Functional analysis of host and viral determinants for ZAP inhibition

项目摘要/摘要 新出现的病毒，如严重急性呼吸综合征冠状病毒2(SARS-CoV-2) 摧毁了世界。我们迫切需要广泛有效的抗病毒药物，以遏制病毒的传播 下一个新出现的病毒。然而，由于我们对细胞的不完全理解，存在着重大的知识鸿沟 控制或促进病毒复制的途径。 I型干扰素是宿主对病毒感染的主要先天免疫反应，调节 一系列干扰素刺激基因(ISGs)的表达。锌指抗病毒蛋白(ZAP)是其中最重要的 有效的ISG可以阻止各种RNA和DNA病毒的复制。ZAP抑制甲型病毒的产生 高达8个对数，对于干扰素对抗甲型病毒的疗效是不可或缺的。此外，增加的毒力， 人类免疫缺陷病毒1型(HIV-1)和SARS-CoV-2与ZAP识别的病毒逃避有关。 重要的是，高致病性甲型病毒，如基孔肯雅病毒(CHIKV)已经对 ZAP，强调ZAP是病毒致病的关键驱动因素。ZAP被建议通过病毒翻译发挥作用 抑制(甲型病毒和黄病毒)和病毒RNA降解(其他病毒)，然而，ZAP如何阻止病毒 复制和病毒如何逃避ZAP识别仍然知之甚少。 我们最近朝着这个目标取得了两个重要进展：第一，我们证明了ZAP病毒翻译 抑制需要宿主因子TRIM25，一种E3泛素连接酶。TRIM25介导的ZAP抗病毒机制是 创新性的，独立于RIG-I、IRF3和干扰素。使用一种新的“基质捕获”方法，我们现在 确定了许多在翻译和RNA过程中具有兴奋作用的细胞蛋白质为TRIM25 交互作用和潜在的底物。这些初步发现将泛素化过程与病毒联系起来。 首次通过ZAP抑制翻译，并为抗病毒治疗提供细胞靶点。第二，我们 确定了甲型病毒对ZAP敏感性的差异。高毒力的甲型病毒，如CHIKV 在最近的疫情中，数百万受感染的人可以通过他们的病毒逃避或抵消ZAP识别 非结构基因区。发现逃避和拮抗的病毒策略将识别可用药的病毒 针对中和ZAP能力减弱的疫苗株并向其提供信息。 综上所述，我们的结果使我们假设ZAP招募了TRIM25来泛化和 调节导致病毒翻译抑制的细胞因素，高致病性甲型病毒已经进化 逃避和/或拮抗ZAP抗病毒活性的策略。我们建议阐明这一机制。 TRIM25介导的ZAP翻译抑制(AIM 1)及病毒对ZAP抗性机制的研究 (目标2)。这些目标的成功将促进我们对干扰素途径如何与细胞 阻断病毒复制和推动病毒致病的过程，并提供有希望的宿主和病毒靶点 甲型病毒和其他病毒感染的治疗干预。

PROJECT SUMMARY/ABSTRACT Emerging viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have devastated the world. We are desperately in need of broadly acting antivirals that can curb the spread of the next emerging virus. However, a major knowledge gap exists due to our incomplete understanding of the cellular pathways that control or facilitate viral replication. Type I interferon (IFN) is the principal host innate immune response to viral infection and regulates the expression of an array of IFN-stimulated genes (ISGs). Zinc finger antiviral protein (ZAP) is one of the most potent ISGs that blocks the replication of diverse RNA and DNA viruses. ZAP attenuates alphavirus production by up to 8 logs and is indispensable for IFN efficacy against alphaviruses. Moreover, increased virulence of human immunodeficiency virus 1 (HIV-1) and SARS-CoV-2 associates with viral evasion from ZAP recognition. Importantly, highly pathogenic alphaviruses such as chikungunya virus (CHIKV) have developed resistance to ZAP, highlighting ZAP as a critical driver of viral pathogenesis. ZAP is proposed to act through viral translational inhibition (alphavirus and flavivirus) and viral RNA degradation (other viruses), however, how ZAP blocks viral replication and how viruses evade from ZAP recognition are still poorly understood. We recently made two important advances towards this goal: First, we showed that ZAP viral translational inhibition requires the host factor TRIM25, an E3 ubiquitin ligase. TRIM25-mediated ZAP antiviral mechanism is innovative, and independent of RIG-I, IRF3, and IFN. Using a novel “substrate trapping” approach we have now identified numerous cellular proteins with exciting roles in translation and RNA processes to be TRIM25 interactors and potential substrates. These preliminary findings connect the ubiquitination process to viral translational suppression by ZAP for the first time and provide cellular targets for antiviral therapy. Second, we identified differences in alphavirus sensitivity to ZAP. Highly virulent alphaviruses such as CHIKV that have infected millions of people in recent epidemics can evade from or counteract ZAP recognition through their viral non-structural gene region. Discovery of viral strategies for evasion and antagonism will identify druggable viral targets and inform development of vaccine strains with weakened ability to counteract ZAP. Taken together, our results have led us to hypothesize that ZAP recruits TRIM25 to ubiquitinate and modulate cellular factors leading to viral translational inhibition, and highly pathogenic alphaviruses have evolved strategies to escape and/or antagonize ZAP antiviral activity. We propose to elucidate the mechanism of TRIM25-mediated ZAP translational inhibition (Aim 1) and determine the mechanisms of viral resistance to ZAP (Aim 2). Success of these Aims will advance our understanding of how the IFN pathway co-opts cellular processes to block viral replication and drive viral pathogenesis, and provide promising host and viral targets for therapeutic intervention of alphavirus and other virus infections.