Virus-mediated compartmentalization of the host translational machinery.
Virus-mediated compartmentalization of the host translational machinery.
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DOI：
10.1128/mbio.01463-14
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发表时间：
2014-09-16
期刊：
影响因子：
6.4
通讯作者：
Parker JS
中科院分区：
文献类型：
--
作者：
Desmet EA;Anguish LJ;Parker JS
病毒需要宿主的转译装置来合成病毒蛋白。因此，抑制翻译的宿主应激反应机制是病毒必须克服的一个重大障碍。在这里，我们报告了一种策略，即哺乳动物正肠病毒在病毒诱导的包含体中划分翻译机制，称为病毒工厂（VF）。VF是呼肠孤病毒复制和组装的位点，但被认为不含核糖体。假设病毒mrna离开VF，通过细胞机器进行翻译，蛋白质重新进入工厂参与组装。在这里，我们使用核脲基化来观察感染细胞中的活性翻译。这些研究表明，活性翻译发生在VF内，并且翻译起始、延伸、终止和再循环所需的核糖体亚基和蛋白质定位于工厂。有趣的是，我们观察到43S起始前复合体（PIC）的成分主要集中在工厂边缘，这表明在VF内存在空间和/或动态的翻译组织。同样，病毒单链RNA结合蛋白σNS定位于工厂边缘，具有从工厂边缘延伸的短距离管泡状染色模式，并与内质网（ER）标记共定位。与这些共定位研究一致，σNS被发现与真核翻译起始因子3亚基A （eIF3A）和核糖体亚基pS6R相关。综上所述，这些发现表明σNS在病毒工厂中将43S PIC机制招募到病毒转译的主要位点。病原体介导的翻译装置区隔化提供了一种新的机制，病毒可以通过这种机制避免宿主的翻译抑制。病毒缺乏生物合成能力，依赖宿主合成蛋白质。这种依赖性要求病毒进化出一种机制，迫使宿主的翻译机制在面对持续的抑制全局蛋白质合成的细胞应激反应时合成病毒蛋白。呼肠孤病毒在称为病毒工厂的细胞质内含物内复制和组装。然而，病毒蛋白的合成被认为发生在细胞质中。为了确定病毒翻译的位点，我们采用了一种基于显微镜的方法，使用核嘌呤mycyylation来检测活性翻译。在这里，我们报告了主动翻译发生在病毒工厂内，翻译因子在工厂内被划分。此外，我们发现呼肠孤病毒非结构蛋白σNS在工厂边缘与43S起始前复合物结合，表明σNS在翻译中起作用。总之，病毒诱导的宿主翻译机制的区隔化代表了病毒将病毒蛋白质合成与病毒复制和组装在时空上偶联的一种策略。
Viruses require the host translational apparatus to synthesize viral proteins. Host stress response mechanisms that suppress translation, therefore, represent a significant obstacle that viruses must overcome. Here, we report a strategy whereby the mammalian orthoreoviruses compartmentalize the translational machinery within virus-induced inclusions known as viral factories (VF). VF are the sites of reovirus replication and assembly but were thought not to contain ribosomes. It was assumed viral mRNAs exited the VF to undergo translation by the cellular machinery, and proteins reentered the factory to participate in assembly. Here, we used ribopuromycylation to visualize active translation in infected cells. These studies revealed that active translation occurs within VF and that ribosomal subunits and proteins required for translation initiation, elongation, termination, and recycling localize to the factory. Interestingly, we observed components of the 43S preinitiation complex (PIC) concentrating primarily at factory margins, suggesting a spatial and/or dynamic organization of translation within the VF. Similarly, the viral single-stranded RNA binding protein σNS localized to the factory margins and had a tubulovesicular staining pattern that extended a short distance from the margins of the factories and colocalized with endoplasmic reticulum (ER) markers. Consistent with these colocalization studies, σNS was found to associate with both eukaryotic translation initiation factor 3 subunit A (eIF3A) and the ribosomal subunit pS6R. Together, these findings indicate that σNS functions to recruit 43S PIC machinery to the primary site of viral translation within the viral factory. Pathogen-mediated compartmentalization of the translational apparatus provides a novel mechanism by which viruses might avoid host translational suppression. Viruses lack biosynthetic capabilities and depend upon the host for protein synthesis. This dependence requires viruses to evolve mechanisms to coerce the host translational machinery into synthesizing viral proteins in the face of ongoing cellular stress responses that suppress global protein synthesis. Reoviruses replicate and assemble within cytoplasmic inclusions called viral factories. However, synthesis of viral proteins was thought to occur in the cytosol. To identify the site(s) of viral translation, we undertook a microscopy-based approach using ribopuromycylation to detect active translation. Here, we report that active translation occurs within viral factories and that translational factors are compartmentalized within factories. Furthermore, we find that the reovirus nonstructural protein σNS associates with 43S preinitiation complexes at the factory margins, suggesting a role for σNS in translation. Together, virus-induced compartmentalization of the host translational machinery represents a strategy for viruses to spatiotemporally couple viral protein synthesis with viral replication and assembly.