Institute of Virology and Immunology IVI, Bern, Switzerland.
Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
Elife. 2019 Jan 11;8:e42037. doi: 10.7554/eLife.42037.
Positive-sense RNA viruses hijack intracellular membranes that provide niches for viral RNA synthesis and a platform for interactions with host proteins. However, little is known about host factors at the interface between replicase complexes and the host cytoplasm. We engineered a biotin ligase into a coronaviral replication/transcription complex (RTC) and identified >500 host proteins constituting the RTC microenvironment. siRNA-silencing of each RTC-proximal host factor demonstrated importance of vesicular trafficking pathways, ubiquitin-dependent and autophagy-related processes, and translation initiation factors. Notably, detection of translation initiation factors at the RTC was instrumental to visualize and demonstrate active translation proximal to replication complexes of several coronaviruses. Collectively, we establish a spatial link between viral RNA synthesis and diverse host factors of unprecedented breadth. Our data may serve as a paradigm for other positive-strand RNA viruses and provide a starting point for a comprehensive analysis of critical virus-host interactions that represent targets for therapeutic intervention.
正链 RNA 病毒劫持细胞内的膜，为病毒 RNA 合成提供小生境，并为与宿主蛋白相互作用提供平台。然而，对于复制酶复合物与宿主细胞质之间界面的宿主因子知之甚少。我们将生物素连接酶工程化到冠状病毒复制/转录复合物 (RTC) 中，并鉴定了 >500 种构成 RTC 微环境的宿主蛋白。用 RTC 附近的每种 siRNA 沉默宿主因子，证明了囊泡运输途径、泛素依赖性和自噬相关过程以及翻译起始因子的重要性。值得注意的是，在 RTC 检测到翻译起始因子对于可视化和证明几种冠状病毒的复制复合物附近的活性翻译至关重要。总的来说，我们在病毒 RNA 合成和前所未有的广泛宿主因子之间建立了空间联系。我们的数据可以作为其他正链 RNA 病毒的范例，并为全面分析关键的病毒-宿主相互作用提供起点，这些相互作用是治疗干预的目标。
