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理中的作用。冠状病毒是阳性链RNA病毒，引起一系列疾病，从相对轻度的呼吸道疾病（由于HCOV-NL63，229E或OC43引起的症状和普通感染）到人类中严重的急性呼吸综合征（SARS）暴发。迄今为止，还没有有效的疫苗或抗病毒药物可以限制任何冠状病毒感染的发病机理。显然，我们需要识别和理解有助于有效的病毒复制和发病机理的病毒和宿主因素，以帮助开发新的疗法。以前，我们将SARS-COV的木瓜蛋白酶样蛋白酶（PLPRO）确定为病毒复制和发病机理的关键决定因素。在冠状病毒复制过程中，输入基因组RNA被翻译成产生复制酶多蛋白，必须通过病毒蛋白乳木瓜样和3C样蛋白酶对其进行处理，以产生复制复制复合物。我们表明，PLPRO使用LXGG的共有识别位点在三个位点进行复制酶多蛋白，类似于通过去泛素化酶（DUB）识别的共有序列。我们解决了PLPRO的X射线晶体结构，并证明它确实是配音。然而，其病毒配音在发病机理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，并且可能不需要全干扰素拮抗作用。我们假设帕帕因样冠状动脉蛋白酶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，并且这些位点的鉴定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的蛋白酶样蛋白酶的活性来研究冠状病毒蛋白酶样蛋白酶的生物学。我们的具体目的是：1）识别类似木瓜蛋白酶的蛋白酶域内的位点，这些蛋白酶域中对于多蛋白加工，去泛素化和去乙酸酯活性至关重要； 2）确定对干扰素拮抗作用至关重要的冠状动脉瘤病毒样蛋白酶中的残基，并鉴定与PLP相互作用以阻断先天免疫力的细胞蛋白； 3）确定与蛋白酶样蛋白酶及其结合伴侣之间蛋白质 - 蛋白质相互作用有关的结构域，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依赖性的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类似木瓜蛋白酶的蛋白酶特异性的关键部位，并为病毒机制提供新的见解，以发病和逃避先天免疫反应。 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促进严重的急性呼吸综合症（SARS）。我们建议使用生化和分子方法研究SARS冠状病毒蛋白酶，并确定该蛋白质的潜在“致命弱”。我们研究的结果将使我们能够鉴定病毒蛋白酶中可能针对抗病毒药物开发或修改以改善疫苗开发的部位。