Vaccinia virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts
Vaccinia virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts
复制标题
DOI：
10.1128/jvi.79.3.1623-1634.2005
复制
发表时间：
2005-02-01
影响因子：
5.4
通讯作者：
Chang, W
中科院分区：
文献类型：
--
作者：
Chung, CS;Huang, CY;Chang, W
痘苗病毒可感染来自不同宿主的多种哺乳动物细胞，但病毒侵入的机制尚不清楚。成熟的细胞内痘苗病毒含有几种包膜蛋白，介导病毒粒子吸附到细胞表面的糖胺多聚糖上；然而，结合的病毒粒子如何启动病毒粒子进入细胞尚不清楚。在这项研究中，我们利用生化和荧光成像技术研究了质膜脂筏在成熟的细胞内痘苗病毒感染过程中的重要性。一种破坏牛排的药物，甲基-β-环糊精，在不影响病毒粒子附着的情况下抑制了痘苗病毒的脱壳，表明含胆固醇的脂筏对于病毒粒子渗透到哺乳动物细胞是必不可少的。为了提供病毒与脂筏联系的直接证据，我们在病毒感染后立即从细胞中分离出不溶于洗涤剂的糖脂富集膜，并证明在细胞膜脂筏部分中存在几种病毒包膜蛋白A14、A17L和D8L，而包膜蛋白H3L不存在。当病毒粒子在4℃下附着在细胞上时，这种联系不会发生，只有当病毒粒子在37℃时发生穿透时才能观察到这种联系。免疫荧光显微镜还显示，病毒包膜蛋白的细胞表面染色与质膜上的脂筏标记GM1共存，这与生化分析一致。最后，缺乏H3L、D8L或A27L蛋白的突变病毒仍然与脂筏相关，这表明牛痘病毒粒子通过糖胺多聚糖的初始附着不是形成脂筏所必需的。
Vaccinia virus infects a wide variety of mammalian cells from different hosts, but the mechanism of virus entry is not clearly defined. The mature intracellular vaccinia virus contains several envelope proteins mediating virion adsorption to cell surface glycosaminoglycans; however, it is not known how the bound virions initiate virion penetration into cells. For this study, we investigated the importance of plasma membrane lipid rafts in the mature intracellular vaccinia virus infection process by using biochemical and fluorescence imaging techniques. A raft-disrupting drug, methyl-beta-cyclodextrin, inhibited vaccinia virus uncoating without affecting virion attachment, indicating that cholesterol-containing lipid rafts are essential for virion penetration into mammalian cells. To provide direct evidence of a virus and lipid raft association, we isolated detergent-insoluble glycolipid-enriched membranes from cells immediately after virus infection and demonstrated that several viral envelope proteins, A14, A17L, and D8L, were present in the cell membrane lipid raft fractions, whereas the envelope H3L protein was not. Such an association did not occur after virions attached to cells at 4degreesC and was only observed when virion penetration occurred at 37degreesC. Immunofluorescence microscopy also revealed that cell surface staining of viral envelope proteins was colocalized with GM1, a lipid raft marker on the plasma membrane, consistent with biochemical analyses. Finally, mutant viruses lacking the H3L, D8L, or A27L protein remained associated with lipid rafts, indicating that the initial attachment of vaccinia virions through glycosaminoglycans is not required for lipid raft formation.