Leege Tobias, Fuchs Walter, Granzow Harald, Kopp Martina, Klupp Barbara G, Mettenleiter Thomas C
Institutes of Molecular Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.
J Virol. 2009 Jan;83(2):896-907. doi: 10.1128/JVI.01842-08. Epub 2008 Nov 12.
The conserved membrane-associated tegument protein pUL11 and envelope glycoprotein M (gM) are involved in secondary envelopment of herpesvirus nucleocapsids in the cytoplasm. Although deletion of either gene had only moderate effects on replication of the related alphaherpesviruses herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PrV) in cell culture, simultaneous deletion of both genes resulted in a severe impairment in virion morphogenesis of PrV coinciding with the formation of huge inclusions in the cytoplasm containing nucleocapsids embedded in tegument (M. Kopp, H. Granzow, W. Fuchs, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 78:3024-3034, 2004). To test whether a similar phenotype occurs in HSV-1, a gM and pUL11 double deletion mutant was generated based on a newly established bacterial artificial chromosome clone of HSV-1 strain KOS. Since gM-negative HSV-1 has not been thoroughly investigated ultrastructurally and different phenotypes have been ascribed to pUL11-negative HSV-1, single gene deletion mutants were also constructed and analyzed. On monkey kidney (Vero) cells, deletion of either pUL11 or gM resulted in ca.-fivefold-reduced titers and 40- to 50%-reduced plaque diameters compared to those of wild-type HSV-1 KOS, while on rabbit kidney (RK13) cells the defects were more pronounced, resulting in ca.-50-fold titer and 70% plaque size reduction for either mutant. Electron microscopy revealed that in the absence of either pUL11 or gM virion formation in the cytoplasm was inhibited, whereas nuclear stages were not visibly affected, which is in line with the phenotypes of corresponding PrV mutants. Simultaneous deletion of pUL11 and gM led to additive growth defects and, in RK13 cells, to the formation of large intracytoplasmic inclusions of capsids and tegument material, comparable to those in PrV-DeltaUL11/gM-infected RK13 cells. The defects of HSV-1DeltaUL11 and HSV-1DeltaUL11/gM could be partially corrected in trans by pUL11 of PrV. Thus, our data indicate that PrV and HSV-1 pUL11 and gM exhibit similar functions in cytoplasmic steps of virion assembly.
保守的膜相关被膜蛋白pUL11和包膜糖蛋白M（gM）参与疱疹病毒核衣壳在细胞质中的二次包膜过程。尽管单独缺失这两个基因中的任何一个对相关的甲型疱疹病毒1型单纯疱疹病毒（HSV-1）和伪狂犬病病毒（PrV）在细胞培养中的复制只有中等程度的影响，但同时缺失这两个基因会导致PrV病毒体形态发生严重受损，同时在细胞质中形成巨大的包涵体，其中含有嵌入被膜的核衣壳（M. Kopp、H. Granzow、W. Fuchs、B. G. Klupp和T. C. Mettenleiter，《病毒学杂志》78:3024 - 3034，2004年）。为了测试HSV-1中是否会出现类似的表型，基于新建立的HSV-1 KOS株细菌人工染色体克隆构建了gM和pUL11双缺失突变体。由于尚未对gM阴性的HSV-1进行全面的超微结构研究，并且已将不同的表型归因于pUL11阴性的HSV-1，因此还构建并分析了单基因缺失突变体。在猴肾（Vero）细胞上，与野生型HSV-1 KOS相比，单独缺失pUL11或gM导致滴度降低约五倍，空斑直径减小40%至50%，而在兔肾（RK13）细胞上，缺陷更为明显，每个突变体导致滴度降低约50倍，空斑大小减小70%。电子显微镜显示，在缺失pUL11或gM的情况下，细胞质中的病毒体形成受到抑制，而核阶段未受到明显影响，这与相应的PrV突变体表型一致。同时缺失pUL11和gM导致累加的生长缺陷，并且在RK13细胞中，形成了大量包含衣壳和被膜物质的胞质内包涵体，类似于PrV-ΔUL11/gM感染的RK13细胞中的包涵体。HSV-1ΔUL11和HSV-1ΔUL11/gM的缺陷可以通过PrV的pUL11在反式中部分纠正。因此，我们的数据表明PrV和HSV-1的pUL11和gM在病毒体组装的细胞质步骤中表现出相似的功能。
