Vanslyke J K, Hruby D E
Department of Microbiology, Oregon State University, Corvallis 97331-3804.
Virology. 1994 Feb;198(2):624-35. doi: 10.1006/viro.1994.1074.
Proteolytic processing of vaccinia virus core proteins is an essential step in the formation of mature virions and occurs during the process of virion morphogenesis. In order to investigate how the vaccinia virus (VV) structural proteins become integrated into virus particles during normal maturation, immunological reagents were generated against the three major VV core proteins 4a, 4b, and 25K and their precursor molecules P4a, P4b, and P25K. These sera were used in conjunction with immunofluorescent and immunogold labeling of VV-infected tissue culture cells. The immunofluorescent results indicated that all three core precursors and their cleavage products were localized to virosomes. As the infection progressed, punctate staining with these sera became spread throughout the cytoplasm which suggested that individual virion particles were being recognized. Immunoelectron microscopy showed that the core proteins were localized to the center of both immature and mature virus particles. This result was in contrast to the situation observed using antisera directed against L65, a protein previously implicated in the assembly of the viral membrane. Immunogold staining of L65 showed that it was initially located along the inner side of the immature virion membrane and remained with the membrane even as the viroplasm began to condense toward the center of the virus particle. In order to determine whether the core protein localization observed was the result of precursors, products, or both, a synthetic peptide strategy was used to generate an antiserum that recognized only P4a in immunoprecipitation reactions. Immunogold labeling with this reagent indicated that P4a was found in the viroplasm of immature particles and in low levels in the mature virion. Intracellular localization of core and L65 proteins during virion morphogenesis is discussed.
痘苗病毒核心蛋白的蛋白水解加工是成熟病毒粒子形成过程中的一个关键步骤，且发生在病毒粒子形态发生过程中。为了研究痘苗病毒（VV）结构蛋白在正常成熟过程中是如何整合到病毒颗粒中的，我们制备了针对三种主要的VV核心蛋白4a、4b和25K及其前体分子P4a、P4b和P25K的免疫试剂。这些血清与感染VV的组织培养细胞的免疫荧光和免疫金标记结合使用。免疫荧光结果表明，所有三种核心前体及其裂解产物都定位于病毒小体。随着感染的进展，用这些血清进行的点状染色扩散到整个细胞质中，这表明单个病毒粒子被识别。免疫电子显微镜显示，核心蛋白定位于未成熟和成熟病毒粒子的中心。这一结果与使用针对L65的抗血清所观察到的情况形成对比，L65是一种先前被认为参与病毒膜组装的蛋白质。L65的免疫金染色显示，它最初位于未成熟病毒粒子膜的内侧，即使病毒质开始向病毒粒子中心浓缩时，它仍与膜结合。为了确定所观察到的核心蛋白定位是前体、产物还是两者的结果，我们采用了合成肽策略来制备一种仅在免疫沉淀反应中识别P4a的抗血清。用该试剂进行的免疫金标记表明，P4a存在于未成熟粒子的病毒质中，在成熟病毒粒子中的含量较低。本文讨论了病毒粒子形态发生过程中核心蛋白和L65蛋白的细胞内定位。
