A Hyperactive Kunjin Virus NS3 Helicase Mutant Demonstrates Increased Dissemination and Mortality in Mosquitoes

Abstract

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.

Conserved motifs in the flavivirus NS3 RNA helicase enzyme

“…S412A Kunjin increased cellular death via decreased NADH and intracellular ATP levels compared with WT Kunjin. S412A Kunjin also decreased and delayed viral infectivity in HEK293T cells, which is consistent with previous results of decreased viral genome replication activity (Du Pont, Davidson, et al, 2020;Du Pont, Sexton, et al, 2020). These results suggest that position 4 in Motif V effects cellular death induced from viral infection (Du Pont, Sexton, et al, 2020).…”

“…S412A Kunjin also decreased and delayed viral infectivity in HEK293T cells, which is consistent with previous results of decreased viral genome replication activity (Du Pont, Davidson, et al, 2020;Du Pont, Sexton, et al, 2020). These results suggest that position 4 in Motif V effects cellular death induced from viral infection (Du Pont, Sexton, et al, 2020). Therefore, we can suggest that flaviviruses have evolved to reduce helicase unwinding activity to reduce viral infection induced cellular death so they can continue to propagate and spread from host to host.…”

“…The first generation of S412A Kunjin was recovered, while T408A Kunjin was not recovered. The unrecovered T408A Kunjin is consistent with the decreased viral genome replication results seen in the previous study (Du Pont, Davidson, et al, 2020;Du Pont, Sexton, et al, 2020). S412A Kunjin was tested for cell viability and viral infectivity in cell culture.…”

“…Positions 4 and 8 were mutated to alanine in Kunjin virus, a WNV subtype, due to an established viral infection protocol with Kunjin virus (Du Pont, Sexton, et al, 2020). In Kunjin virus, position 4 is T408 and position 8 is S412.…”

“…In Kunjin virus, position 4 is T408 and position 8 is S412. These two mutated viruses, T408A Kunjin and S412A Kunjin, were generated from a novel bacterium-free viral launch system (Du Pont, Sexton, et al, 2020). The first generation of S412A Kunjin was recovered, while T408A Kunjin was not recovered.…”