Brack A R, Dijkstra J M, Granzow H, Klupp B G, Mettenleiter T C
Institutes of Molecular and Cellular Virology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany.
J Virol. 1999 Jul;73(7):5364-72. doi: 10.1128/JVI.73.7.5364-5372.1999.
Glycoprotein M (gM), the product of the UL10 gene of pseudorabies virus (PrV), is one of the few nonessential glycoproteins conserved throughout the Herpesviridae. In contrast to wild-type PrV strains, the UL10 gene product of the attenuated PrV vaccine strain Bartha (PrV-Ba) is not modified by N-glycans due to a mutation in the DNA sequence encoding the consensus N-glycosylation motif. To assay function of the UL10 protein in PrV-Ba, a UL10-deletion mutant (PrV-Ba-UL10(-)) was isolated. Surprisingly, in contrast to gM-deleted wild-type PrV, PrV-Ba-UL10(-) was severely impaired in plaque formation, inducing only foci of very few infected RK13, Vero, and PSEK cells and tiny plaques on MDBK cells. Since this effect was significantly more dramatic than in wild-type PrV, additional mutations known to be present in PrV-Ba were analyzed for their contribution to this phenotype. trans-complementation of the mutated PrV-Ba UL21 or gC protein by the wild-type version had no influence on the observed phenotype. In contrast, complementation of the gE/gI deletion rescued the phenotype. The synergistic effect of deletions in gE/gI and gM on plaque size was verified by construction of a gE/I/M triple mutant derived from wild-type PrV which exhibited the same phenotype. The dramatic effect of deletion of gM on plaque size in a gE/I- virus background was mainly attributable to a function of gM, and not of the gM/gN complex, as shown by analysis of a gE/I/N triple mutant. Interestingly, despite the strong effect on plaque size, penetration was not significantly impaired. In noncomplementing cells infected with the gE/I/M triple mutant, electron microscopy showed absence of secondary envelopment in the cytoplasm but occurrence of intracytoplasmic accumulations of nucleocapsids in association with electron dense material, presumably tegument proteins. These structures were not observed after infection of cells expressing either gE/I or gM. We suggest that gE/I and gM are required for late stages in virion morphogenesis prior to final envelopment in the cytoplasm.
糖蛋白M（gM）是伪狂犬病病毒（PrV）UL10基因的产物，是整个疱疹病毒科中少数几个非必需糖蛋白之一。与野生型PrV毒株不同，减毒PrV疫苗株Bartha（PrV-Ba）的UL10基因产物由于编码共有N-糖基化基序的DNA序列发生突变，未被N-聚糖修饰。为了检测PrV-Ba中UL10蛋白的功能，分离出了一个UL10缺失突变体（PrV-Ba-UL10(-)）。令人惊讶的是，与缺失gM的野生型PrV不同，PrV-Ba-UL10(-)在噬斑形成方面严重受损，仅诱导极少数感染的RK13、Vero和PSEK细胞形成病灶，在MDBK细胞上形成微小噬斑。由于这种效应比野生型PrV中更为显著，因此对已知存在于PrV-Ba中的其他突变对该表型的贡献进行了分析。野生型版本对突变的PrV-Ba UL21或gC蛋白进行反式互补对观察到的表型没有影响。相反，gE/gI缺失的互补挽救了该表型。通过构建源自野生型PrV的gE/I/M三突变体验证了gE/gI和gM缺失对噬斑大小的协同效应，该三突变体表现出相同的表型。如对gE/I/N三突变体的分析所示，在gE/I病毒背景下，gM缺失对噬斑大小的显著影响主要归因于gM的功能，而非gM/gN复合物的功能。有趣的是，尽管对噬斑大小有强烈影响，但病毒穿透并未受到显著损害。在用gE/I/M三突变体感染的非互补细胞中，电子显微镜显示细胞质中没有二次包膜，但存在与电子致密物质（可能是被膜蛋白）相关的核衣壳胞质内聚集物。在感染表达gE/I或gM的细胞后未观察到这些结构。我们认为，gE/I和gM是病毒粒子在细胞质中最终包膜之前形态发生后期所必需的。
