Identifying the targets of virus-induced PARPs during SARS-CoV-2 infection

项目总结 冠状病毒(CoV)是一大类正链rna病毒，能够引起严重的 人类疾病和死亡，SARS-CoV-2大流行就是例证。先天免疫 对冠状病毒感染的反应包括多聚ADP核糖聚合酶(PARP)和冠状病毒 冠状病毒大结构域(MAC1)，分别从蛋白质中添加和去除ADP-核糖。在 由于缺乏MAC1酶的活性，冠状病毒在面对先天免疫应答时复制能力较差 在包括SARS-CoV-2在内的几种动物感染模型中，几乎没有疾病。这些结果表明 PARP介导的ADP核糖化在限制冠状病毒诱导的疾病和支持我们的中枢 假设PARPS针对宿主和病毒蛋白，以及这些蛋白的翻译后修饰 靶标(MAR化)诱导一种限制病毒复制的抗病毒状态。尽管显然重要的是 PARP酶在驱动冠状病毒感染的转归中的作用，关于其确切的作用机制仍存在很大的认识空白 这场战斗在冠状病毒感染期间展开，最值得注意的是i)哪些PAP严重参与了这场战斗；以及 Ii)哪些细胞或病毒蛋白在感染过程中被ADP核糖化。功能冗余，类似NAD+ 结合位点、低蛋白丰度和逆转其影响的病毒酶使其具有挑战性 在冠状病毒感染期间确定单个PAP的直接靶点。 这项建议的目的是确定在SARS-CoV-2期间PAP的特定ADP核糖化靶点 将揭示病毒限制的新机制的感染。这一目标将通过 具体目标如下：1)鉴定SARS-CoV-2过程中表达和影响的PARPS蛋白 2)使用化学遗传学和邻近标记识别病毒和细胞PARP靶标。这 这项工作具有创新性，因为我们将首次应用基于NAD+的化学蛋白质组学、化学 遗传学和BioID邻近标记以揭示影响MARylating Parp的特定靶点 SARS-CoV-2感染。此外，我们有一个独特的工具，一种缺乏对抗PARP能力的病毒 活性，以帮助我们识别生理上相关的PARP靶蛋白。我们的理由是 在SARS-CoV-2感染过程中识别单个PARP的靶点将确定病毒的新机制 限制，这将极大地扩大ADP-核糖化的图景及其如何影响病毒 复制。结合我们在化学、PARP/ADP-核糖和冠状病毒生物学方面的专业知识，我们可以 准备应对这些挑战，并做出描述PARP介导的新靶点的开创性发现 ADP-核糖化以及它们如何在SARS-CoV-2感染期间驱动抗病毒先天免疫反应。我们 预计在感染期间识别数十种ADP核糖化蛋白，这将如何改变 在磷酸化或泛素化之外，PTM可以影响病毒感染的结果，并将 为抗病毒治疗提供新的途径。

PROJECT SUMMARY Coronaviruses (CoVs) are a large class of positive-strand RNA viruses that are capable of causing severe human disease and death, as is exemplified by the pandemic outbreak of SARS-CoV-2. The innate immune response to coronavirus infection includes a battle between poly-ADP-ribose polymerases (PARPs) and the coronavirus macrodomain (Mac1), which add and remove ADP-ribose from proteins, respectively. In the absence of Mac1 enzyme activity, CoVs replicate poorly in the face of the innate immune response and cause little to no disease in several animal models of infection, including SARS-CoV-2. These results demonstrate the power of PARP-mediated ADP-ribosylation to limit CoV-induced disease and support our central hypothesis that PARPs target host and viral proteins, and that the post-translational modification of these targets (MARylation) induces an antiviral state that limits virus replication. Despite the clear importance of PARP enzymes in driving the outcome of a CoV infection, a large gap in knowledge remains as to exactly how this battle plays out during CoV infections, most notably i) what PARPs are heavily involved in this battle; and ii) what cellular or viral proteins are ADP-ribosylated during infection. Functional redundancy, similar NAD+ binding sites, low protein abundance, and viral enzymes that reverse their effects have made it challenging to identify direct targets of individual PARPs during coronavirus infection. The objective of this proposal is to identify specific ADP-ribosylated targets of PARPs during a SARS-CoV-2 infection that will uncover novel mechanisms of virus restriction. This objective will be resolved with the following specific aims: 1) Identify PARPs proteins that are expressed during and impact SARS-CoV-2 infection, and 2) Identify viral and cellular PARP targets using chemical genetics and proximity labeling. This work is innovative because we will apply, for the first time, NAD+-based chemical proteomics, chemical genetics, and BioID proximity labeling to uncover the specific targets of of MARylating PARPs that impact SARS-CoV-2 infection. Furthermore, we have a unique tool, a virus that lacks the ability to counter PARP activity, to aid in our identification of physiologically relevant PARP target proteins. Our rationale is that identifying the targets of individual PARPs during SARS-CoV-2 infection will define novel mechanisms of virus restriction that will dramatically expand the landscape of ADP-ribosylation and how it can impact virus replication. Together, with our combined expertise in chemistry, PARP/ADP-ribose, and CoV biology, we are poised to address these challenges and make seminal discoveries describing novel targets of PARP-mediated ADP-ribosylation and how they can drive antiviral innate immune responses during SARS-CoV-2 infection. We anticipate identifying dozens of ADP-ribosylated proteins during infection, which will alter the landscape of how PTMs, outside of phosphorylation or ubiquitination, can impact the outcomes of virus infections, and will provide new avenues for antiviral therapy.