The unwinding of dsRNA intermediates is critical for the replication of flavivirus RNA genomes. This activity is provided by the C-terminal helicase domain of viral nonstructural protein 3 (NS3). As a member of the superfamily 2 (SF2) helicases, NS3 requires 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 conducted by our group have identified Motif V as a potential “communication hub” for this energy transduction pathway. To investigate the role of Motif V in this process, here we combined molecular dynamics, biochemistry, and virology approaches. We tested Motif V mutations in both the replicon and recombinant protein systems to investigate viral genome replication, RNA-binding affinity, ATP hydrolysis activity, and helicase-mediated unwinding activity. We found that the T407A and S411A substitutions in NS3 reduce viral replication and increase the helicase-unwinding turnover rates by 1.7- and 3.5-fold, respectively, suggesting that flaviviruses may use suboptimal NS3 helicase activity for optimal genome replication. Additionally, we used simulations of each mutant 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 results help define the linkage between ATP hydrolysis and helicase activities within NS3 and provide insight into the biophysical mechanisms for ATPase-driven NS3 helicase function.

Background: BST-2 forms covalently linked dimers and inhibits virus release. Results: Cysteine residues are not required for BST-2 homodimerization but are critical for antiviral activity. Conclusion: Cysteine residues in the BST-2 ectodomain stabilize a structure capable of tethering virions to host cells. Significance: Understanding the molecular basis of BST-2 virus-tethering activity will help us understand how cells try to minimize spreading infections.

Human BST-2/tetherin is a host factor that inhibits the release of enveloped viruses, including HIV-1, HIV-2, and SIV, from the cell surface by tethering viruses to the host cell membrane. BST-2 has an α-helical ectodomain that forms disulfide-linked dimers between two monomers forming a coiled coil. The ectodomain contains three cysteine residues that can participate in disulfide bond formation and are critical for viral tethering. The role of the disulfides in viral tethering is unknown but proposed to be for maintaining the dimer. We explored the role of the disulfides in the structure of BST-2 using experimental, biophysical methods. To understand the role of the disulfides in viral tethering, we used a new approach in viral tethering, steered molecular dynamics. We find that the disulfides coordinate the unfolding of the BST-2 monomers, which adds tensile strength to the coiled coil. Structural differences between oxidized and reduced BST-2 are apparent during unfolding, showing the monomers slide past each other in the absence of the disulfides. We found no evidence to support dissociation of the dimer upon reduction of the disulfide bonds. Moreover, the structure of BST-2 in the absence of the disulfides is similar to that of the oxidized form of BST-2, supporting previous X-ray crystallography and cellular work that showed the disulfides are not required for expression of BST-2. These data provide new insights into viral tethering by using novel techniques in the analysis of BST-2 to give amino acid level insight into functions of BST-2.

Flaviviruses are a major health concern because over half of the world population is at risk of infection and there are very few antiviral therapeutics to treat diseases resulting from infection. Replication is an essential part of the flavivirus survival. One of the viral proteins, NS3 helicase, is critical for unwinding the double stranded RNA intermediate during flaviviral replication.The helicase performs the unwinding of the viral RNA intermediate structure in an ATP-dependent manner. NS3 helicase is a member of the Viral/DEAHlike subfamily of the superfamily 2 helicase containing eight highly conserved structural motifs (I, Ia, II, III, IV, IVa, V, and VI) localized between the ATPbinding and RNA-binding pockets. Of these structural motifs only three are well characterized for function in flaviviruses (I, II, and VI). The roles of the other structural motifs are not well understood for NS3 helicase function, but comparison of NS3 with other superfamily 2 helicases within the viral/DEAHlike, DEAH/RHA, and DEAD-box subfamilies can be used to elucidate the roles of these structural motifs in the flavivirus NS3 helicase. This review aims to summarize the role of each conserved structural motif within flavivirus NS3 in RNA helicase function.

The unwinding of double-stranded RNA intermediates is critical for replication and packaging of flavivirus RNA genomes. This unwinding activity is achieved by the ATP-dependent nonstructural protein 3 (NS3) helicase. In previous studies, we investigated the mechanism of energy transduction between the ATP and RNA binding pockets using molecular dynamics simulations and enzymatic characterization. Our data corroborated the hypothesis that Motif V is a communication hub for this energy transduction. More specifically, mutations T407A and S411A in Motif V exhibit a hyperactive helicase phenotype leading to the regulation of translocation and unwinding during replication. However, the effect of these mutations on viral infection in cell culture and in vivo is not well understood. Here, we investigated the role of Motif V in viral replication using T407A and S411A West Nile virus (Kunjin subtype) mutants in cell culture and in vivo. We were able to recover S411A Kunjin but unable to recover T407A Kunjin. Our results indicated that S411A Kunjin decreased viral infection, and increased cytopathogenicity in cell culture as compared to WT Kunjin. Similarly, decreased infection rates in surviving S411A-infected Culex quinquefasciatus mosquitoes were observed, but S411A Kunjin infection resulted in increased mortality compared to WT Kunjin. Additionally, S411A Kunjin increased viral dissemination and saliva positivity rates in surviving mosquitoes compared to WT Kunjin. These data suggest that S411A Kunjin increases pathogenesis in mosquitoes. Overall, these data indicate that NS3 Motif V may play a role in the pathogenesis, dissemination, and transmission efficiency of Kunjin virus. IMPORTANCE Kunjin and West Nile viruses belong to the arthropod-borne flaviviruses, which can result in severe symptoms including encephalitis, meningitis, and death. Flaviviruses have expanded into new populations and emerged as novel pathogens repeatedly in recent years demonstrating they remain a global threat. Currently, there are no approved anti-viral therapeutics against either Kunjin or West Nile viruses. Thus, there is a pressing need for understanding the pathogenesis of these viruses in humans. In this study, we investigate the role of the Kunjin virus helicase on infection in cell culture and in vivo. This work provides new insight into how flaviviruses control pathogenesis and mosquito transmission through the nonstructural protein 3 helicase.