van Eijl H, Hollinshead M, Smith G L
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, United Kingdom.
Virology. 2000 May 25;271(1):26-36. doi: 10.1006/viro.2000.0260.
Vaccinia virus gene A36R encodes a 45-kDa protein that is conserved in orthopoxviruses. A virus lacking the A36R protein formed a small plaque, was unable to induce the polymerization of actin tails, and was avirulent in vivo. Here we present a further characterization of the A36R protein by in vitro transcription and translation and analysis of infected cells by confocal microscopy and immunoelectron microscopy of cryosections using a monoclonal antibody raised against the C-terminal domain of the A36R protein. Translation of the A36R mRNA in vitro produced a protein of the same size whether or not the translation reaction was performed in the presence of canine pancreatic microsomes. However, the polypeptide synthesized in the presence of microsomes was associated integrally with the membrane and was sensitive to digestion by exogenous protease without permeabilization of the membrane with detergent, indicating that the majority of the protein is exposed on the outside of the vesicle. Consistent with this, immunofluorescent analysis of virus-infected cells demonstrated that the C-terminal domain of A36R was not exposed on the cell surface but was detected once the cell membrane was permeabilized. Immunoelectron microscopy of cryosections of infected cells showed that the protein was absent from IMV particles but present on intracellular enveloped virus (IEV) particles, predominantly on the cytosolic face of the IEV outer membrane. Where cell-associated enveloped virus (CEV) particles were attached to the cell surface, the A36R protein was detected only on the cytosolic surface of the plasma membrane where the virus particle remained attached to the cell and not elsewhere on the plasma membrane or on the CEV particle. A36R and actin copurified with EEV particles due to the association of fragments of cellular membranes with the EEV particles. Therefore, A36R represents the first example of a virus-encoded protein that is present on IEV but not CEV particles.
痘苗病毒基因A36R编码一种45 kDa的蛋白质，该蛋白质在正痘病毒中保守。缺乏A36R蛋白的病毒形成小噬斑，无法诱导肌动蛋白尾的聚合，并且在体内无致病性。在这里，我们通过体外转录和翻译以及使用针对A36R蛋白C末端结构域的单克隆抗体对冷冻切片进行共聚焦显微镜和免疫电子显微镜分析，对A36R蛋白进行了进一步表征。无论翻译反应是否在犬胰腺微粒体存在下进行，A36R mRNA的体外翻译都产生相同大小的蛋白质。然而，在微粒体存在下合成的多肽与膜整体相关，并且在不使用去污剂使膜通透的情况下对外源蛋白酶消化敏感，这表明大多数蛋白质暴露在囊泡外部。与此一致的是，对病毒感染细胞的免疫荧光分析表明，A36R的C末端结构域未暴露在细胞表面，但在细胞膜通透后可检测到。对感染细胞冷冻切片的免疫电子显微镜分析表明，IMV颗粒中不存在该蛋白，但存在于细胞内包膜病毒（IEV）颗粒上，主要存在于IEV外膜的胞质面。当细胞相关包膜病毒（CEV）颗粒附着在细胞表面时，仅在病毒颗粒仍附着在细胞上的质膜胞质表面检测到A36R蛋白，而在质膜的其他位置或CEV颗粒上未检测到。由于细胞膜片段与EEV颗粒的关联，A36R和肌动蛋白与EEV颗粒共纯化。因此，A36R代表了一种病毒编码蛋白的第一个例子，该蛋白存在于IEV颗粒上但不存在于CEV颗粒上。
