Imaging the Alphavirus Exit Pathway
Imaging the Alphavirus Exit Pathway
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DOI：
10.1128/jvi.00592-14
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发表时间：
2014-06-01
影响因子：
5.4
通讯作者：
Kielian, Margaret
中科院分区：
文献类型：
--
作者：
Martinez, Maria Guadalupe;Snapp, Erik-Lee;Kielian, Margaret
甲病毒是小包膜RNA病毒，具有高度组织化的结构，排斥宿主细胞蛋白。它们含有一个内部核衣壳和一个病毒E2和E1跨膜蛋白的外部晶格。甲病毒从质膜（PM）萌发，但甲病毒组装和出芽的过程和动力学尚不清楚。在这里，我们生成了在E2包膜蛋白上带有荧光蛋白标记的Sindbis病毒（sinv），并利用它们来表征病毒在活细胞中的组装和出芽。在病毒感染过程中，E2在PM上的局部斑块和丝状突起中富集。这些e2标记的斑块和延伸包含了所有的病毒结构蛋白。相关光镜和电镜研究证实，斑块和延伸与病毒出芽结构共定位，而光镜显示它们排除了自由扩散的PM标记蛋白。排除需要E2蛋白与衣壳蛋白相互作用，这是病毒出芽的关键步骤，并且与包膜蛋白在细胞表面的固定化有关。病毒感染诱导了两种不同类型的延伸：微管蛋白阴性延伸，长度约为2至4 μ m，不含PM标记；微管蛋白阳性延伸，长度约为10 μ m，含有PM标记，可以将病毒颗粒转移到未感染的细胞中。在未出芽的SINV突变体感染的细胞中，微管蛋白阳性延伸选择性地减少。总之，我们的数据支持甲病毒感染诱导PM和细胞骨架重组的模型，导致病毒从专门的位点出芽。
Alphaviruses are small enveloped RNA viruses with highly organized structures that exclude host cell proteins. They contain an internal nucleocapsid and an external lattice of the viral E2 and E1 transmembrane proteins. Alphaviruses bud from the plasma membrane (PM), but the process and dynamics of alphavirus assembly and budding are poorly understood. Here we generated Sindbis viruses (SINVs) with fluorescent protein labels on the E2 envelope protein and exploited them to characterize virus assembly and budding in living cells. During virus infection, E2 became enriched in localized patches on the PM and in filopodium-like extensions. These E2-labeled patches and extensions contained all of the viral structural proteins. Correlative light and electron microscopy studies established that the patches and extensions colocalized with virus budding structures, while light microscopy showed that they excluded a freely diffusing PM marker protein. Exclusion required the interaction of the E2 protein with the capsid protein, a critical step in virus budding, and was associated with the immobilization of the envelope proteins on the cell surface. Virus infection induced two distinct types of extensions: tubulin-negative extensions that were similar to 2 to 4 mu m in length and excluded the PM marker, and tubulin-positive extensions that were >10 mu m long, contained the PM marker, and could transfer virus particles to noninfected cells. Tubulin-positive extensions were selectively reduced in cells infected with a nonbudding SINV mutant. Together, our data support a model in which alphavirus infection induces reorganization of the PM and cytoskeleton, leading to virus budding from specialized sites.