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 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（Human noroviruses, huNoV）是全球范围内引发非细菌性急性胃肠炎最常见的致病原，其中尤以基因群II型4型（GII.4）变异株为甚。由开放阅读框1（ORF1）多聚蛋白编码的病毒非结构（NS）蛋白可诱导形成包含病毒复制位点的囊泡簇。目前学界对这类复制细胞器的超微结构，以及单个NS蛋白在其生物发生过程中的具体贡献仍知之甚少。本研究将诺如病毒ORF1多聚蛋白表达诱导的超微结构改变，与鼠诺如病毒（MNV）感染细胞所引发的变化进行了对比。ORF1蛋白表达诱导的特征性膜改变，与MNV感染细胞中观察到的现象高度相似，均表现为囊泡聚集，这类囊泡大概率源自内质网（ER），涵盖单膜囊泡（SMVs）、双膜囊泡（DMVs）与多膜囊泡（MMVs）。通过电子断层扫描技术开展的深入分析显示，多膜囊泡的形成机制与小核糖核酸病毒已报道的过程类似，即通过管状结构包裹单膜囊泡而来。对与绿色荧光蛋白（GFP）融合的GII.4型NS1-2、NS3及NS4蛋白进行表达，并结合关联光镜-电镜技术分析后发现，不同蛋白可诱导出各具特征的膜改变：NS1-2可诱导滑面内质网膜增殖，形成长管状结构，该过程会受假定的NS1-2水解酶结构域活性中心突变的影响；NS3定位于脂滴（LDs）周围的内质网膜，并可诱导卷曲膜的形成，而NS4诱导的卷曲膜结构更为显著。值得注意的是，NS4是唯一一种单独表达即可诱导单膜囊泡与双膜囊泡形成的GII.4型蛋白。本研究首次完成了诺如病毒GII.4型诱导的囊泡簇的超微结构分析，结果表明其形态与生物发生过程与小核糖核酸病毒最为相似。本研究进一步确认NS4是人类诺如病毒膜改变形成的关键因子，并构建了NS1-2、NS3与NS4的推定膜拓扑结构模型，以期为后续相关研究提供指导。