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

识别 SARS-CoV-2 感染期间病毒诱导的 PARP 的靶标

• 批准号: 10573499
• 负责人: Michael S Cohen
• 金额: $ 23.8万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-12 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10573499
• 关键词: 2019-nCoV; ADP ribosylation; Address; Adenosine Diphosphate Ribose; Animal Model; Antibodies; Antiviral Response; Antiviral Therapy; Attenuated; Automobile Driving; Binding; Binding Sites; Biology; COVID-19 outbreak; Cessation of life; Chemicals; Chemistry; Complement; Coronavirus; Coronavirus Infections; Development; Disease; Enzymes; Future; Genes; Goals; Host Defense; Human; Individual; Infection; Innate Immune Response; Interferon Type I; Interferons; Knowledge; Label; Mediating; Methods; Murine hepatitis virus; Mutagenesis; Natural Immunity; Nicotinamide adenine dinucleotide; Outcome; Pathogenesis; Phosphorylation; Photoaffinity Labels; Physiological; Play; Poly(ADP-ribose) Polymerases; Positioning Attribute; Post-Translational Protein Processing; Proteins; Proteomics; RNA Viruses; Reporting; Role; SARS-CoV-2 infection; SARS-CoV-2 inhibitor; Seminal; Signal Pathway; Signal Transduction; Site; Site-Directed Mutagenesis; System; Time; Ubiquitination; Viral; Viral Proteins; Viral hepatitis; Virus; Virus Diseases; Virus Replication; Work; analog; chemical genetics; defense response; enzyme activity; experimental study; genetic approach; human disease; in vivo; innovation; interest; knock-down; mutant; novel; overexpression; pandemic disease; protein expression; tool

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.

项目摘要 冠状病毒（CoV）是一大类正链RNA病毒，其能够引起严重的冠状病毒感染。 人类疾病和死亡，如SARS-CoV-2大流行爆发所示。先天免疫 对冠状病毒感染的反应包括聚ADP-核糖聚合酶（PARP）和 冠状病毒宏结构域（Mac 1），其分别从蛋白质添加和去除ADP-核糖。在 由于缺乏Mac 1酶活性，CoV在先天免疫反应和原因面前复制较差 在包括SARS-CoV-2在内的几种动物感染模型中几乎没有疾病。这些结果证明 PARP介导的ADP核糖基化限制CoV诱导的疾病并支持我们的中枢神经系统的能力。 假设PARP靶向宿主和病毒蛋白，并且这些蛋白的翻译后修饰 靶点（MARylation）诱导限制病毒复制的抗病毒状态。尽管很明显， PARP酶在驱动冠状病毒感染的结果，知识的巨大差距仍然是如何确切地 这场战斗在CoV感染期间展开，最值得注意的是i）什么PARP大量参与这场战斗;以及 ii）在感染期间哪些细胞或病毒蛋白质被ADP-核糖基化。功能冗余，类似NAD+ 结合位点、低蛋白丰度和逆转其作用的病毒酶使得 识别冠状病毒感染期间单个PARP的直接靶点。 该建议的目的是确定在SARS-CoV-2过程中PARP的特异性ADP核糖基化靶点。 感染将揭示病毒限制的新机制。这一目标将通过 以下具体目标：1）鉴定在SARS-CoV-2期间表达并影响SARS-CoV-2的PARP蛋白 感染，和2）使用化学遗传学和邻近标记鉴定病毒和细胞PARP靶标。这 这项工作是创新的，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，我们有一种独特的工具，一种缺乏对抗PARP能力的病毒 活性，以帮助我们鉴定生理学相关的PARP靶蛋白。我们的理由是， 在SARS-CoV-2感染过程中识别单个PARP的靶点将定义病毒的新机制， 限制，这将大大扩大ADP-核糖基化的景观，以及它如何影响病毒 复制的我们在化学、PARP/ADP-核糖和CoV生物学方面的综合专业知识， 准备应对这些挑战，并做出开创性的发现，描述PARP介导的新靶点。 ADP-核糖基化以及它们如何在SARS-CoV-2感染期间驱动抗病毒先天免疫应答。我们 预计在感染过程中识别出数十种ADP核糖基化蛋白，这将改变感染过程中ADP核糖基化蛋白的分布。 除了磷酸化或泛素化，PTM可以影响病毒感染的结果， 为抗病毒治疗提供了新的途径。