Reovirus Z-RNAs activate ZBP1 dependent necroptosis

摘要 呼肠孤病毒（ReoV）是一种肠道dsRNA病毒，其可以感染广泛的哺乳动物并诱导肠道和 由于感染期间引起的细胞死亡而引起的自身免疫性疾病。ReoV诱导的机制 这种细胞死亡尚不清楚。几十年来，ReoV被认为主要引起感染细胞的凋亡。最近， Danthi实验室已经表明ReoV也可以激活坏死性凋亡，这是一种不依赖于半胱天冬酶炎症 依赖于激酶RIPK 3及其底物MLKL的程序性细胞死亡。坏死性下垂具有高度免疫原性 因为它导致损伤相关分子模式（DAMP）的释放，病原体相关的 分子模式（PAMPs）和细胞内细胞因子，而细胞凋亡在免疫学上更“沉默”。的 在ReoV感染过程中，坏死性凋亡和细胞凋亡都被激活， 这种激活的免疫学后果在很大程度上是未知的。我发现先天免疫传感器 Z-DNA结合蛋白1（ZBP 1）在ReoV感染期间启动细胞凋亡和坏死性凋亡。我也 发现ReoV在感染的细胞中产生Z-RNA，这是双螺旋RNA的左手构象。 细胞此外，我发现ZBP 1在ReoV过程中被K63介导的多聚泛素连接修饰， 感染. 总之，这些新的发现使我们能够假设（1）ReoV产生细胞质Z-RNA， ZBP 1的活化配体;（2）ZBP 1泛素化对于ZBP 1活化、RIPK 3的接合和 RIPK 3下游的凋亡和坏死性凋亡的执行;和（3）ZBP 1触发的坏死性凋亡驱动 通过促进病毒释放和诱导肠道中的有害炎症来促进病毒致病。在这 根据该提议，我们将通过鉴定ReoV产生的ZBP 1的Z-RNA配体来测试这些假设（Aim 1.1）， 确定K63泛素化对ZBP 1激活的机制和贡献（目标1.2），并研究 ZBP 1激活的ReoV体内感染过程中宿主防御和发病机制的作用，区分 细胞凋亡和坏死性凋亡对这些结果的贡献（目的2）。我们还将删除ZBP 1及其 肠上皮细胞中的下游途径效应物，肠道中的主要ReoV靶点，以确定 ZBP 1触发的细胞死亡对ReoV传播和发病的选择性作用。

ABSTRACT Reovirus (ReoV) is an enteric dsRNA virus which can infect a wide range of mammals and induce intestinal and autoimmune diseases due to the cell death elicited during infection. The mechanisms by which ReoV induces such cell death are unclear. For decades ReoV was thought to cause mainly apoptosis in infected cells. Recently, the Danthi lab has shown that ReoV can also activate necroptosis, a caspase-independent form of inflammatory programmed cell death reliant on the kinase RIPK3 and its substrate MLKL. Necroptosis is highly immunogenic because it results in the release of damage-associated molecular patterns (DAMPs), pathogen-associated molecular patterns (PAMPs), and intracellular cytokines, whereas apoptosis is more immunologically ‘silent’. The mechanisms by which both necroptosis and apoptosis are activated during ReoV infection, as well as the immunological consequences of this activation, are largely unknown. I have found that the innate immune sensor Z-DNA Binding Protein 1 (ZBP1) initiates both apoptosis and necroptosis during ReoV infections. I have also discovered that ReoV generates Z-RNA, the left-handed conformation of double-helical (ds)RNA, in infected cells. Additionally, I have found that ZBP1 is modified by K63-mediated polyubiquitin linkages during ReoV infections. Together, these new findings allow us to hypothesize that (1) ReoV produces cytoplasmic Z-RNAs, which are activating ligands for ZBP1; (2) ZBP1 ubiquitination is essential for ZBP1 activation, engagement of RIPK3, and execution of both apoptosis and necroptosis downstream of RIPK3; and (3) ZBP1-triggered necroptosis drives virus pathogenesis by both promoting virus release and inducing detrimental inflammation in the gut. In this proposal, we will test these hypotheses by identifying the ReoV-generated Z-RNA ligands for ZBP1 (Aim 1.1), determining the mechanism and contribution of K63 ubiquitination to ZBP1 activation (Aim 1.2), and investigating the role ZBP1-activated to host defense and pathogenesis during ReoV infections in vivo, distinguishing between the contributions of apoptosis and necroptosis to these outcomes (Aim 2). We will also delete ZBP1 and its downstream pathway effectors in intestinal epithelial cells, the primary ReoV targets in the gut, to determine the selective role of ZBP1-triggered cell death to ReoV spread and pathogenesis.