Profiling Serine Hydrolase Activity At The Virus-Host Interface

病毒-宿​​主界面丝氨酸水解酶活性分析

• 批准号: 9085225
• 负责人: Juan C. de la Torre
• 金额: $ 24.06万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-15 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9085225
• 关键词: Accounting; Active Sites; Acute; Affect; Amides; Antiviral Agents; Antiviral Response; Antiviral Therapy; Arenavirus; Arenavirus Infections; Biological; Biology; Cell Separation; Cells; Chemicals; Collaborations; Detection; Development; Enzymes; Esters; Family; Gel; Genes; Goals; Health; Hepatitis C virus; Human; Immune; Immune response; In Vitro; Infection; Integration Host Factors; Investigation; Knowledge; Label; Life Cycle Stages; Lipids; Lymphocytic choriomeningitis virus; Mammals; Mass Spectrum Analysis; Modification; Monitor; Morbidity - disease rate; Mus; Nature; Non-Insulin-Dependent Diabetes Mellitus; Parasites; Pathogenesis; Pathology; Peptide Hydrolases; Peptides; Physiological Processes; Play; Population; Proteins; Proteome; Readiness; Recombinants; Regulation; Reporter; Role; Serine; Serine Hydrolase; Source; Spleen; System; Technology; Testing; Transcript; Translating; Translations; Viral; Viral Proteins; Virus; Virus Diseases; Virus Replication; activity-based protein profiling; base; biodefense; biological systems; chemoproteomics; combat; environmental change; enzyme activity; follow-up; gel electrophoresis; human disease; improved; in vivo; inhibitor/antagonist; liquid chromatography mass spectrometry; magnetic beads; member; mortality; mutant; new therapeutic target; novel; novel strategies; novel therapeutics; novel virus; protein profiling; small molecule; thioester; viral resistance; virus host interaction

 DESCRIPTION (provided by applicant): Viruses causing morbidity and mortality in humans frequently subvert the development of an effective host immune response, which results in unrestricted virus multiplication and associated pathological manifestations. Infection of the mouse with the prototypic arenavirus LCMV provides us with a superb experimental system for the investigation of virus-host interactions contributing to both host's control of virus multiplication and viral evasion from the host antiviral response. Moreover, the significance of arenaviruses in human health and biodefense readiness, together with the limited existing armamentarium to treat arenavirus infections, highlight the importance of developing novel countermeasures to combat arenavirus infections. Antiviral therapies have been primarily focused on targeting the activity of viral gene products, an approach often compromised by the rapid selection of inhibitor-escape viral mutants. Viruses utilize and manipulate host cell factors for their multiplication and to modulate host immune responses towards favoring their survival. These host factors represent attractive and underutilized targets for antiviral therapy. Current major approaches to uncover these host factors do not account for dynamic changes in protein activity during infection. Activity-based protein profiling (ABPP) is a novel approach that permits to monitor the effects of viral infections on the functional state of the host proteome to identify novel virus-host protein interactions that affect virus propagation and pathogenesis. This exploratory R21 application will use ABPP to identify and quantify changing activity during acute and persistent infection of mice with LCMV of Serine Hydrolases (SHs), one of the largest and most diverse enzyme classes known to play important roles in many physiological processes including viral infection. For this we will complete the following specific aims: Aim 1. Characterize global spleenic SHs activity in mice during acute and persistent LCMV infection. We will use ABPP to identify changing SH activities in spleen during acute and persistent LCMV infection of the mouse. These studies will help us to begin to decipher the role of distinct SHs during viral infection. Aim 2. Determine the cellular distribution of spleenic SHs activities that re altered during acute and persistent LCMV infection. We will employ a cre-expressing recombinant LCMV to infect the mT/mG reporter mouse, which will facilitate the detection and separation of infected (GFP+) and non-infected (RFP+) cells within distinct purified immune cell populations from LCMV-infected mice. SH activities in infected and non-infected cells within purified cellular populations will be characterized by gel and mass spectrometry-based approaches as in Aim 1. Selected identified SHs will be functionally characterized regarding their roles in the regulation of the host response to LCMV infection, and specific steps of the LCMV life cycle. Results from these studies will help us to assess the biological implications of the observed changes.

 描述（应用程序提供）：引起人类发病率和死亡率的病毒经常颠覆有效的宿主免疫响应，从而导致不受限制的病毒繁殖和相关的病理表现。用原型体育症病毒LCMV感染小鼠为我们提供了一个出色的实验系统，用于研究病毒 - 宿主相互作用，这既导致宿主对病毒乘法的控制和宿主抗病毒反应的病毒进化。此外，体育症病毒在人类健康和生物浓度准备方面的重要性，以及有限的治疗竞技病毒感染的有限的Armamentarium强调了开发新颖的对策以对抗Arenavirus感染的重要性。抗病毒疗法主要集中在靶向病毒基因产物的活性上，这种方法通常受到抑制剂 - 埃斯卡普病毒突变体的快速选择而损害。病毒利用和操纵宿主细胞因子 为了繁殖并调节宿主对其生存的免疫反应。这些宿主因素代表了抗病毒治疗的有吸引力和未充分利用的靶标。当前发现这些宿主因子的主要方法无法解释感染过程中蛋白质活性的动态变化。基于活动的蛋白质分析（ABPP）是一种允许的新方法 监测病毒感染对宿主蛋白质组功能状态的影响以识别 影响病毒传播和发病机理的新型病毒宿主蛋白相互作用。此探索性R21应用将使用ABPP来识别和量化与LCMV丝氨酸水解酶（SHS）的急性和持续感染期间变化的活动，这是已知在包括病毒感染在内的许多物理过程中起重要作用的最大，最多样化的酶类别之一。为此，我们将完成以下特定目的：目标1。在急性和持续性LCMV感染期间，将小鼠全球SLENIC SHS活性表征。我们将使用ABPP在急性和持续的LCMV感染过程中识别SLEN中不断变化的SH活动。这些研究将有助于我们开始破译病毒感染中不同SH的作用。 AIM 2。确定在急性和持续性LCMV感染过程中改变的弹shs活性的细胞分布。我们将采用表达CRE的重组LCMV来感染MT/MG报告基因小鼠，这将促进感染（GFP+）的检测和分离（GFP+）和未感染的（RFP+）细胞，在与LCMV感染的小鼠中不同的纯化的Immunocell种群中。如AIM 1中的AIM 1一样，纯化细胞种群中感染和未感染细胞中的SH活性将以凝胶为特征，基于质谱的方法。所选的SHS在调节宿主对LCMV感染的响应中的作用以及LCMV生命周期的特定步骤的作用在功能上表征。这些研究的结果将有助于我们评估观察到的变化的生物学意义。