ATPase coupling in the processive RNA helicase NS3 from hepatitis C virus

Abstract

Hepatitis C virus (HCV) NS3 motor protein contains both RNA helicase and serine protease activities that are vital to HCV replication in infected cells. NS3 belongs to the DExD/H‐box family of RNA helicases, which recognize short structured stretches of RNA and utilize ATP to remodel RNA and RNA‐protein complex structures. In contrast, NS3 is hypothesized to processively unwind the + and ‐ strands of the entire HCV RNA genome during viral replication, which likely requires that NS3 ATPase activity is tightly coupled to helicase translocation. To characterize the coupling of NS3 ATPase and translocation activities, we utilized equilibrium binding, steady‐state and transient kinetic biophysical methods. We compared a series of RNA substrates and found that RNA length affects both the concentration‐dependence and maximal rate of the RNA‐stimulated ATPase activity. Using transient kinetics, we demonstrated that the steady‐state rate of ATPase cycling is partially limited by a nucleotide‐dependent isomerization of the NS3‐RNA complex. On shorter RNAs, translocating NS3 protomers fall off the 5′ end of the nucleic acid track, and undergo this isomerization upon rebinding the ssRNA in order to restart productive translocation and rapid RNA‐stimulated ATPase. NS3 with an intact protease domain engages more productively with the RNA lattice, which increases apparent processivity of the ssRNA translocase.