Membrane alterations induced by nonstructural proteins of human norovirus

Abstract

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.

Fat, fight, and beyond: The multiple roles of lipid droplets in infections and inflammation

“…Several (+)RNA viruses use host LDs at different steps of their life cycle, such as reovirus, rotavirus, norovirus, Junín virus, and poliovirus, as well as various members of the Flaviviridae . These viruses manipulate lipid metabolism for efficient viral morphogenesis, and inhibition of lipid metabolism and/or interference with LD homeostasis decreases viral replication and/or assembly …”

“…The LD structural proteins (PLIN1 and PLIN2/ADRP) colocalize with the rotavirus nonstructural proteins NSP2 and NSP5 and with the structural proteins VP1, VP2, and VP6 . The NS3 protein of human norovirus (huNoV) was tightly associated with LDs and induced convoluted membranes, structures responsible for viral replication . Moreover, the µ1 outer capsid protein of reoviruses was also reported on LDs …”

“…141 The NS3 protein of human norovirus (huNoV) was tightly associated with LDs and induced convoluted membranes, structures responsible for viral replication. 142 Moreover, the µ1 outer capsid protein of reoviruses was also reported on LDs. 140 Notwithstanding being an important viral replication site, LDs can also be an important energy and building block source for viral replication and assembly.…”

Nucleotide triphosphatase and RNA chaperone activities of murine norovirus NS3

“…S2), suggesting that these intrinsic disordered regions probably provide the RNA-binding function and affect the RNA chaperone activity. A model of the putative membrane association of NS1-2, NS3 and NS4 was recently proposed [44], where NS3 may harbour its amphipathic a-helix at the NTD tethering the protein to membrane.…”

Demarcation of Viral Shelters Results in Destruction by Membranolytic GTPases: Antiviral Function of Autophagy Proteins and Interferon‐Inducible GTPases

“…Extrapolating from other pathogens targeted by the GTPases, a straightforward potential mechanism is that the GTPases may disrupt pre‐formed RCs through vesiculation (Figure ). However, the small size of the viral RC complicates this possibility . The average size of vesicles created during the IRG/GBP attack on the PVM of T. gondii is nearly as large as the entire replication compartment of MNV .…”