NS5 polymerase stimulates RNA duplex unwinding by the Orthoflavivirus NS3 helicase

The NS3 helicases from the Flaviviridae family of viruses exhibit nucleotide-hydrolysis-dependent, nucleic-acid-unwinding activity. The RNA unwinding activity for NS3 helicases from the Orthoflavivirus genus has not been fully explored and contrasts with NS3 helicase from hepatitis C virus (HCV) of the Hepacivirus genus, which has thus far served as the prototypical model enzyme from this family of viruses. To begin to understand the functional differences between flavivirus NS3 helicases, we first developed an expression and purification system for full-length untagged NS3 protein from West Nile virus and Zika virus. Both enzymes exhibit RNA-stimulated ATPase activity and are dependent on the nucleoside triphosphatase active site of the enzyme. Unlike HCV NS3, Orthoflavivirus NS3s do not efficiently pre-assemble on a 3'-ssRNA-tailed dsRNA substrate in the absence of Mg²⁺-ATP, which is a prerequisite for the formation of a productive HCV NS3-RNA complex that can exhibit a rapid burst of RNA unwinding. Instead, to observe RNA unwinding by West Nile virus and Zika virus NS3s, low Mg²⁺-ATP concentrations are required at a time coincident when NS3 encounters the RNA substrate. In addition, we find that Orthoflavivirus NS3s require translocation beyond the displaced strand to completely unwind a dsRNA substrate. Last, we find that Orthoflavivirus NS5 stimulates the ability of NS3 to unwind dsRNA. These results suggest that functional differences exist between the flavivirus NS3 helicases and illuminate that Orthoflavivirus NS3s require a functional interaction with the NS5 protein for coordination of its activity, as it is believed these two proteins constitute the viral replicase.