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 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.

人类诺如病毒（huNoV）是全世界非细菌性急性肠胃炎的最常见原因，尤其是II型基因组4基因型（GII.4）变体。由ORF1多蛋白编码的病毒非结构（NS）蛋白诱导囊泡簇，其具有病毒复制位点。到目前为止，对这些复制细胞器的超微结构或单个NS蛋白对其生物发生的贡献知之甚少。我们比较了由诺如病毒ORF1多蛋白表达诱导的超微结构变化与鼠诺如病毒（MNV）感染后诱导的超微结构变化。由ORF1表达诱导的特征性膜改变类似于在MNV感染的细胞中发现的那些，包括可能由内质网（ER）形成的囊泡积累，其中包括单个膜囊泡（SMV），双膜囊泡（DMV）和多膜囊泡（MMV）。使用电子断层扫描技术进行的深入分析表明，MMV起源于包裹的SMV，其管状结构类似于小核糖核酸病毒报道的机制。当通过相关光镜和电子显微镜分析时，与GFP融合的GII.4 NS1-2，NS3和NS4的表达显示出明显的膜改变。NS1-2的表达诱导了平滑的ER膜的增殖，形成了长管状结构，该结构受假定的NS1-2水解酶结构域活性中心突变的影响。NS3与脂滴（LDs）周围的ER膜相关，并诱导了回旋膜的形成，这在NS4情况下更为明显。有趣的是，NS4是唯一的GII。单独表达时能够诱导SMV和DMV形成的4种蛋白质。我们的工作提供了诺如病毒GII.4诱导的囊泡簇的首次超微结构分析，并表明它们的形态和生物发生与小核糖核酸病毒最相似。我们进一步确定了NS4是huNoV膜改变形成的关键因素，并提供了NS1-2，NS3和NS4的假定膜拓扑模型来指导未来的研究。