Cellular RNA-binding Zinc Finger Proteins in Viral Infection: Understanding the Rules of Engagement

项目摘要 人类免疫缺陷病毒-1（HIV-1）和严重急性呼吸系统综合征等病毒 冠状病毒-2（SARS-CoV 2）是正在进行的流行病的病原体。了解主机病毒 相互作用对对抗这些病毒至关重要。蛋白质组学方法已经鉴定出 靶向病毒蛋白;然而，我们缺乏关于靶向病毒RNA（ZAP）或调节 在感染期间宿主RNA（TTP、MCPIP 1和Roquin-1）。有趣的是，已知的RNA结合的短名单 效应子共享共同的CCCH型锌指（ZnF）基序。 与其他细胞ZnF蛋白不同，大多数CCCH型ZnF蛋白结合RNA而不是DNA， 所有RNA结合ZnF蛋白的近40%。此外，这些蛋白质中的许多蛋白质的注释很差， 功能协调发展的我建议，身份不明的CCCH ZnF蛋白调节宿主和病毒的RNA在感染过程中。 通过系统的siRNA筛选，我将确定CCCH-ZnF蛋白，调节复制， 两种临床相关病毒：人类免疫缺陷病毒1型（HIV-1）和人类冠状病毒OC 43 （HCoV-OC 43）。我已经确定了令人兴奋的CCCH-ZnF蛋白，它们具有显著的有益或 对HIV-1和HCoV-OC 43复制的有害作用。虽然每种病毒都有独特的命中率，但有7种病毒是在同一时间内被发现的。 CCCH-ZnF蛋白在HIV-1和HCoV-OC 43筛选中是共享的，这表明其中一些蛋白在HIV-1和HCoV-OC 43筛选中是共享的。 这些因子可能是广泛的抗病毒因子。这项拟议中的研究旨在了解RNA结合的要求 通过ZnF基序在这些宿主-病毒相互作用中的作用，并将使用转录组学，蛋白质组学和显微镜 阐明详细作用机制的方法。 初步结果证明了这种快速和稳健的筛选方法用于鉴定CCCH-ZnF（和 其它ZnF型）因子参与不同病毒家族的复制。此外，我将建立一个有效的， 简化的工作流程，以确定最有希望的命中机制的探索。我的工作将揭示 参与病毒复制并将分子功能分配给细胞CCCH的新型宿主因子网络 ZnF蛋白。

Project Summary Viruses such as human immunodeficiency virus-1 (HIV-1) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) are the causative pathogens of on-going pandemics. Understanding host-virus interactions is essential to combatting these viruses. Proteomic approaches have identified host proteins that target viral proteins; however, we lack information regarding host factors that target viral RNA (ZAP) or regulate host RNAs (TTP, MCPIP1, and Roquin-1) during infection. Interestingly, the short list of known RNA-binding effectors shares a common CCCH-type zinc finger (ZnF) motif. Unlike other cellular ZnF proteins, most CCCH-type ZnF proteins bind RNA rather than DNA and account for nearly 40% of all RNA-binding ZnF proteins. Furthermore, many of these proteins have poorly annotated functions. I propose that unidentified CCCH ZnF proteins regulate host and viral RNAs during infection. Through systematic siRNA screening, I will identify CCCH-ZnF proteins that modulate the replication of two clinically relevant viruses: human immunodeficiency virus type 1 (HIV-1) and human coronavirus OC43 (HCoV-OC43). I have already identified exciting CCCH-ZnF proteins that have significant beneficial or deleterious effects on HIV-1 and HCoV-OC43 replication. While unique hits were identified for each virus, seven CCCH-ZnF proteins are shared hits in both HIV-1 and HCoV-OC43 screens, suggesting that some of these factors may be broadly antiviral. The proposed research seeks to understand the requirement for RNA-binding by ZnF motif(s) in these host-virus interactions and will use transcriptomics, proteomics, and microscopy approaches to elucidate detailed mechanisms of action. Preliminary results justify this rapid and robust screening method for identification of CCCH-ZnF (and other ZnF-type) factors involved in replication of diverse virus families. Furthermore, I will establish an efficient, streamlined workflow to determine the most promising hits for mechanistic exploration. My work will uncover novel host factors networks that are involved in virus replication and assign molecular functions to cellular CCCH ZnF proteins.