Forstová J, Krauzewicz N, Wallace S, Street A J, Dilworth S M, Beard S, Griffin B E
Department of Virology, Royal Postgraduate Medical School, London, United Kingdom.
J Virol. 1993 Mar;67(3):1405-13. doi: 10.1128/JVI.67.3.1405-1413.1993.
The polyomavirus minor late capsid antigen, VP2, is myristylated on its N-terminal glycine, this modification being required for efficient infection of mouse cells. To study further the functions of this antigen, as well as those of the other minor late antigen, VP3, recombinant baculoviruses carrying genes for VP1, VP2, and VP3 have been constructed and the corresponding proteins have been synthesized in insect cells. A monoclonal antibody recognizing VP1, alpha-PyVP1-A, and two monoclonal antibodies against the common region of VP2 and VP3, alpha-PyVP2/3-A and alpha-PyVP2/3-B, have been generated. Reactions of antibodies with antigens were characterized by indirect immunofluorescence, immunoprecipitation, and immunoblot analysis. Immunofluorescent staining of mouse cells infected with polyomavirus showed all antigens to be localized in nuclei. When the late polyomavirus proteins were expressed separately in insect cells, however, only VP1 was efficiently transported into the nucleus; VP2 was localized discretely around the outside of the nucleus, and VP3 exhibited a diffused staining pattern in the cytoplasm. Coexpression of VP2, or VP3, with VP1 restored nuclear localization. Immunoprecipitation of infected mouse cells with either anti-VP1 or anti-VP2/3 antibodies precipitated complexes containing all three species, consistent with the notion that VP1 is necessary for efficient transport of VP2 and VP3 into the nucleus. Purified empty capsid-like particles, formed in nuclei of insect cells coinfected with all three baculoviruses, contained VP2 and VP3 proteins in amounts comparable to those found in empty capsids purified from mouse cells infected with wild-type polyomavirus. Two-dimensional gel analysis of VP1 species revealed that coexpression with VP2 affects posttranslational modification of VP1.
多瘤病毒次要晚期衣壳抗原VP2在其N端甘氨酸上发生肉豆蔻酰化，这种修饰是有效感染小鼠细胞所必需的。为了进一步研究该抗原以及另一种次要晚期抗原VP3的功能，构建了携带VP1、VP2和VP3基因的重组杆状病毒，并在昆虫细胞中合成了相应的蛋白质。已产生一种识别VP1的单克隆抗体α-PyVP1-A以及两种针对VP2和VP3共同区域的单克隆抗体α-PyVP2/3-A和α-PyVP2/3-B。通过间接免疫荧光、免疫沉淀和免疫印迹分析对抗体与抗原的反应进行了表征。用多瘤病毒感染的小鼠细胞的免疫荧光染色显示所有抗原都定位于细胞核中。然而，当多瘤病毒晚期蛋白在昆虫细胞中单独表达时，只有VP1能有效地转运到细胞核中；VP2离散地定位于细胞核外部周围，而VP3在细胞质中呈现弥散的染色模式。VP2或VP3与VP1共表达可恢复核定位。用抗VP1或抗VP2/3抗体对感染的小鼠细胞进行免疫沉淀，沉淀出包含所有三种蛋白的复合物，这与VP1是VP2和VP3有效转运到细胞核所必需的观点一致。在同时感染所有三种杆状病毒的昆虫细胞核中形成的纯化空衣壳样颗粒含有VP2和VP3蛋白，其含量与从感染野生型多瘤病毒的小鼠细胞中纯化的空衣壳中的含量相当。VP1蛋白的二维凝胶分析表明，与VP2共表达会影响VP1的翻译后修饰。
