Rouiller I, Brookes S M, Hyatt A D, Windsor M, Wileman T
Division of Immunology, Pirbright Laboratories, Institute for Animal Health, Surrey, England.
J Virol. 1998 Mar;72(3):2373-87. doi: 10.1128/JVI.72.3.2373-2387.1998.
African swine fever (ASF) virus is a large DNA virus that shares the striking icosahedral symmetry of iridoviruses and the genomic organization of poxviruses. Both groups of viruses have a complex envelope structure. In this study, the mechanism of formation of the inner envelope of ASF virus was investigated. Examination of thin cryosections by electron microscopy showed two internal membranes in mature intracellular virions and all structural intermediates. These membranes were in continuity with intracellular membrane compartments, suggesting that the virus gained two membranes from intracellular membrane cisternae. Immunogold electron microscopy showed the viral structural protein p17 and resident membrane proteins of the endoplasmic reticulum (ER) within virus assembly sites, virus assembly intermediates, and mature virions. Resident ER proteins were also detected by Western blotting of isolated virions. The data suggested the ASF virus was wrapped by the ER. Analysis of the published sequence of ASF virus (R. J. Yanez et al., Virology 208:249-278, 1995) revealed a reading frame, XP124L, that encoded a protein predicted to translocate into the lumen of the ER. Pulse-chase immunoprecipitation and glycosylation analysis of pXP124L, the product of the XP124L gene, showed that pXP124L was retained in the ER lumen after synthesis. When analyzed by immunogold electron microscopy, pXP124L localized to virus assembly intermediates and fully assembled virions. Western blot analysis detected pXP124L in virions isolated from Percoll gradients. The packaging of pXP124L from the lumen of the ER into the virion is consistent with ASF virus being wrapped by ER cisternae: a mechanism which explains the presence of two membranes in the viral envelope.
非洲猪瘟（ASF）病毒是一种大型DNA病毒，具有与虹彩病毒惊人的二十面体对称性以及痘病毒的基因组结构。这两类病毒都有复杂的包膜结构。在本研究中，对ASF病毒内膜包膜的形成机制进行了研究。通过电子显微镜对冷冻超薄切片的检查显示，在成熟的细胞内病毒粒子以及所有结构中间体中存在两层内膜。这些膜与细胞内膜隔室相连，表明病毒从细胞内膜池中获得了两层膜。免疫金电子显微镜显示，病毒结构蛋白p17和内质网（ER）的驻留膜蛋白存在于病毒装配位点、病毒装配中间体和成熟病毒粒子中。通过对分离出的病毒粒子进行蛋白质印迹分析也检测到了内质网驻留蛋白。数据表明ASF病毒被内质网包裹。对已发表的ASF病毒序列（R. J. Yanez等人，《病毒学》208:249 - 278，1995年）进行分析，发现一个阅读框XP124L，其编码的一种蛋白质预计会转运到内质网腔中。对XP124L基因产物pXP124L进行脉冲追踪免疫沉淀和糖基化分析表明，pXP124L在合成后保留在内质网腔中。通过免疫金电子显微镜分析时，pXP124L定位于病毒装配中间体和完全装配好的病毒粒子中。蛋白质印迹分析在从Percoll梯度中分离出的病毒粒子中检测到了pXP124L。pXP124L从内质网腔包装到病毒粒子中，这与ASF病毒被内质网池包裹是一致的：这一机制解释了病毒包膜中存在两层膜的现象。
