Abstract:
African horse sickness virus (AHSV) is an orbivirus in the Reoviridae family that causes severe disease in horses, with major economic implications. The viral genome consists of ten double-stranded RNA segments, encoding seven structural viral proteins (VP) plus four non-structural (NS) proteins. The non-structural proteins serve to enhance the viral life cycle by influencing viral morphogenesis, replication or egress. In orbiviruses, NS2 multimers form dense cytoplasmic matrices termed viral inclusion bodies (VIBs). It has until recently been assumed that the VIBs of AHSV merely serve as replication factories and sites wherein virus particle assembly occurs. However, various different viruses have been shown to compartmentalise virus protein synthesis within cytoplasmic replication factories, which is believed to contribute to enhancing the viral life cycle. Little is known about how AHSV utilises the host translational machinery for its replicative advantage.
In this study, the distribution and morphology of AHSV VIBs were characterised at different stages of the replication cycle. The VIBs were shown to increase both in size and abundance up until a certain time point after infection. The formation of the VIBs from NS2 precursors was characterised by making use of plasmid-expressed fluorescent NS2-eGFP. Preliminary results from live-cell imaging revealed the formation of the VIBs to be a dynamic process, involving the progressive coalescence of small NS2 foci in a random fashion.
Using a biochemical assay followed by confocal microscopy analyses, it was investigated whether the VIBs are sites of protein synthesis. Our results showed that active translation was substantially enhanced within the VIBs of AHSV-4, AHSV-5 and BTV-10, surpassing the intensities of cytoplasmic protein synthesis. This indicates virus-mediated compartmentalisation of translation within the VIBs as a broad functionality amongst the orbiviruses.
We next determined whether different eukaryotic ribosomal components and translation initiation factors localise to the VIBs of AHSV. Interestingly, we observed differential distribution patterns of these components, indicating distinct sub-structural domains of the VIBs. The ribosomal component L11 and initiation factor eIF3θ localised throughout the VIBs, with elevated intensities towards the central regions, whereas ribosomal component S3 and initiation factor eIF4E localised to the VIB perimeters. These localisation patterns were shown to be solely mediated by NS2.
These results indicate a novel functionality of the VIBs, by serving as hubs for virus protein synthesis. It still remains to be elucidated how translation is orchestrated within the VIBs, however our preliminary results could indicate to the potential for sub-domains of the VIBs being involved in specific functions regarding translation. Overall, this indication of translation compartmentalisation within the VIBs could elucidate a strategy whereby AHSV enhances the viral infection cycle.
