Rodríguez J R, Risco C, Carrascosa J L, Esteban M, Rodríguez D
Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus Universidad Autónoma, Madrid, Spain.
J Virol. 1998 Feb;72(2):1287-96. doi: 10.1128/JVI.72.2.1287-1296.1998.
Early stages in vaccinia virus (VV) assembly involve the recruitment of cellular membranes from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) to virus factories (or virosomes). The key viral factors involved in this process are not yet known. We have previously identified and characterized two viral proteins, of 21 kDa (A17L gene) and 15 kDa (A14L gene), that associate with tubulovesicular elements related to the ERGIC and are localized in viral membranes at all stages of virion assembly. We showed that the 21-kDa protein is not responsible for the recruitment of membranes from the ERGIC to viral factories. However, it appears to be essential for the organization of viral membranes. In this investigation we have generated a VV recombinant, VVindA14L, in which the expression of the A14L gene is inducibly regulated by the Escherichia coli lacI operator-repressor system. Repression of 15-kDa protein synthesis has a dramatic effect on virus yields and severely impairs plaque formation. Compared to wild-type VV, reduced amounts of 15-kDa protein are produced in VVindA14L-infected cells in the presence of IPTG (isopropyl-beta-D-thiogalactoside), and this correlates with a small-plaque phenotype and reduced VVindA14L yields under these conditions. In the absence of the 15-kDa protein, early and late viral protein syntheses proceed normally; however, proteolytic cleavage of the major core precursors is inhibited. Electron microscopic examination of cells infected with VVindA14L under nonpermissive conditions reveals the presence of numerous membranous elements that look like unfinished or disassembled crescents interspersed between electron-dense masses. These abnormal membrane elements are usually well separated from the surfaces of the dense structures. These findings show that the 15-kDa protein is essential for VV morphogenesis and indicate that this polypeptide is necessary both for the correct assembly of viral crescents and for their stable attachment to the surfaces of viral factories.
痘苗病毒（VV）组装的早期阶段涉及从内质网-高尔基体中间区室（ERGIC）募集细胞膜至病毒工厂（或病毒小体）。参与此过程的关键病毒因子尚不清楚。我们之前已鉴定并表征了两种病毒蛋白，分别为21 kDa（A17L基因）和15 kDa（A14L基因），它们与ERGIC相关的小管状囊泡元件相关联，并在病毒粒子组装的各个阶段定位于病毒膜上。我们发现21 kDa蛋白并非负责将ERGIC的膜募集至病毒工厂。然而，它似乎对病毒膜的组织至关重要。在本研究中，我们构建了一种VV重组体VVindA14L，其中A14L基因的表达受大肠杆菌lacI操纵子-阻遏物系统的诱导调控。抑制15 kDa蛋白的合成对病毒产量有显著影响，并严重损害噬斑形成。与野生型VV相比，在异丙基-β-D-硫代半乳糖苷（IPTG）存在的情况下，VVindA14L感染的细胞中产生的15 kDa蛋白量减少，这与小噬斑表型以及在此条件下VVindA14L产量降低相关。在缺乏15 kDa蛋白的情况下，病毒早期和晚期蛋白合成正常进行；然而，主要核心前体的蛋白水解切割受到抑制。在非允许条件下对VVindA14L感染的细胞进行电子显微镜检查发现，存在许多膜状元件，看起来像未完成或拆解的月牙体，散布于电子致密物质之间。这些异常的膜元件通常与致密结构的表面分隔良好。这些发现表明15 kDa蛋白对VV形态发生至关重要，并表明该多肽对于病毒月牙体的正确组装及其与病毒工厂表面的稳定附着均必不可少。
