N-terminal 1–50 sequence of NS4A, we demonstrated that the presence of NS4A allows NS3hel to conserve energy in the course of oligonucleotide substrate unwinding. Using NS4A mutants, we also determined that the C-terminal acidic EELPD/E motif of NS4A, which appears to be functionally similar to the acidic EFDEMEE motif of hepatitis C virus (HCV) NS4A, is essential for regulating the ATPase activity of NS3hel. We concluded that, similar to HCV NS4A, NS4A of WNV acts as a cofactor for NS3hel and allows helicase to sustain the unwinding rate of the viral RNA under conditions of ATP deficiency.


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