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)和严重急性呼吸综合征等病毒 冠状病毒-2(SARS-CoV2)是持续流行的病原体。了解主机病毒 相互作用是对抗这些病毒的关键。蛋白质组学方法已经鉴定出宿主蛋白 靶向病毒蛋白；然而，我们缺乏关于靶向病毒RNA(ZAP)或调节的宿主因素的信息 感染期间的宿主RNA(TTP、MCPIP1和Roquin-1)。有趣的是，已知的RNA结合的简短清单 效应器有一个共同的CCCH-型锌指(ZnF)基序。 与其他细胞内的锌氟蛋白不同，大多数CCCH型锌氟蛋白结合的是RNA而不是DNA 近40%的与RNA结合的锌氟蛋白。此外，这些蛋白质中的许多都没有很好的注释 功能。我认为在感染过程中，未知的CCCH ZF蛋白调节宿主和病毒的RNA。 通过系统的siRNA筛选，我将确定CCCH-ZF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OC43 (hCoV-OC43)。我已经确定了令人兴奋的CCCH-ZF蛋白，它们具有显著的益处或 对HIV-1和HCoV-OC43复制的有害影响。虽然每种病毒都确定了唯一的命中，但有七种 CCCH-ZnF蛋白在HIV-1和HCoV-OC43筛查中都有相同的命中率，这表明其中一些 这些因素可能在很大程度上是抗病毒的。这项拟议的研究试图了解RNA结合的要求 通过锌F基序(S)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，这种快速和稳健的筛选方法可用于CCCH-ZnF的鉴定(和 其他锌F类)因子参与了不同病毒家族的复制。此外，我将建立一个高效的， 简化了工作流程，以确定最有希望的机械勘探命中。我的工作将揭开 参与病毒复制并为细胞CCCH分配分子功能的新型宿主因子网络 锌氟蛋白。

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.