Localization of actin in moloney murine leukemia virus by immunoelectron microscopy
Localization of actin in moloney murine leukemia virus by immunoelectron microscopy
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DOI：
10.1006/viro.1999.9803
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发表时间：
1999-07-20
期刊：
影响因子：
3.7
通讯作者：
Pager, J
中科院分区：
文献类型：
--
作者：
Nermut, MV;Wallengren, K;Pager, J
使用免疫电子显微镜检测野生型（wt）莫洛尼鼠白血病病毒（MoMuLV）和仅表达MoMuV gag多蛋白的重组塞姆利基森林病毒产生的病毒样颗粒（VLP）中的肌动蛋白。金免疫标记揭示了肌动蛋白在脱脂VLP和脱脂WT病毒颗粒表面上的存在。每个VLP的胶体金颗粒数量的统计学评价揭示了大范围的值和具有少量金颗粒的VLP的患病率。肌动蛋白的标记后，失去了长期治疗的VLP与1% Nonidet-P40，高pH缓冲液，或凝溶胶蛋白。gag蛋白p15（MA）和p12和p30（CA）的抗体的金免疫标记是丰富的，不受VLP或野生型病毒与1%的Nonidet或凝溶胶蛋白处理。用洗涤剂和醛固定剂的混合物处理的VLP显示出比没有固定剂更均匀和一致的肌动蛋白标记。阴性染色或重金属阴影显示脱脂VLP的球形表面。金免疫标记VLP的立体显微照片显示，p15 gag和p12 gag与球状突起相关。去脂化的VLP也被p30 gag的抗体很好地标记，这表明gag壳允许p30 gag的抗体进入，因此不是紧密堆积的结构。肌动蛋白结合蛋白膜突蛋白和埃兹蛋白的标记是阴性的野生型病毒和VLP。VLP样品中肌动蛋白的高斯分布的缺失表明肌动蛋白不是结构蛋白，并且其在MuLV病毒颗粒中的存在可能是偶然的。然而，这并不排除肌动蛋白在病毒颗粒的运输、组装、出芽或释放中的任何可能作用，这些事件发生在细胞质或质膜上。肌动蛋白在VLP的网站进行了讨论，目前的知识的分子组织的MuLV盖壳。(C)北京：科学出版社.
Immunoelectron microscopy was used to detect actin in wild-type (wt) Moloney murine leukemia virus (MoMuLV) and in virus-like particles (VLP) produced by recombinant Semliki Forest virus expressing only the MoMuV gag polyprotein. Gold immunolabeling revealed the presence of actin on the surface of delipidized VLP and delipidized wt virus particles. Statistical evaluation of the number of colloidal gold particles per VLP revealed a large range of Values and a prevalence of VLP with small numbers of gold particles. Labeling for actin was lost after prolonged treatment of VLP with 1% Nonidet-P40, high-pH buffer, or gelsolin. Gold immunolabeling with antibodies to gag proteins p15(MA) and p12 and p30 (CA) was abundant and was not affected by treatment of VLP or wt virus with 1% Nonidet or gelsolin. VLP treated with a mixture of detergent and aldehyde fixatives showed more uniform and consistent labeling for actin than without fixatives. Negative staining or heavy metal shadowing revealed a globular surface of delipidized VLP. Stereomicrographs of gold-immunolabeled VLP showed that p15gag and p12gag were associated with the globular projections. Delipidized VLP were also well labeled with antibody to p30gag, which indicated that the gag shell permitted access of antibodies to p30gag and was therefore not a closely packed structure. Labeling for actin-binding proteins moesin and ezrin was negative in both the wt virus and the VLP. The absence of Gaussian distribution of actin in the sample of VLP suggests that actin is not a structural protein and its presence in MuLV virus particles may be fortuitous. This, however, does not rule out any possible role of actin in transport, assembly, budding, or release of virus particles, events which take place in the cytoplasm or at the plasma membrane. The site of actin in VLP is discussed in relation to the present knowledge of the molecular organization of the MuLV gag shell. (C) 1999 Academic Press.