Mutational analysis of the West Nile Virus NS4B protein
Jason Alan Wicker
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West Nile virus (WNV) is a member of the genus Flavivirus in the family Flaviviridae. The WNV genome is a positive-sense RNA molecule approximately 11kb in length encoding a single polyprotein that is cleaved by a combination of viral and host proteases to produce three structural and seven nonstructural proteins. The NS4B protein is a small hydrophobic protein approximately 27kD in size that is hypothesized to participate both in the viral replication complex and evasion of host innate immune defenses. The objective of this dissertation was to investigate the role of the NS4B protein in viral cell multiplication and mouse virulence phenotypes by studying recombinant mutant viruses encoding amino acid substitutions of selected residues within the NS4B protein. The first aim of this project used protein modeling and phylogenetic analysis of the NY99 WNV NS4B protein in comparison to NS4B proteins from other flavivirus and WNV strains to identify amino acid residues with a theoretical probability of contributing to the function of NS4B. The second aim utilized site-directed mutagenesis of a WNV NY99 infectious clone to introduce amino acid substitutions into the NS4B protein primarily targeting a highly conserved N-terminal domain, the variable central hydrophobic region, and the four cysteine residues. Out of fourteen recombinant viruses encoding engineered substitutions, two highly attenuated mutant viruses were identified (C102S and P38G/T116I viruses) that exhibited temperature-sensitive and mouse attenuation (greater than 10,000,000-fold compared to wild-type) phenotypes. The third aim investigated the putative underlying molecular mechanisms responsible for the attenuation of the C102S and P38G/T116I viruses. Both NS4B mutants exhibited reduced multiplication kinetics both in mice and in murine macrophage and dendritic cell types critical for mediating the antiviral immune response. In addition, preliminary data identified a series of genes by DNA microarray analysis that exhibited differential expression in wild-type WNV-infected cells compared to C102S mutant-infected cells that may be involved in viral manipulation of cellular processes. This study has for the first time demonstrated the role of the NS4B protein as mediator of WNV temperature-sensitive and mouse attenuation phenotypes and has led to the identification of putative molecular mechanisms that may be involved.