Intercellular Extensions Are Induced by the Alphavirus Structural Proteins and Mediate Virus Transmission.
Intercellular Extensions Are Induced by the Alphavirus Structural Proteins and Mediate Virus Transmission.
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DOI：
10.1371/journal.ppat.1006061
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发表时间：
2016-12
期刊：
影响因子：
6.7
通讯作者：
Kielian M
中科院分区：
文献类型：
--
作者：
Martinez MG;Kielian M
甲型病毒是一种高度组织化的包膜RNA病毒，其内部核衣壳被包含E2和E1跨膜蛋白的膜所包围。甲型病毒的萌发发生在质膜上，需要E2的细胞质结构域与衣壳蛋白的相互作用。在这里，我们使用WT甲型病毒和Sindbis病毒，其中E2被融合到一个荧光蛋白中来表征病毒从宿主细胞中的退出。我们的结果表明，甲型病毒感染引起宿主细胞细胞骨架的显着改变，并导致稳定的细胞间延伸的形成，这些延伸仅来自受感染的细胞。细胞间延伸较长(~gt；10μM)，含有肌动蛋白和微管蛋白，与相邻细胞形成扁平接触，但不调节细胞间膜或细胞质的连续性。受体下调研究表明，稳定延伸的形成不需要病毒受体，并且延伸促进了病毒在细胞之间的传播到受体耗尽的细胞。病毒突变实验表明，延伸的形成需要E2-衣壳的相互作用，而不是活性颗粒的萌发，而感染的细胞间传播需要产生融合活性的病毒颗粒。蛋白质表达研究表明，即使在没有病毒感染的情况下，病毒结构蛋白本身也会诱导细胞间延伸，并且这些延伸优先针对不表达的细胞。总之，我们的结果确定了甲型病毒细胞间传播的机制，并定义了它所需的关键病毒蛋白相互作用。甲型病毒是一组小包膜RNA病毒，包括一些重要的人类病原体，如基孔肯雅病毒和导致致命脑炎的病毒。基孔肯雅病毒最近在包括美洲在内的世界许多国家出现，它在那里造成了重大疫情。迫切需要针对这些病毒的疫苗和抗病毒战略，有关甲型病毒感染途径的基本信息将有助于针对关键步骤。在这里，我们描述了甲型病毒感染细胞的变化，使其能够将病毒传播给邻近的未感染细胞。受感染的细胞形成长长的延伸，与邻近的细胞接触，并介导病毒在细胞间的传播。这种病毒传播机制可能有助于保护病毒免受宿主抗体的中和。令人惊讶的是，病毒结构蛋白的表达本身就诱导了这些细胞间的延伸，这些细胞优先针对不表达的细胞。我们使用这个系统来定义衣壳和包膜蛋白之间的关键相互作用，这是形成延伸所必需的。
Alphaviruses are highly organized enveloped RNA viruses with an internal nucleocapsid surrounded by a membrane containing the E2 and E1 transmembrane proteins. Alphavirus budding takes place at the plasma membrane and requires the interaction of the cytoplasmic domain of E2 with the capsid protein. Here we used WT alphaviruses and Sindbis virus in which E2 was fused to a fluorescent protein to characterize virus exit from host cells. Our results show that alphavirus infection induced striking modifications of the host cell cytoskeleton and resulted in the formation of stable intercellular extensions that emanated exclusively from the infected cell. The intercellular extensions were long (> 10 μM), contained actin and tubulin, and formed flattened contacts with neighboring cells, but did not mediate membrane or cytoplasmic continuity between cells. Receptor down-regulation studies indicated that formation of stable extensions did not require the virus receptor, and that extensions promoted cell-to-cell virus transmission to receptor-depleted cells. Virus mutant experiments demonstrated that formation of extensions required the E2-capsid interaction but not active particle budding, while intercellular transmission of infection required the production of fusion-active virus particles. Protein expression studies showed that even in the absence of virus infection, the viral structural proteins alone induced intercellular extensions, and that these extensions were preferentially targeted to non-expressing cells. Together, our results identify a mechanism for alphavirus cell-to-cell transmission and define the key viral protein interactions that it requires. Alphaviruses are a group of small enveloped RNA viruses that include a number of important human pathogens such as Chikungunya virus and viruses that cause fatal encephalitis. Chikungunya virus emerged recently in a number of countries worldwide including the Americas, where it has caused major outbreaks. Vaccines and anti-viral strategies for these viruses are urgently needed, and basic information on the alphavirus infection pathway will help in targeting critical steps. Here we describe the changes in the alphavirus-infected cell that allow it to transmit virus to neighboring uninfected cells. Infected cells form long extensions that contact neighboring cells and mediate cell-to-cell virus transmission. This mechanism of virus transmission may help to shield virus from neutralization by host antibodies. Surprisingly, expression of the viral structural proteins alone induces these intercellular extensions, which preferentially target non-expressing cells. We used this system to define a critical interaction of the capsid and envelope protein that is required for formation of extensions.
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通讯作者：
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