Functional characterization of the enteroviral degradome

项目摘要 肠道病毒是人类病原体，在多个器官中复制并引起多种疾病， 包括胃肠炎、肺炎、心肌炎和脑炎。目前，人们对如何做到这一点知之甚少 肠道病毒改变受感染细胞的生物学。这个拟议的项目，这是建立在我们最近的 一份记载肠道病毒蛋白酶切割靶向的细胞蛋白的出版物将阐明 肠道病毒蛋白酶在感染过程中改变宿主细胞环境中的作用。 迄今为止，对切割靶点子集的初步研究已经产生了两种候选蛋白质， LSM 14 A和IRF 2BP 2激活先天性免疫信号传导并抑制培养细胞中的病毒复制， 细胞我们的机制研究已经开始揭示这些蛋白质如何限制病毒的分子细节 感染以及它们的先天免疫功能是否是抑制病毒所必需的。这些初步 这些发现为描述其他宿主靶点并确定其调节能力奠定了基础 肠病毒感染。 我们已经建立了一个新的方法和试剂的工具包，将使拟议的工作。 例如，我们实施了一个创新的实验系统， 多能干细胞衍生的无间充质肠上皮细胞，其对以下高度敏感： 肠道病毒，并通过触发强大的先天免疫程序对感染作出反应。同样，我们有 产生的人细胞系含有干扰素刺激的基因，这些基因在其内源基因座处用 荧光蛋白，允许实时监测响应病毒感染的免疫诱导。 我们将使用这些和其他工具来研究宿主靶向LSM 14 A的分子细节， 激活先天性免疫信号传导并抑制病毒感染（目的1）; 肠病毒生命周期（目标2）;以及生成功能重要靶标的全面列表 在肠道病毒感染和/或抗病毒先天免疫中起作用的蛋白质（Aim 3）。 这些目标的完成将揭示宿主对肠道病毒复制的限制因素以及它们的作用。 行动机制。更广泛地说，该项目将促进对肠道病毒的理解， 发病机制，并可能为广泛作用的预防和治疗策略的设计提供信息 对抗肠道病毒感染。

Project Summary Enteroviruses are human pathogens that replicate in multiple organs and cause a variety of diseases, including gastroenteritis, pneumonia, myocarditis, and encephalitis. Currently, little is known about how enteroviruses alter the biology of infected cells. This proposed project, which is founded on our recent publication documenting the cellular proteins that enteroviral proteases target for cleavage, will elucidate the role of enteroviral proteases in changing the host cell enviornment during infection. The initial investigations of a subset of cleavage targets have so far yielded two candidate proteins, LSM14A and IRF2BP2, which activated innate immune signaling and inhibited viral replication in cultured cells. Our mechanistic studies have begun to reveal the molecular details of how these proteins limit virus infection and whether their innate immune functions are required for viral inhibition. These preliminary findings have built the foundation to characterize other host targets and determine their ability to regulate enterovirus infection. We have established a toolkit of novel methodologies and reagents that will enable the proposed work. For example, we have implemented an innovative experimental system that consists of human induced pluripotent stem cell-derived mesenchyme-free intestinal epithelial cells, which are highly susceptible to enteroviruses and respond to infection by triggering a robust innate immune program. Similarly, we have generated human cell lines containing interferon-stimulated genes tagged at their endogenous loci with fluorescent proteins, allowing real-time monitoring of immune induction in response to viral infections. We will employ these and other tools to investigate the molecular details of how the host target LSM14A activates innate immune signaling and inhibit viral infections (Aim 1); dissect the function of IRF2BP2 in the enterovirus lifecycle (Aim 2); and generate a comprehensive list of functionally important target proteins that have roles in enterovirus infection and/or antiviral innate immunity (Aim 3). Completion of these aims will reveal host restriction factors against enteroviral replication as well as their mechanisms of action. More broadly, this project will advance the understanding of enteroviral pathogenesis and potentially inform the design of broadly acting preventive and therapeutic strategies against enteroviral infections.