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: 8099729
• 负责人: Susan C. Baker
• 金额: $ 60.36万
• 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 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8099729
• 关键词: Active Sites; Affect; Amino Acids; Animal Model; Animals; Antiviral Agents; Binding; Biochemical; Biological Assay; Biology; Cell Culture Techniques; Common Cold; Complex; Consensus; Consensus Sequence; Coronavirus; Coronavirus Infections; Croup; Deubiquitinating Enzyme; Deubiquitination; Development; Disease Outbreaks; Docking; Engineering; Enzymes; Exhibits; Genomics; Goals; Homology Modeling; Human; Immune response; Integration Host Factors; Interferon-beta; Interferons; Intervention; Kinetics; Ligand Binding; Lung diseases; Mediating; Methods; Modeling; Molecular; Mus; Mutagenesis; Mutation; Natural Immunity; Papain; Pathogenesis; Pathway interactions; Peptide Hydrolases; Phenotype; Polyproteins; Predisposition; Process; Protease Domain; Protein Binding; Proteins; RNA; RNA Viruses; Reporter; Research; Roentgen Rays; Role; Sequence Homology; Severe Acute Respiratory Syndrome; Sindbis Virus; Site; Site-Directed Mutagenesis; Specificity; Structure; Substrate Specificity; Surface; Symptoms; Testing; Thermodynamics; Translating; Transmembrane Domain; Ubiquitin; Vaccines; Viral; Viral Pathogenesis; Virus; Virus Diseases; Virus Replication; cellular targeting; design; drug development; improved; insight; mouse model; mutant; new therapeutic target; novel; novel therapeutics; positional cloning; protein protein interaction; public health relevance; replicase; research study; response; ubiquitin isopeptidase; vaccine development; virus pathogenesis

DESCRIPTION (provided by applicant): The long term goal of our research is to determine the role of viral protease and interferon antagonism activity in coronavirus replication and pathogenesis. Coronaviruses are positive strand RNA viruses which cause a range of illness, from relatively mild respiratory disease (croup and common cold like symptoms due to HCoV- NL63, 229e or OC43) to severe acute respiratory syndrome (SARS) outbreaks in humans. To date, there are no effective vaccines or antiviral drugs to limit the pathogenesis of any coronavirus infection. Clearly, we need to identify and understand the viral and host factors that contribute to efficient viral replication and pathogenesis to aid in the development of new therapeutics. Previously, we identified the papain-like protease (PLpro) of SARS-CoV as a critical determinant of viral replication and pathogenesis. During coronavirus replication, the input genomic RNA is translated to produce a replicase polyprotein which must be processed by viral papain-like and 3C-like proteases to generate the replication complex. We showed that PLpro processes the replicase polyprotein at three sites using a consensus recognition site of LXGG, similar to the consensus sequence recognized by de-ubiquitinating enzymes (DUBs). We solved the X-ray crystal structure of PLpro and demonstrated that it is indeed a DUB. However, the role of its viral DUB activity in pathogenesis remains to be determined. In addition, we found that PLpro exhibits interferon antagonism activity and that catalytic activity may not be required for full interferon antagonism. We hypothesize that distinct residues within the papain-like protease domain of coronaviruses are critical for mediating substrate specificity and interferon antagonism and that identification of these sites will provide novel targets for antiviral intervention. Here, we propose to investigate the biology of coronavirus papain-like proteases by comparing and contrasting the activity of papain-like proteases of SARS-CoV, HCoV-NL63, and murine coronavirus A59. Our specific aims are to: 1) Identify sites within the papain-like protease domains that are critical for polyprotein processing, deubiquitinating and deISGylating activity; 2) Determine residues within coronavirus papain-like proteases that are important for interferon antagonism and identify cellular proteins that interact with PLPs to block innate immunity; 3) Determine the domains, amino acids, and binding energies involved in protein-protein interactions between papain-like proteases and their binding partners; and 4) Determine if mutation of the PLpro or PLP2 domain alters evasion of ubiquitin- or ISG15-dependent innate immune responses. The results from this research will allow us to identify critical sites that mediate specificity of papain-like proteases and provide new insight into viral mechanisms for pathogenesis and evasion of the innate immune response. PUBLIC HEALTH RELEVANCE: The goal of our project is to determine how specific parts of a virus contribute to causing severe acute respiratory syndrome (SARS). We propose to use biochemical and molecular methods to study the SARS coronavirus protease and identify potential "Achilles' heels" of this protein. The results from our research will allow us to identify sites in the viral protease that may be targeted for antiviral drug development or modified to improve vaccine development.

