The unwinding of double-stranded RNA intermediates is a critical component for the replication of flavivirus RNA genomes. This function is achieved by the C-terminal helicase domain of nonstructural protein 3 (NS3). As a member of the superfamily 2 (SF2) helicases, NS3 is known to require the binding and hydrolysis of ATP/NTP to translocate along and unwind double-stranded nucleic acids. However, the mechanism of energy transduction between the ATP and RNA binding pockets is not well understood. Previous molecular dynamics simulations published by our group have identified Motif V as a potential "communication hub" for this energy transduction pathway. In order to investigate the role of Motif V in this process, a study combining molecular dynamics, biochemistry and virology has been employed.Mutations of Motif V were tested in both replicon and recombinant protein systems to investigate viral genome replication, RNA binding affinity, ATP hydrolysis activity and helicase unwinding activity. Using these analyses, we found that T407A and S411A in Motif V demonstrated increased turnover rates, suggesting that the mutations causes the helicase to unwind dsRNA more quickly than WT. Additionally, simulations of each mutant were used to probe structural changes within NS3 caused by each mutation. These simulations indicate that Motif V controls communication between the ATP binding pocket and the helical gate. These data help define the linkage between ATP hydrolysis and helicase activity within NS3 and provide insight into the biophysical mechanisms for ATPase driven NS3 helicase function.
Arthropod-borne flaviviruses, such as yellow fever virus, Japanese encephalitis virus, Zika virus, WestNile virus (WNV) and dengue virus, are a major health concern in the tropical and subtropical regions of the world (1, 2). Infection from these viruses cause disease symptoms ranging from flu-like illness to encephalitis, hemorrhagic fever, coma, and potentially death (3). Over half of the world population is at risk for infection from one or more of these viruses (4, 5). Dengue virus, specifically, infects around 50 million people each year, and of those individuals, 20,000 contract dengue hemorrhagic fever leading to their mortality (6). Additionally, WNV over the past 20 years within the 48 continental United States has around 50,000 clinical infections with a 5% mortality rate (7-9). Currently, there are no approved antiviral drugs for treating flaviviral infections and the vaccines in circulation, like the yellow fever vaccine, are not readily available worldwide for most flaviviruses (10-12). In order to develop new antiviral treatments (drugs and vaccines) for these viruses, a fundamental understanding of how these flaviviruses replicate is required.