Tracking and elucidating Alphavirus-host protein interactions
Tracking and elucidating Alphavirus-host protein interactions
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DOI：
10.1074/jbc.m603980200
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发表时间：
2006-10-06
影响因子：
4.8
通讯作者：
MacDonald, Margaret R.
中科院分区：
文献类型：
--
作者：
Cristea, Ileana M.;Carroll, John-William N.;MacDonald, Margaret R.
病毒感染引起宿主细胞的深刻变化。在这里，我们探讨了在哺乳动物细胞感染的过程中，甲病毒辛德毕斯和宿主因子的蛋白质之间的相互作用。使用突变体病毒表达的病毒nsP 3蛋白标记的绿色荧光蛋白（GFP），我们直接观察nsP 3的定位和分离nsP 3相互作用的蛋白在感染后的不同时间。这些结果表明，宿主因子招募到含有nsP 3的复合物是时间依赖性的，具有特定的早期和持续的G3 BP招募和14-3-3蛋白的后期招募。GFP标记的G3 BP的表达允许在辛德比斯感染的细胞中相互分离nsP 3，以及在未感染和感染的细胞中鉴定新的G3 BP相互作用蛋白。值得注意的相互作用包括核孔复合物组分，其与G3 BP的相互作用在辛德毕斯感染后减少。这表明，G3 BP是一个核转运因子，如先前假设的那样，病毒感染可能会改变RNA转运。免疫电子显微镜显示，Sindbis nsP 3的一部分是本地化的核膜，这表明一个可能的网站G3 BP招聘nsP 3含复合物。我们的研究结果表明，使用一个标准的绿色荧光蛋白标签，既跟踪病毒蛋白定位和阐明特定的病毒宿主相互作用，随着时间的推移，在感染的哺乳动物细胞的效用。
Viral infections cause profound alterations in host cells. Here, we explore the interactions between proteins of the Alphavirus Sindbis and host factors during the course of mammalian cell infection. Using a mutant virus expressing the viral nsP3 protein tagged with green fluorescent protein (GFP) we directly observed nsP3 localization and isolated nsP3-interacting proteins at various times after infection. These results revealed that host factor recruitment to nsP3-containing complexes was time dependent, with a specific early and persistent recruitment of G3BP and a later recruitment of 14-3-3 proteins. Expression of GFP-tagged G3BP allowed reciprocal isolation of nsP3 in Sindbis infected cells, as well as the identification of novel G3BP-interacting proteins in both uninfected and infected cells. Noteworthy interactions include nuclear pore complex components whose interactions with G3BP were reduced upon Sindbis infection. This suggests that G3BP is a nuclear transport factor, as hypothesized previously, and that viral infection may alter RNA transport. Immunoelectron microscopy showed that a portion of Sindbis nsP3 is localized at the nuclear envelope, suggesting a possible site of G3BP recruitment to nsP3-containing complexes. Our results demonstrate the utility of using a standard GFP tag to both track viral protein localization and elucidate specific viral-host interactions over time in infected mammalian cells.