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（由适用提供）：我们研究的目的是确定病毒蛋白酶在冠状病毒的复制和发病机理中的作用（COV）。 COV是一个阳性链RNA病毒的家族，包括严重的急性呼吸综合征（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。 Previously, we dissected the multifunctional nature of CoV papain-like proteins (PLPs) and found that CoV PLPs clear the viral replicase polyprotein, act as deubiquitinases (DUBs) and deISGylases (deISGs) by removing ubiquitin (Ub) or ISG15 conjugated to lysine retention on proteins, and that PLPs can antagonize the innate immune response, likely通过除外信号分子。使用详细的生化和PLP-UB共晶体结构分析，我们在COV PLP中确定了在体外和基于细胞的测定中影响酶活性不同的结果。我们的最初研究是使用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）类蛋白酶样蛋白酶。我们假设多功能PLP/DUB活性有助于病毒发病机理，并且有选择地破坏DUB活性将允许活化先天免疫学并减少病毒发病机理。为了检验这一假设，我们将确定修饰的PLP/DUB酶活性是否会改变病毒复制，先天免疫响应或发病机理。我们将使用反向遗传学来生成编码具有不同酶促曲线的PLP的鼠COV，例如DUB防御性，去乙二醇缺乏或多活跃蛋白酶。这些新型病毒将在细胞培养和小鼠中评估病毒RNA合成的动力学，感染性病毒的产生以及先天免疫调查的激活动力学。为了将这些研究扩展到其他COV，我们将确定包括BAT COV PLP在内的α-和Beta-CoV类木瓜样蛋白酶的酶促（EP）和酶促指纹（EF）。我们将从10种不同的COV物种中表达PLP结构域，并确定每种酶的肽裂解活性，去泛素化活性，去乙酸活性和赖氨酸 - 链接偏好。借助此轮廓，我们将使用现有和新的X射线结构合并来指导诱变实验，以破坏DUB活性并确定与DUB活性减少相关的指纹。我们还将确定差异活性在确定蝙蝠细胞中先天免疫反应中的作用。另外，我们确定了 COV ADP-ribose-1“ - 磷酸酶（ADRP）结构域与PLP的相互作用，我们将确定修改这种相互作用对酶活性，病毒复制和发病机理的影响。这些研究将揭示有关病毒蛋白酶/DUB活性的新信息，这些信息将用于设计抗病毒疗法和疫苗的抗病毒疗法和其他蛋白酶/蛋白酶和其他蛋白酶和其他研究。