Hacking at the cellular level; How do viruses subvert intracellular networks for viral RNA genome trafficking within infected cells?

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Nairoviruses are a group of insect-borne RNA viruses that include the extreme human pathogen Crimean-Congo haemorrhagic fever virus (CCHFV), which is listed as a priority pathogen in the WHO 'Research and Development Blueprint'. No vaccines or therapies are currently available to prevent or treat CCHFV disease, and this represents an urgent unmet need. Nairoviruses are enveloped viruses that internalise into cells within endosomes, and following endosome-escape, their RNA genomes transit to a specific destination. This transit does not occur by random diffusion - instead nairoviruses subvert cellular process to move their genomes to a site known as a viral factory. As its name suggests, the factory is the site of intense anabolic activity, where viral components are mass-produced, destined for assembly into new viruses. Formation of the virus factory is a multi-step process, critical for infection and disease. However, no detailed information of how the nairovirus genome transits to the factory site, or factory structure, is currently available. This project will fill this knowledge gap, providing potential cellular and viral targets for future anti-viral therapies, as well as providing detailed information of critical host-pathogen interactions.Using the closely related yet non-pathogenic Hazara nairovirus (HAZV) as a model for CCHFV, this project will identify cellular pathways and components that are hijacked by nairoviruses to allow their journey from the endosome to the virus factory, as well as determine the location and ultrastructure of the factory itself. To achieve these aims, we will first use reverse genetics to introduce site-specific mutations within the HAZV RNA genome to develop a 'tool-kit' of engineered infectious HAZV variants bearing genetically encoded fluorescent and epitope tags, which will allow visualization and tracking of all HAZV RNA and protein components within cells on the entry pathway. Next, using our previously generated RNAi and CRISPR-based gene knock-down/knock-out screen data (Fuller et al, 2020), we will determine cell factor involvement by visualizing impaired RNA genome transit in cells with disrupted protein function, resulting from genetic ablation or use of specific inhibitors or expression of dominant-negatives. Finally, we will visualize virus factory organisation using electron microscopy observation of sectioned cells, using state-of-the-art cryo-focused ion beam, alongside high-resolution light microscopy techniques such as STED. We recently characterised for the first time the location and content of the hantavirus replication factory using light and electron microscopy (Davies et al, 2020) and similar techniques will be adopted here.