描述（由申请人提供）：我们研究的长期目标是确定病毒蛋白酶和干扰素拮抗活性在冠状病毒复制和发病机制中的作用。冠状病毒是正链 RNA 病毒，可引起一系列疾病，从相对轻微的呼吸道疾病（HCoV-NL63、229e 或 OC43 引起的哮吼和普通感冒症状）到人类严重急性呼吸综合征 (SARS) 爆发。迄今为止，还没有有效的疫苗或抗病毒药物来限制任何冠状病毒感染的发病机制。显然，我们需要识别和了解有助于有效病毒复制和发病机制的病毒和宿主因素，以帮助开发新疗法。此前，我们发现 SARS-CoV 的木瓜蛋白酶样蛋白酶（PLpro）是病毒复制和发病机制的关键决定因素。在冠状病毒复制过程中，输入的基因组 RNA 被翻译产生复制酶多蛋白，该多蛋白必须由病毒类木瓜蛋白酶和类 3C 蛋白酶加工以生成复制复合物。我们表明，PLpro 使用 LXGG 的共有识别位点在三个位点处理复制酶多蛋白，类似于去泛素化酶 (DUB) 识别的共有序列。我们解析了 PLpro 的 X 射线晶体结构，并证明它确实是一个 DUB。然而，其病毒 DUB 活性在发病机制中的作用仍有待确定。此外，我们发现 PLpro 表现出干扰素拮抗活性，并且完全干扰素拮抗可能不需要催化活性。我们假设冠状病毒的木瓜蛋白酶样蛋白酶结构域内的不同残基对于介导底物特异性和干扰素拮抗作用至关重要，并且这些位点的识别将为抗病毒干预提供新的靶标。在这里，我们建议通过比较和对比 SARS-CoV、HCoV-NL63 和鼠冠状病毒 A59 的类木瓜蛋白酶的活性来研究冠状病毒类木瓜蛋白酶的生物学。我们的具体目标是： 1) 鉴定对多蛋白加工、去泛素化和去糖基化活性至关重要的木瓜蛋白酶结构域内的位点； 2) 确定冠状病毒木瓜蛋白酶样蛋白酶内对干扰素拮抗作用很重要的残基，并鉴定与 PLP 相互作用以阻断先天免疫的细胞蛋白； 3) 确定类木瓜蛋白酶与其结合配偶体之间蛋白质-蛋白质相互作用所涉及的结构域、氨基酸和结合能； 4) 确定 PLpro 或 PLP2 结构域的突变是否会改变泛素或 ISG15 依赖性先天免疫反应的逃避。这项研究的结果将使我们能够确定介导木瓜蛋白酶样蛋白酶特异性的关键位点，并为病毒发病机制和逃避先天免疫反应的机制提供新的见解。 公共卫生相关性：我们项目的目标是确定病毒的特定部分如何导致严重急性呼吸综合征 (SARS)。我们建议利用生化和分子方法来研究SARS冠状病毒蛋白酶，并找出该蛋白潜在的“阿喀琉斯之踵”。我们的研究结果将使我们能够确定病毒蛋白酶中可能用于抗病毒药物开发或修饰以改进疫苗开发的位点。