ASSEMBLY OF VACCINIA VIRUS - EFFECTS OF RIFAMPIN ON THE INTRACELLULAR-DISTRIBUTION OF VIRAL PROTEIN P65
ASSEMBLY OF VACCINIA VIRUS - EFFECTS OF RIFAMPIN ON THE INTRACELLULAR-DISTRIBUTION OF VIRAL PROTEIN P65
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DOI：
10.1128/jvi.68.2.1103-1114.1994
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发表时间：
1994-02-01
影响因子：
5.4
通讯作者：
DOMS, RW
中科院分区：
文献类型：
--
作者：
SODEIK, B;GRIFFITHS, G;DOMS, RW
牛痘病毒的细胞质组装被抗生素利福平可逆地阻断，导致部分膜界定的利福平小体在感染细胞中积累。耐利福平痘苗病毒突变体的 D13L 基因具有点突变，该基因由晚期启动子控制并表达 65 kDa 的蛋白质，称为 p65。为了进一步表征利福平抑制机制和 p65 在病毒组装中的功能，我们产生了针对该蛋白的抗体。免疫反应性 p65 在感染后期表达，其表达及其周转均不受利福平影响。病毒相关的 p65 只能用变性去污剂从纯化的病毒颗粒中提取，这表明它是病毒的一个组成部分。免疫荧光研究表明 p65 定位于病毒组装位点。此外，免疫电子显微镜显示 p65 与病毒新月体以及球形、未成熟的病毒体相关，在这两种情况下主要在内表面或凹表面上。在利福平存在的情况下，p65 被发现存在于与利福平小体不同的大的细胞质包涵体中。仅在利福平阻断逆转后，利福平体本身才被 p65 抗体标记，主要标记在药物去除后迅速形成的病毒新月体上。我们认为 p65 在病毒新月体和未成熟病毒体形成过程中充当内部支架，类似于其他病毒的基质蛋白。
The cytoplasmic assembly of vaccinia virus is reversibly blocked by the antibiotic rifampin, leading to the accumulation of partially membrane-delineated rifampin bodies in infected cells. Rifampin-resistant vaccinia virus mutants have point mutations in the D13L gene, which is controlled by a late promoter and expresses a 65-kDa protein, designated p65. To further characterize the mechanism of rifampin inhibition and the function of p65 in virus assembly, we raised antibodies to this protein. Immunoreactive p65 was expressed at late times of infection, and neither its expression nor its turnover was affected by rifampin. Virus-associated p65 could be extracted only with denaturing detergents from purified virions, suggesting that it is an integral viral component. Immunofluorescence studies showed that p65 is localized to the sites of virus assembly. Also, immunoelectron microscopy showed p65 to be associated with viral crescents as well as spherical, immature virions, in both cases predominantly on the inner or concave surface. In the presence of rifampin, p65 was found in large, cytoplasmic inclusion bodies that were distinct from rifampin bodies. The rifampin bodies themselves were labeled with p65 antibodies only after reversal of the rifampin block, predominantly on the viral crescents which rapidly formed following removal of the drug. We propose that p65 functions as an internal scaffold in the formation of viral crescents and immature virions, analogously to the matrix proteins of other viruses.