Visualising influenza virus replication using a click chemistry approach

Influenza A viruses (IAVs) have a significant impact on public health through seasonal epidemics. Moreover, the sporadic emergence of zoonotic and pandemic strains represents an additional global threat. Understanding how novel IAV strains adapt to infect and transmit between humans is essential for effective surveillance and may provide rationale for anti-viral therapeutics. IAVs replicate their RNA genomes in the host cell nucleus. Host proteins, such as the proviral factor ANP32, are co-opted to support viral replication and transcription, whilst other inhibitory factors act to restrict. Ultimately the outcome of infection in different species depends on compatibility with these factors, however the precise nuclear localisation of these interactions is not clearly defined. It is not known whether viral RNA synthesis takes place at discrete sites, or whether the virus modifies the cell to produce viral factories in a similar manner to other viruses. We are utilising a click chemistry approach to investigate the spatial details of IAV replication and to probe the differences in nuclear localisation between human-adapted vs poorly-adapted avian origin IAVs, to gain insight into mechanisms of host restriction. Influenza genomes labelled with the click reagent 5-ethynyl uridine (5-EU) remain infectious and can be visualised through the cycloaddition of azide-tagged fluorescent dyes. Utilising this tool, the co-localisation of incoming viral particles with nuclear sub-domains, as well as key host factors, are being examined. Moreover, the localisation of human- vs avian-adapted virus in human and avian cells, as well as cell lines lacking key host factors, are being compared.