Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America.
Mass Spectrometry and Proteomics Resource Laboratory (MSPRL), FAS Division of Science, Harvard University, Cambridge, Massachusetts, United States of America.
PLoS Pathog. 2022 Oct 28;18(10):e1010906. doi: 10.1371/journal.ppat.1010906. eCollection 2022 Oct.
As ultimate parasites, viruses depend on host factors for every step of their life cycle. On the other hand, cells evolved multiple mechanisms of detecting and interfering with viral replication. Yet, our understanding of the complex ensembles of pro- and anti-viral factors is very limited in virtually every virus-cell system. Here we investigated the proteins recruited to the replication organelles of poliovirus, a representative of the genus Enterovirus of the Picornaviridae family. We took advantage of a strict dependence of enterovirus replication on a host protein GBF1, and established a stable cell line expressing a truncated GBF1 fused to APEX2 peroxidase that effectively supported viral replication upon inhibition of the endogenous GBF1. This construct biotinylated multiple host and viral proteins on the replication organelles. Among the viral proteins, the polyprotein cleavage intermediates were overrepresented, suggesting that the GBF1 environment is linked to viral polyprotein processing. The proteomics characterization of biotinylated host proteins identified multiple proteins previously associated with enterovirus replication, as well as more than 200 new factors recruited to the replication organelles. RNA metabolism proteins, many of which normally localize in the nucleus, constituted the largest group, underscoring the massive release of nuclear factors into the cytoplasm of infected cells and their involvement in viral replication. Functional analysis of several newly identified proteins revealed both pro- and anti-viral factors, including a novel component of infection-induced stress granules. Depletion of these proteins similarly affected the replication of diverse enteroviruses indicating broad conservation of the replication mechanisms. Thus, our data significantly expand the knowledge of the composition of enterovirus replication organelles, provide new insights into viral replication, and offer a novel resource for identifying targets for anti-viral interventions.
作为终极寄生虫，病毒在其生命周期的每一步都依赖于宿主因素。另一方面，细胞进化出了多种检测和干扰病毒复制的机制。然而，我们对几乎每一种病毒-细胞系统中促进和抗病毒因子的复杂组合的理解非常有限。在这里，我们研究了脊髓灰质炎病毒（属于小核糖核酸病毒科肠道病毒属）复制细胞器中募集的蛋白质。我们利用肠道病毒复制严格依赖宿主蛋白 GBF1 的特性，建立了一种稳定表达与 APEX2 过氧化物酶融合的截断 GBF1 的细胞系，该细胞系在抑制内源性 GBF1 后有效支持病毒复制。该构建体在复制细胞器上生物素化了多种宿主和病毒蛋白。在病毒蛋白中，多蛋白切割中间体过表达，表明 GBF1 环境与病毒多蛋白加工有关。生物素化宿主蛋白的蛋白质组学特征鉴定了多个先前与肠道病毒复制相关的蛋白，以及 200 多个新募集到复制细胞器的因子。RNA 代谢蛋白，其中许多蛋白通常定位于细胞核内，构成了最大的组，强调了大量核因子释放到感染细胞的细胞质中，并参与病毒复制。对几个新鉴定蛋白的功能分析揭示了既促进又抑制病毒的因子，包括感染诱导应激颗粒的一个新组成部分。这些蛋白的耗竭同样影响多种肠道病毒的复制，表明复制机制具有广泛的保守性。因此，我们的数据显著扩展了肠道病毒复制细胞器的组成知识，为病毒复制提供了新的见解，并为鉴定抗病毒干预的靶点提供了新的资源。
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