Investigating the mechanism of genome encapsidation for Avian Nephritis Virus

Avian astroviruses (AAstVs) cause growth suppression syndromes that place large economic burdens on the global poultry industry. The vast inter-serotype antigenic diversity of these +ssRNA viruses has hindered the production of vaccines, and the emergence of increasingly pathogenic strains means an improved understanding of AAstV biology is urgently required to develop novel control strategies. A growing number of +ssRNA viruses are reported to use packaging signal (PS)-mediated assembly, where multiple RNA stem-loops (SL) across the genome act cooperatively to facilitate efficient genome encapsidation and virion assembly. This mechanism and its sequence-specific characteristics are apparently conserved between strain variants, potentially presenting an ideal antiviral target. Therefore, the existence of PSs in avian nephritis virus (ANV), a prominent AAstV, was explored.
To guide expression of ANV capsid protein (CP) for identification of PSs by RNA SELEX, the CP structure was investigated by computational modelling. ANV CP showed significant structural homology to human astrovirus CP, and an RNA SL was predicted to interact with the interior surface of the capsid by binding-specific substructure homology to panicum mosaic virus. Expression of CP was trialled in plant, avian-derived, and bacterial systems, with intact CP only generated in E. coli which was then used for further study. Whilst magnesium ions induced capsomer-like formation by ANV CP, these appeared assembly-incompetent as neither additional magnesium nor viral RNA promoted capsid assembly. In the absence of protein suitable for SELEX, ANV was recovered by reverse genetics for an alternate NGS approach to identify enriched nucleotides within virions. RNA structure analysis at three identified positions revealed a PS-like consensus SL motif. Synonymous mutation of the most conserved SL resulted in delayed accumulation of extracellular virions, indicating an involvement in encapsidation. This thesis presents the first evidence for PS-mediated assembly by ANV, which may be a mechanism employed across the Astroviridae.