Functional analysis of host and viral determinants for ZAP inhibition

项目总结/摘要 新出现的病毒，如严重急性呼吸道综合征冠状病毒2（SARS-CoV-2）， 摧毁了世界我们迫切需要广泛作用的抗病毒药物，以遏制这种疾病的传播。 下一个新病毒然而，由于我们对细胞的不完全理解， 控制或促进病毒复制的途径。 I型干扰素（IFN）是宿主对病毒感染的主要先天性免疫应答，并调节免疫应答。 IFN刺激基因（ISG）阵列的表达。锌指抗病毒蛋白（ZAP）是目前发现的最具生物学活性的抗病毒蛋白之一。 有效的ISG，可阻断多种RNA和DNA病毒的复制。ZAP减弱甲病毒产生 高达8个对数，是IFN对抗甲病毒功效所不可或缺的。此外， 人类免疫缺陷病毒1（HIV-1）和SARS-CoV-2与病毒逃避ZAP识别有关。 重要的是，高致病性甲病毒如基孔肯雅病毒（CHIKV）已经发展出对甲病毒的抗性。 ZAP，强调ZAP是病毒发病机制的关键驱动因素。ZAP被认为是通过病毒翻译来发挥作用的。 抑制（甲病毒和黄病毒）和病毒RNA降解（其他病毒），然而，ZAP如何阻断病毒 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：首先，我们表明ZAP病毒翻译 抑制需要宿主因子TRIM 25，一种E3泛素连接酶。TRIM 25介导的ZAP抗病毒机制是 创新，独立于RIG-I，IRF 3和IFN。使用一种新的“衬底捕获”方法，我们现在 鉴定了许多在翻译和RNA过程中具有令人兴奋的作用的细胞蛋白质为TRIM 25 相互作用物和潜在底物。这些初步发现将泛素化过程与病毒 ZAP的翻译抑制作用，并为抗病毒治疗提供细胞靶点。二是 确定了甲病毒对ZAP敏感性的差异。高毒性甲病毒，如CHIKV， 在最近的流行病中，数百万感染者可以通过他们的病毒逃避或抵消ZAP识别。 非结构基因区病毒逃避和拮抗策略的发现将鉴定出可药物化的病毒 靶向并告知对抗ZAP能力减弱的疫苗株的开发。 综上所述，我们的结果使我们假设ZAP招募TRIM 25来泛素化， 调节导致病毒翻译抑制的细胞因子，并且高致病性甲病毒已经进化 逃避和/或拮抗ZAP抗病毒活性的策略。我们建议阐明的机制 TRIM 25介导的ZAP翻译抑制（Aim 1），并确定病毒对ZAP耐药的机制 (Aim 2）。这些目标的成功将促进我们对IFN途径如何选择细胞的理解。 阻断病毒复制和驱动病毒发病机制的过程，并提供有希望的宿主和病毒靶点， 甲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.