Functional characterization of the enteroviral degradome

项目摘要 肠道病毒是在多个器官中复制并导致各种疾病的人类病原体， 包括胃肠炎、肺炎、心肌炎和脑炎。目前，人们对此知之甚少。 肠道病毒改变了受感染细胞的生物学特性。这个拟议的项目是建立在我们最近的 记录肠道病毒蛋白酶切割目标的细胞蛋白的出版物将澄清 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。 到目前为止，对切割靶子集的初步研究已经产生了两个候选蛋白质， 激活天然免疫信号和抑制病毒复制的LSM14A和IRF2BP2 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 以及他们的先天免疫功能是否需要病毒抑制。这些初步的 这些发现为确定其他宿主目标的特征并确定它们的调控能力奠定了基础 肠道病毒感染。 我们已经建立了一套新的方法和试剂工具包，将使拟议的工作成为可能。 例如，我们实现了一个创新的实验系统，它由人类诱导的 多能干细胞来源的无间充质肠上皮细胞，它们对 肠道病毒，并通过触发强大的先天免疫程序对感染做出反应。同样，我们有 产生的含有干扰素刺激基因的人类细胞系，这些基因在其内源基因座上标记为 荧光蛋白，允许实时监测对病毒感染的免疫诱导。 我们将使用这些和其他工具来调查宿主如何靶向LSM14A的分子细节 激活先天免疫信号和抑制病毒感染(目标1)；剖析IRF2BP2在 肠道病毒生命周期(目标2)；并生成功能上重要目标的全面清单 在肠道病毒感染和/或抗病毒先天免疫中起作用的蛋白质(目标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 行动机制。更广泛地说，这个项目将促进对肠道病毒的理解 发病机制，并有可能为设计广泛有效的预防和治疗策略提供参考 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.