Mechanisms of viral proteases in coronavirus replication and pathogenesis

病毒蛋白酶在冠状病毒复制和发病机制中的机制

• 批准号: 9096719
• 负责人: Susan C. Baker
• 金额: $ 74.12万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9096719
• 关键词: Adenosine Diphosphate Ribose; Affect; Amino Acid Sequence; Animals; Antiviral Agents; Antiviral Therapy; Attenuated; Biochemical; Biological Assay; Body Weight decreased; Cell Culture Techniques; Cells; Chiroptera; Cleaved cell; Coronavirus; Data; Disease; Disease Outbreaks; Educational process of instructing; Enzymes; FDA approved; Family; Fingerprint; Funding; Goals; Hand; Health; Human; Immune; Immune response; In Vitro; Intervention; Kinetics; Liver; Lysine; Middle East Respiratory Syndrome Coronavirus; Modeling; Monitor; Murine hepatitis virus; Mus; Mutagenesis; Mutate; Mutation; Natural Immunity; Nature; Papain; Pathogenesis; Pathology; Peptide Hydrolases; Peptides; Phosphoric Monoester Hydrolases; Pneumonia; Polyproteins; Production; Protease Domain; Proteins; RNA Viruses; RNA chemical synthesis; Research; Roentgen Rays; Role; Severe Acute Respiratory Syndrome; Signaling Molecule; Site-Directed Mutagenesis; Structure; System; Testing; Ubiquitin; Vaccines; Viral; Viral Load result; Viral Pathogenesis; Virus; Virus Replication; Work; base; cytokine; design; in vitro activity; molecular dynamics; novel virus; pandemic disease; pathogen; preference; replicase; research study; response; reverse genetics; temperature sensitive mutant; viral RNA

 DESCRIPTION (provided by applicant): The goal of our research is to determine how viral proteases function in the replication and pathogenesis of coronaviruses (CoVs). CoVs are a family of positive strand RNA viruses and include Severe Acute Respiratory Syndrome (SARS) CoV and Middle East Respiratory Syndrome (MERS) CoV which are significant human pathogens with pandemic potential. Previously, we dissected the multifunctional nature of CoV papain-like proteases (PLPs) and found that CoV PLPs cleave the viral replicase polyprotein, act as deubiquitinases (DUBs) and deISGylases (deISGs) by removing ubiquitin (Ub) or ISG15 conjugated to lysine residues on proteins, and that PLPs can antagonize the innate immune response, likely by deubiquitylating signaling molecules. Using detailed biochemical and PLP-Ub co-crystal structural analysis, we identified residues within CoV PLPs that differentially affec enzymatic activity in vitro and in cell-based assays. Our initial studies were performed using SARS-CoV PLpro. Here we provide preliminary in vitro and structural data demonstrating that these results can be extended to the BSL-2 model coronavirus mouse hepatitis virus (MHV-A59) papain-like protease. We hypothesize that multifunctional PLP/DUB activity contributes to viral pathogenesis and that selectively disrupting DUB activity will allow activation of innate immunity and reduced viral pathogenesis. To test this hypothesis, we will determine if a modified PLP/DUB enzymatic activity alters viral replication, innate immune response or pathogenesis. We will use reverse genetics to generate murine CoVs encoding PLPs with distinct enzymatic profiles such as DUB deficient, deISGylation deficient, or hyperactive protease. These novel viruses will be evaluated in cell culture and in mice for kinetics of viral RNA synthesis, production of infectious virus, and kinetics of activation of innate immune responses. To extend these studies to other CoVs, we will determine the enzymatic profile (EP) and enzymatic fingerprint (EF) of alpha- and beta-CoV papain-like proteases including bat CoV PLPs. We will express the PLP domain from 10 different CoV species and determine the peptide cleavage activity, deubiquitinating activity, deISGylating activity, and lysine-linkage preferences for each enzyme. With this profile in hand, we will use existing and new X-ray structures combined to guide mutagenesis experiments to differentially disrupt DUB activity and identify the fingerprint associated with reduced DUB activity. We will also determine the role of differential activity in regulating the innate immune response in bat cells. Also, we identified an interaction of the CoV ADP-ribose-1"-phosphatase (ADRP) domain with PLP and we will determine the effect of modifying this interaction on enzymatic activity, viral replication and pathogenesis. These studies will reveal new information on viral protease/DUB activity that will be useful for designing antiviral therapies and vaccines for coronaviruses and other protease/DUB-encoding viruses.

 描述（由申请人提供）：我们研究的目标是确定病毒蛋白酶如何在冠状病毒（CoV）的复制和发病机制中发挥作用。CoV是正链RNA病毒家族，包括严重急性呼吸综合征（SARS）CoV和中东呼吸综合征（MERS）CoV，它们是具有大流行潜力的重要人类病原体。先前，我们剖析了CoV木瓜蛋白酶样蛋白酶（PLPs）的多功能性质，并发现CoV PLPs切割病毒复制酶多聚蛋白，通过去除与蛋白质上的赖氨酸残基缀合的泛素（Ub）或ISG 15作为去泛素化酶（DUB）和去ISGylases（deISG），并且PLPs可以拮抗先天免疫应答，可能通过去泛素化信号分子。使用详细的生物化学和PLP-Ub共晶体结构分析，我们确定了CoV PLPs中的残基，这些残基在体外和基于细胞的测定中差异地影响酶活性。我们的初步研究是使用SARS-CoV PLpro进行的。在这里，我们提供了初步的体外和结构数据表明，这些结果可以扩展到BSL-2模型冠状病毒小鼠肝炎病毒（MHV-A59）木瓜蛋白酶样蛋白酶。我们假设多功能PLP/DUB活性有助于病毒发病机制，选择性破坏DUB活性将允许激活先天免疫和减少病毒发病机制。为了检验这一假设，我们将确定修饰的PLP/DUB酶活性是否改变病毒复制、先天免疫应答或发病机制。我们将使用反向遗传学来产生编码具有不同酶谱的PLP的鼠CoV，所述酶谱例如DUB缺陷、去ISGylation缺陷或高活性蛋白酶。将在细胞培养物和小鼠中评价这些新型病毒的病毒RNA合成动力学、感染性病毒的产生和先天免疫应答激活动力学。为了将这些研究扩展到其他CoV，我们将确定α-和β-CoV木瓜蛋白酶样蛋白酶（包括蝙蝠CoV PLPs）的酶谱（EP）和酶指纹（EF）。我们将表达来自10种不同CoV物种的PLP结构域，并确定每种酶的肽切割活性、去泛素化活性、去ISGylating活性和赖氨酸连接偏好。有了这个配置文件在手，我们将使用现有的和新的X射线结构相结合，以指导诱变实验，以差异破坏DUB活性，并确定与DUB活性降低相关的指纹。我们还将确定差异活性在调节蝙蝠细胞先天免疫反应中的作用。另外，我们发现了一个 我们将研究CoV ADP-核糖-1”-磷酸酶（ADRP）结构域与PLP的相互作用，并确定修饰这种相互作用对酶活性、病毒复制和发病机制的影响。这些研究将揭示有关病毒蛋白酶/DUB活性的新信息，这将有助于设计针对冠状病毒和其他蛋白酶/DUB编码病毒的抗病毒疗法和疫苗。