Profiling Serine Hydrolase Activity At The Virus-Host Interface

 描述(申请人提供)：导致人类发病和死亡的病毒经常破坏有效宿主免疫反应的发展，从而导致病毒不受限制的增殖和相关的病理表现。小鼠感染典型的ArenaVirus LCMV为我们提供了一个极好的实验系统，用于研究病毒与宿主的相互作用，有助于宿主控制病毒增殖和病毒逃避宿主的抗病毒反应。此外，ArenaVirus在人类健康和生物防御准备方面的重要性，以及现有治疗ArenaVirus感染的有限设施，突显了开发抗击ArenaVirus感染的新对策的重要性。抗病毒治疗主要集中在针对病毒基因产物的活性，这一方法经常受到快速选择抑制剂逃逸病毒突变体的影响。病毒利用和操纵宿主细胞因子 用于它们的繁殖和调节宿主的免疫反应以利于它们的生存。这些宿主因素代表了抗病毒治疗的有吸引力和未得到充分利用的靶点。目前发现这些宿主因素的主要方法不能解释感染期间蛋白质活性的动态变化。基于活性的蛋白质图谱(ABPP)是一种新的方法，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 影响病毒繁殖和致病机制的新的病毒-宿主蛋白相互作用。这一探索性的R21应用将使用ABPP来识别和量化丝氨酸水解酶LCMV(SHS)在小鼠急性和持续感染期间的活性变化，SHS是已知的最大和最多样化的酶类别之一，在包括病毒感染在内的许多生理过程中发挥重要作用。为此，我们将完成以下具体目标：目的1.表征急性和持续LCMV感染过程中小鼠全脾SHS的活性。我们将使用ABPP来确定在小鼠急性和持续LCMV感染期间脾中SH活性的变化。这些研究将帮助我们开始破译不同的SHS在病毒感染中的作用。目的2.确定急性和持续性LCMV感染过程中脾SHS活性变化的细胞分布。我们将使用表达cre的重组LCMV感染mT/mg报告小鼠，这将有助于从感染LCMV的小鼠中分离出不同的纯化免疫细胞群中的感染(GFP+)和未感染(RFP+)细胞。在纯化的细胞群体中，感染细胞和未感染细胞中的SH活性将通过凝胶和质谱学方法进行表征，如目标1所示。选定的已鉴定的SHS将根据它们在调节宿主对LCMV感染的反应中的作用以及LCMV生命周期的具体步骤进行功能表征。这些研究的结果将帮助我们评估所观察到的变化的生物学意义。

 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.