Latrobe University1 Peter Doherty Institute, Melbourne University2
Noroviruses (NoV) are positive sense RNA viruses from the Caliciviridae family and are responsible for up to 90% of non-bacterial gastroenteritis cases worldwide. However, Human NoV (HuNoV) is difficult to study due to the lack of a reliable tissue culture system or small animal model, hence little is known about its replication. The recently discovered and closely related Murine NoV (MNV) has been a useful model for Norovirus research.
Here we show that the NS3 protein, derived from MNV, is intimately associated with the MNV replication complex and the viral replication intermediate dsRNA. We have observed that when expressed individually both MNV NS3 and NS3 encoded by the HuNoV Norwalk virus, induced the formation of distinct vesicle-like structures that did not co-localise specifically with distinct markers to cellular organelles, but were high in cholesterol content and showed some interaction with b-tubulin. We showed that both NS3 proteins co-localise in membranes counterstained with filipin, an indicator of cholesterol content, and co-localised with flotillin and stomatin, proteins known to associate with sphingolipid and cholesterol-rich micro-domains within the endocytic pathway. Cholesterol depletion by Lovastatin treatment invoked a rearrangement of the NS3 vesicular structures, indicating that their formation is dependent on continual cholesterol biosynthesis. Utilising live-cell confocal microscopy we observed that the NS3 structures were highly motile and dynamic and that their movement was dependent on intact microtubules.
These results begin to interrogate the functions of NoV proteins during infection and contribute to our understanding of NoV replication and its pathogenesis, in particular the NS3 expression and highlight the conserved properties of the NoV protein between genogroups.