EXTRACELLULAR VACCINIA VIRUS FORMATION AND CELL-TO-CELL VIRUS TRANSMISSION ARE PREVENTED BY DELETION OF THE GENE ENCODING THE 37,000-DALTON OUTER ENVELOPE PROTEIN
EXTRACELLULAR VACCINIA VIRUS FORMATION AND CELL-TO-CELL VIRUS TRANSMISSION ARE PREVENTED BY DELETION OF THE GENE ENCODING THE 37,000-DALTON OUTER ENVELOPE PROTEIN
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DOI：
10.1128/jvi.65.11.5910-5920.1991
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发表时间：
1991-11-01
影响因子：
5.4
通讯作者：
MOSS, B
中科院分区：
文献类型：
--
作者：
BLASCO, R;MOSS, B
传染性痘苗病毒颗粒有两种类型：细胞内裸病毒粒子和细胞外包膜病毒粒子（EEV）。为了确定包膜形式的牛痘病毒的生物学作用，我们产生并鉴定了EEV形成缺陷的突变体。该策略包括将编码EEV外包膜特异性37,000-Da蛋白（VP37）的基因F13L与选择性抗生素抗性标记大肠杆菌gpt基因进行同源重组。然而，最初的实验表明，这种突变是致命的或阻止斑块的形成。通过采用由转染细胞的细胞内病毒的高倍数传代组成的方案，然后限制稀释克隆，我们成功地分离了所需的突变体，该突变体在产生斑块和细胞外病毒方面存在缺陷，但在细胞内产生正常数量的裸病毒粒子。电镜检查表明，与野生型不同，突变病毒颗粒既没有被高尔基衍生膜包裹，也没有与细胞表面相关。VP37的缺失并没有阻止病毒血凝素向质膜的转运，但却破坏了低ph和抗体介导的细胞融合。这些结果表明VP37是EEV形成所必需的，并且在牛痘病毒的局部细胞间传播中也起着关键作用，可能是通过附着在细胞膜上或从细胞膜上释放的包膜病毒粒子。相比之下，一种与VP37基因同源的K4L开放阅读框缺失的突变病毒在形成斑块或EEV方面没有缺陷。
There are two types of infectious vaccinia virus particles: intracellular naked virions and extracellular enveloped virions (EEV). To determine the biological role of the enveloped form of vaccinia virus, we produced and characterized a mutant that is defective in EEV formation. The strategy involved replacement by homologous recombination of the gene F13L, encoding a 37,000-Da protein (VP37) that is specific for the outer envelope of EEV, with a selectable antibiotic resistance marker, the Escherichia coli gpt gene. Initial experiments, however, suggested that such a mutation was lethal or prevented plaque formation. By employing a protocol consisting of high-multiplicity passages of intracellular virus from the transfected cells and then limiting dilution cloning, we succeeded in isolating the desired mutant, which was defective in production of plaques and extracellular virus but made normal amounts of intracellular naked virions. Electron microscopic examination indicated that the mutant virus particles, unlike wild type, were neither wrapped with Golgi-derived membranes nor associated with the cell surface. The absence of VP37 did not prevent the transport of the viral hemagglutinin to the plasma membrane but nevertheless abrogated both low-pH- and antibody-mediated cell fusion. These results indicate that VP37 is required for EEV formation and also plays a critical role in the local cell-to-cell transmission of vaccinia virus, perhaps via enveloped virions attached to or released from the cell membrane. By contrast, a mutated virus with a deletion of the K4L open reading frame, which is a homolog of the VP37 gene, was not defective in formation of plaques or EEV.