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

基本信息

批准号：
8869489
负责人：
Juan C. de la Torre
金额：
$ 28.43万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-06-15 至 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8869489
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 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 virus protein profiling public health relevance small molecule thioester viral resistance virus host interaction

项目摘要

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（由申请人提供）：经常引起人类发病和死亡的病毒会破坏有效宿主免疫反应的发展，从而导致病毒不受限制地增殖和相关的病理表现。用原型沙粒病毒 LCMV 感染小鼠为我们提供了极好的结果。用于研究病毒与宿主相互作用的实验系统，有助于宿主控制病毒增殖和病毒逃避宿主抗病毒反应。此外，沙粒病毒在宿主中的重要性。人类健康和生物防御准备情况，以及治疗沙粒病毒感染的现有设备有限，凸显了开发对抗沙粒病毒感染的新对策的重要性。抗病毒疗法主要集中于针对病毒基因产物的活性，这种方法经常受到病毒基因产物活性的影响。病毒利用和操纵宿主细胞因子快速选择逃避抑制剂的病毒突变体。其增殖并调节宿主免疫反应以利于其生存。这些宿主因子代表了抗病毒治疗中有吸引力且未充分利用的目标。目前发现这些宿主因子的主要方法并不能解释感染期间蛋白质活性的动态变化。分析（ABPP）是一种新颖的方法，它允许监测病毒感染对宿主蛋白质组功能状态的影响，以确定影响病毒传播和发病机制的新型病毒-宿主蛋白相互作用。这种探索性 R21 应用将使用 ABPP 来识别和量化丝氨酸水解酶 (SH) LCMV 小鼠急性和持续感染期间的变化活性，丝氨酸水解酶 (SH) 是最大、最多样化的酶之一。已知在包括病毒感染在内的许多生理过程中发挥重要作用的类别为此，我们将完成以下具体目标：目标 1. 表征小鼠在急性和持续性 LCMV 感染期间的整体脾脏 SH 活性。 ABPP 识别小鼠急性和持续 LCMV 感染期间脾脏中 SH 活性的变化。这些研究将帮助我们开始破译病毒感染期间不同 SH 的作用。目标 2.确定重新改变的脾脏 SH 活性的细胞分布。在急性和持续性 LCMV 感染期间，我们将使用表达 cre 的重组 LCMV 来感染 mT/mG 报告小鼠，这将有助于检测和分离感染的 (GFP+) 和来自 LCMV 感染小鼠的不同纯化免疫细胞群中的未感染 (RFP+) 细胞将通过基于凝胶和质谱的方法来表征纯化细胞群中感染和未感染细胞的 SH 活性。 SH 将在功能上表征它们在调节宿主对 LCMV 感染反应中的作用，并且 LCMV 生命周期的具体步骤将帮助我们评估所观察到的变化的生物学意义。