Membrane alterations induced by nonstructural proteins of human norovirus

Human noroviruses (huNoV) are the most frequent cause of non-bacterial acute gastroenteritis worldwide, particularly genogroup II genotype 4 (GII.4) variants. The viral nonstructural (NS) proteins encoded by the ORF1 polyprotein induce vesical clusters harboring the viral replication sites. Little is known so far about the ultrastructure of these replication organelles or the contribution of individual NS proteins to their biogenesis. We compared the ultrastructural changes induced by expression of norovirus ORF1 polyproteins with those induced upon infection with murine norovirus (MNV). Characteristic membrane alterations induced by ORF1 expression resembled those found in MNV infected cells, consisting of vesicle accumulations likely built from the endoplasmic reticulum (ER) which included single membrane vesicles (SMVs), double membrane vesicles (DMVs) and multi membrane vesicles (MMVs). In-depth analysis using electron tomography suggested that MMVs originate through the enwrapping of SMVs with tubular structures similar to mechanisms reported for picornaviruses. Expression of GII.4 NS1-2, NS3 and NS4 fused to GFP revealed distinct membrane alterations when analyzed by correlative light and electron microscopy. Expression of NS1-2 induced proliferation of smooth ER membranes forming long tubular structures that were affected by mutations in the active center of the putative NS1-2 hydrolase domain. NS3 was associated with ER membranes around lipid droplets (LDs) and induced the formation of convoluted membranes, which were even more pronounced in case of NS4. Interestingly, NS4 was the only GII.4 protein capable of inducing SMV and DMV formation when expressed individually. Our work provides the first ultrastructural analysis of norovirus GII.4 induced vesicle clusters and suggests that their morphology and biogenesis is most similar to picornaviruses. We further identified NS4 as a key factor in the formation of membrane alterations of huNoV and provide models of the putative membrane topologies of NS1-2, NS3 and NS4 to guide future studies.
Author summary Positive-strand RNA viruses induce membrane alterations harboring the viral replication complexes. In the case of human noroviruses (huNoV), the major cause of acute viral gastroenteritis, these are induced by the ORF1 polyprotein, which is post-translationally processed into the functional nonstructural (NS) proteins. Partly due to the lack of efficient cell culture models, little is known so far about membrane alterations induced by huNoV belonging to the most clinically relevant genogroup II, genotype 4 (GII.4), nor about the function of individual NS proteins in their formation. We therefore expressed ORF1 proteins of GII.4 and individual NS proteins in cells to study their contribution to viral replication complex formation. Expression of ORF1 proteins of GII.4 induced vesicular membrane alterations comparable to those found in infected cells and similar to picornaviruses and hepatitis C virus (HCV). GII.4 NS1-2, NS3 and NS4 are contributing to viral membrane alterations. Our work provides new insights into their function in huNoV induced replication complex formation while identifying NS4 as the most important single determinant. This knowledge might provide novel attractive targets for future therapies inhibiting the formation of the membranous viral replication complex, as exemplified by the efficacy of HCV NS5A inhibitors.