Cristea Ileana M, Carroll John-William N, Rout Michael P, Rice Charles M, Chait Brian T, MacDonald Margaret R
Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York 10021, USA.
J Biol Chem. 2006 Oct 6;281(40):30269-78. doi: 10.1074/jbc.M603980200. Epub 2006 Aug 8.
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. Note-worthy 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.
病毒感染会导致宿主细胞发生深刻变化。在此，我们探究了哺乳动物细胞感染过程中，甲病毒辛德毕斯病毒蛋白与宿主因子之间的相互作用。利用表达带有绿色荧光蛋白（GFP）标签的病毒nsP3蛋白的突变病毒，我们在感染后的不同时间直接观察了nsP3的定位，并分离了与nsP3相互作用的蛋白。这些结果表明，宿主因子向含nsP3复合物的募集具有时间依赖性，G3BP在早期有特异性且持续的募集，而14-3-3蛋白则在后期募集。带有GFP标签的G3BP的表达使得在辛德毕斯感染的细胞中能够相互分离出nsP3，同时也能鉴定出未感染和感染细胞中与G3BP相互作用的新蛋白。值得注意的相互作用包括核孔复合体成分，它们与G3BP的相互作用在辛德毕斯感染后减弱。这表明G3BP如先前假设的那样是一种核转运因子，并且病毒感染可能会改变RNA转运。免疫电子显微镜显示，一部分辛德毕斯nsP3定位于核膜，这表明这可能是G3BP募集到含nsP3复合物的一个位点。我们的结果证明了使用标准GFP标签在感染的哺乳动物细胞中追踪病毒蛋白定位并随着时间推移阐明特定病毒-宿主相互作用的实用性。
