3C Protease Polyubiquitylation as a Picornavirus Replication Success Determinant

3C 蛋白酶多泛素化作为小核糖核酸病毒复制成功的决定因素

• 批准号: 8272093
• 负责人: Thomas Glen Lawson
• 金额: $ 35万
• 依托单位: BATES COLLEGE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8272093
• 关键词: Affect; Animal Diseases; Animals; Antiviral Therapy; Attention; Biochemical; Biochemical Process; Biomedical Research; Cell Communication; Cell physiology; Cells; Cellular Structures; Data; Disease; Encephalomyocarditis virus; Failure; Family; Family Picornaviridae; Goals; Grant; Hepatitis A Virus; Human; Individual; Infection; Kinetics; Knowledge; Laboratories; Ligase; Link; Mass Spectrum Analysis; Measures; Mediating; Modeling; Pathway interactions; Peptide Hydrolases; Phase; Picornaviridae Infections; Play; Polyproteins; Polyubiquitin; Preparation; Process; Production; Proteins; RNA; RNA library; Reaction; Regulation; Research; Research Infrastructure; Research Training; Role; Satellite Viruses; Science; Screening procedure; Students; Study Subject; Supporting Cell; System; Testing; Time; Training; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligase Complexes; Viral; Viral Proteins; Virion; Virus; Virus Diseases; Virus Replication; Work; base; career; human disease; infrastructure development; insight; member; multicatalytic endopeptidase complex; operation; pathogen; premature; programs; protein degradation; protein purification; reconstitution; success; ubiquitin-protein ligase; undergraduate student

DESCRIPTION (provided by applicant): A large fraction of the diseases in humans and animals that result from virus infections are caused by members of the picornavirus family. Like all picornaviruses, the encephalomyocarditis virus (EMCV) and hepatitis A virus (HAV) produce a protein called the 3C protease, which is absolutely required for virus replication in infected cells. The 3C proteases produced by EMCV and HAV, at least, are rapidly degraded by the cellular ubiquitin-proteasome system (UPS). The UPS, which is involved in the regulation of many critical cellular processes, operates by modifying proteins to be degraded with chains of the small protein ubiquitin in a process called polyubiquitylation, and the tagged proteins are degraded by the proteasome. The long-term objectives of the proposed project are to clarify the mechanisms by which the 3C proteases are marked for destruction and to discover the functions of the UPS-mediated destruction of the 3C proteases, as they relate to virus replication success. One specific aim of the project is to purify and identify the unknown ubiquitin- protein ligase, or ligases, that recognize the EMCV and HAV 3C proteases as ubiquitylation substrates and determine how the three ubiquitylation pathways known to target the 3C proteases function together to mark these viral proteins for degradation by the proteasome. The strategy to achieve this aim is to use protein purification and mass spectroscopy, along with interfering RNA library screening, to identify the unknown ligase, or ligases. The determination of how 3C protease polyubiquitylation occurs will be accomplished by measuring the kinetics with which the purified, reconstituted pathways catalyze the major phases of the polyubiquitylation process. A second aim is to determine the extent to which UPS-mediated 3C protease degradation in cells infected by EMCV affects virus replication success and the onset of cytopathic effects. The strategy to achieve this aim is to use interfering RNA to specifically inhibit the ubiquitylation pathways involved in 3C protease polyubiquitylation and measure the extent to which this inhibition alters new virion component production rates, infectious progeny virion yield, and the timing of cytopathicity onset. The proposed research is expected to provide a detailed characterization of the polyubiquitylation process by which the EMCV and HAV 3C proteases are tagged for degradation and to demonstrate the extent to which EMCV replication depends upon the UPS-mediated reduction of the viral 3C protease concentration. The findings will facilitate new research into the relationship between the UPS and other picornaviruses and may reveal new targets for antiviral therapies that disrupt the UPS. In addition, because little is known about how multiple protein-ubiquitylating pathways function together to catalyze protein polyubiquitylation, the findings from this research will likey be very useful in helping to explain how other proteins associated with human diseases are targeted and tagged for destruction. The project will support biomedical research infrastructure development because it includes a substantial undergraduate student research training component. PUBLIC HEALTH RELEVANCE: A large fraction of virus-associated diseases in humans is caused by infections with picornaviruses, but the mechanisms by which these viruses are replicated in their hosts are far from understood. The proposed project will provide important new insights into the links between picornavirus infection success and critical host cell components. This is likely in turn to result in the identification of new strategies for treating diseases caused by picornavirus infections.

描述（由申请人提供）：病毒感染引起的人类和动物中的很大一部分是由Picornavirus家族的成员引起的。与所有PICORNAVIRES一样，脑膜炎病毒（EMCV）和丙型肝炎病毒（HAV）产生一种称为3C蛋白酶的蛋白质，这绝对是感染细胞中病毒复制所必需的。 EMCV和HAV产生的3C蛋白酶至少被细胞泛素 - 蛋白酶体系统（UPS）迅速降解。与许多关键细胞过程有关的UPS通过在称为polyupiquitylation的过程中修饰要用小蛋白泛素降解的蛋白质来作用，并通过蛋白酶体降解标记的蛋白质。拟议项目的长期目标是阐明3c蛋白酶被标记为破坏的机制，并发现与病毒复制成功有关的3C蛋白酶的UPS介导的破坏的功能。该项目的一个具体目的是净化和识别未知的泛素 - 蛋白连接酶或连接酶，这些蛋白质连接酶或连接酶识别EMCV和HAV 3C蛋白酶为泛素化底物，并确定三种泛素化途径如何确定靶向3C蛋白的三种泛素化途径，以将这些病毒蛋白用于蛋白质的蛋白质来降解这些病毒蛋白，以通过蛋白质脱发来降解这些病毒蛋白。实现此目的的策略是使用蛋白质纯化和质谱，以及干扰RNA库筛选，以识别未知的连接酶或连接酶。通过测量纯化的，重组的途径催化多泛素化过程的主要阶段的动力学，将实现3C蛋白酶多泛素化的确定。第二个目的是确定由EMCV感染的细胞中UPS介导的3C蛋白酶降解的程度会影响病毒的复制成功和细胞性效应的发作。实现此目的的策略是使用干扰RNA特异性抑制3C蛋白酶多偶然化涉及的泛素化途径，并测量这种抑制作用的程度可以改变新的病毒体成分生产率，感染性后代病毒率产量，以及细胞病性发作的时间。预计拟议的研究将提供对多泛素化过程的详细表征，通过该过程将EMCV和HAV 3C蛋白酶标记为降解，并证明EMCV复制取决于在多大程度上取决于UPS介导的病毒3C蛋白酶的降低。这些发现将促进对UPS与其他Picornaviruses之间关系的新研究，并可能揭示了破坏UPS的抗病毒疗法的新靶标。此外，由于对多种蛋白质泛素化途径如何共同起作用以催化蛋白质多泛素化的知识知之甚少，因此这项研究的发现将在帮助解释与人类疾病相关的其他蛋白质的目标方面非常有用，并且如何将其标记为破坏。该项目将支持生物医学研究基础设施开发，因为它包括大量的本科生研究培训部分。 公共卫生相关性：人类中与病毒相关的疾病的一小部分是由PICORNAVIRES的感染引起的，但是在其宿主中复制这些病毒的机制远非理解。拟议的项目将为Picornavirus感染成功与关键宿主细胞成分之间的联系提供重要的新见解。这反过来又可能导致鉴定由皮纳病毒感染引起的疾病的新策略。