Norovirus (NoV) infections are a significant health burden to society, yet the lack of reliable tissue culture systems has hampered the development of appropriate antiviral therapies. Here we show that the NoV NS3 protein, derived from murine NoV (MNV), is intimately associated with the MNV replication complex and the viral replication intermediate double-stranded RNA (dsRNA). We observed that when expressed individually, MNV NS3 and NS3 encoded by human Norwalk virus (NV) induced the formation of distinct vesicle-like structures that did not colocalize with any particular protein markers to cellular organelles but localized to cellular membranes, in particular those with a high cholesterol content. Both proteins also showed some degree of colocalization with the cytoskeleton marker β-tubulin. Although the distribution of MNV and NV NS3s were similar, NV NS3 displayed a higher level of colocalization with the Golgi apparatus and the endoplasmic reticulum (ER). However, we observed that although both proteins colocalized in membranes counterstained with filipin, an indicator of cholesterol content, MNV NS3 displayed a greater association with flotillin and stomatin, proteins known to associate with sphingolipid- and cholesterol-rich microdomains. Utilizing time-lapse epifluorescence microscopy, we observed that the membrane-derived vesicular structures induced by MNV NS3 were highly motile and dynamic in nature, and their movement was dependent on intact microtubules. These results begin to interrogate the functions of NoV proteins during virus replication and highlight the conserved properties of the NoV NS3 proteins among the seven Norovirus genogroups
