The flavivirus NS3 helicase Motif V controls unwinding function and alters viral pathogenesis in mosquitoes
Date
2020
Authors
Du Pont, Kelly Elizabeth, author
McCullagh, Martin, advisor
Geiss, Brian J., advisor
Szamel, Grzegorz, committee member
Snow, Christopher, committee member
Krummel, Amber, committee member
Ho, Shing, committee member
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Abstract
Over half of the world's population is at risk of flavivirus (e.g. dengue virus, West Nile virus, Japanese Encephalitis virus, and Zika virus) infection making it a global health concern. These specific mosquito-borne flaviviruses are responsible for causing a variety of symptoms and outcomes including flu-like fevers, encephalitis, hemorrhagic fevers, microcephaly, Guillain-Barré syndrome, and death. Unfortunately, vaccines and anti-viral therapeutics are not always effective in protecting against and treating viral infections. Sometimes these therapies cause more severe symptoms through an antibody dependent enhancement. Therefore, there is a pressing need for the development of effective anti-viral therapies against each flavivirus. For the advancement of these interventional strategies, a fundamental understanding of how flaviviruses replicate within hosts, including the mosquito vector, is required. This dissertation investigates how flaviviruses regulate viral replication, pathogenesis and mosquito transmission through the nonstructural protein 3 (NS3) helicase structure and function. A combination of virology, biochemistry, and computational simulations will be utilized to address how NS3 plays a role in viral infection, viral replication, and viral protein structure. An essential aspect of flaviviral genome replication is the unwinding of the double-stranded RNA intermediate via the C-terminal helicase domain of NS3. NS3 helicase translocates along and unwinds the double-stranded nucleic acids in an ATP-dependent manner. However, the mechanism of energy transduction between the ATP- and RNA-binding pockets is not well understood. Previous simulations in the group led us to hypothesize that Motif V is a critical component of the transduction mechanism. Here, we tested Motif V mutations in both sub-genomic replicon and recombinant protein systems to examine viral genome replication, helicase unwinding activity, ATP hydrolysis activity, and RNA binding affinity activity. NS3 helicase mutants, T407A and S411A, indicated reduced viral genome replication and increased turnover rates of helicase unwinding activity by a factor of 1.7 and 3.5 respectively. Additionally, we simulated Motif V mutants to probe the structural changes within NS3 helicase caused by the mutations. These simulations indicate that Motif V controls communication between the ATP-binding pocket and the helical gate. Motif V mutations T407A and S411A exhibit a hyperactive helicase phenotype leading to the regulation of translocation and unwinding during viral genome replication. Next, we utilized T407A and S411A West Nile virus (Kunjin subtype) mutants in cell culture and in vivo to probe the how these mutations play a role in pathogenesis and transmission of flaviviruses. Of the two Kunjin virus mutants, only S411A Kunjin virus was recovered. In cell culture, S411A Kunjin decreased viral infection and increased cytopathogenicity as compared to WT Kunjin. Similarly, in surviving Culex quinquefasciatus mosquitoes, S411A Kunjin decreased infection rates as compared to WT Kunjin, but S411A Kunjin infection increased mortality compared with that of WT Kunjin infection. 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 computational simulation, biochemical assay, and virology data indicate that flavivirus NS3 helicase Motif V may play a role in the pathogenesis, dissemination, and transmission efficiency of Kunjin virus, not just regulation of translocation and unwinding during viral genome replication. The molecular level insights presented in this dissertation provide the fundamental research for understanding how to target specific regions of NS3 helicase for the advancement of anti-viral therapeutics.
Description
Rights Access
Subject
cytopathic effect
mosquito
viral pathogenesis
flaviviruses
allosteric regulation
NS3 helicase