ANTIVIRAL MECHANISMS OF 2-5A-DEPENDENT RNASE L

2‘，5’-寡腺苷(2-5A)合成酶-核糖核酸酶L系统是干扰素诱导的一个很强的系统 帮助高等脊椎动物抵抗RNA和DNA病毒生存的抗病毒途径。 因此，病毒已经获得并进化出多种机制来拮抗OAS-RNaseL。 在过去的5年里，我们建立了核糖核酸酶L在干扰素信号转导、炎症体激活和 自噬。然而，关于核糖核酸酶L如何抑制病毒感染，病毒如何 逃避这些酶，以及宿主如何从核糖核酸酶L的激活中恢复。尤其是，我们对如何 核糖核酸酶L是通过其蛋白激酶样结构域来调节的，核糖核酸酶L是如何介导ADAR1的细胞死亡的 缺乏，核糖核酸酶L抑制物(ABCE1又名RLi)如何发挥作用，以及核糖核酸酶L如何激活NLRP3 炎性小体仍不完整。因此，该项目的拟议续展将系统地 调查关于这个至关重要的宿主抗病毒途径的主要悬而未决的问题。一元化 本申请的主题涉及核糖核酸酶L功能如何被2-5A激活以外的调节，在 特别是通过其蛋白激酶样(PKL)结构域，控制病毒感染和炎症。我们的发现 从而得出这样的假设，即OAS-核糖核酸酶L系统是宿主免疫系统不可分割的一部分，必须 严格监管，以消除病毒，同时限制其有害的促炎作用。我们的特定 目的是：(1)研究核糖核酸酶L的PKL结构域对细胞和病毒的影响 鉴定了小分子抑制剂、增强剂及其衍生物对核糖核酸酶L活性的影响，测定能力 控制病毒感染的小分子，并研究核糖核酸酶L抑制剂对细胞死亡的影响 由ADAR1缺乏症引起。(2)研究ATP结合盒成员ABCE1(RLI)如何限制 核糖核酸酶L活性，我们将准确地确定ABCE1是如何与核糖核酸酶L相互作用来抑制其活性的，建立 病毒感染过程中宿主细胞如何通过caspase对ABCE1的切割增强核糖核酸酶L的活性， 并测定ABCE1通过抑制核糖核酸酶L来对抗双链RNA细胞毒性的能力。 (3)为了探讨核糖核酸酶L激活NLRP3炎症体的机制，我们将对其进行鉴定和表征 核糖核酸酶L裂解NLRP3炎症体的病毒核糖核酸激活剂，确定小核糖核酸的作用 裂解产物对炎症体激活的影响，并测定核糖核酸酶L抑制剂药物对病毒的影响 体内介导性炎症。病毒感染仍然是世界范围内对人类健康的严重威胁。尽管病毒式传播 对策方面，OAS-核糖核酸酶L系统对多种致病病毒有重大影响。 这些研究强调了调节核糖核酸酶L以调节平衡是多么核心和重要 抗病毒活性和细胞死亡之间的关系。人们相信，对病毒和宿主因素的更好理解 调节核糖核酸酶L活性可能最终导致新的和改进的治疗方法 人类人口中既有的和新出现的病毒感染。

The 2',5'-oligoadenylate (2-5A) synthetase (OAS)-RNase L system is a robust interferon (IFN)-inducible antiviral pathway that contributes to the survival of higher vertebrates against both RNA and DNA viruses. Accordingly, viruses have acquired and evolved a multitude of mechanisms to antagonize OAS-RNase L. During the past 5 years, we established roles for RNase L in IFN signaling, inflammasome activation and autophagy. Yet there remains much to be learned about how RNase L suppresses viral infections, how viruses evade these enzymes, and how the host recovers from RNase L activation. In particular, our knowledge of how RNase L is regulated through its protein kinase-like domain, how RNase L mediates cell death from ADAR1 deficiency, how the RNase L inhibitor (ABCE1 aka RLI) functions, and how RNase L activates the NLRP3 inflammasome is still fragmentary. Therefore, the proposed continuation of this project will systematically investigate the major outstanding questions about this critically important host antiviral pathway. The unifying theme of this application concerns how RNase L function is regulated beyond its activation by 2-5A, in particular through its protein kinase-like (PKL) domain, to control viral infections and inflammation. Our findings lead to the hypothesis that the OAS-RNase L system is an integral part of the host immune system that must be tightly regulated to eliminate viruses while also limiting its harmful pro-inflammatory effects. Our specific aims are: (1) To study the impact of the RNase L PKL domain on cells and viruses we will evaluate recently identified small molecule inhibitors, enhancers and their derivatives on RNase L activity, determine the ability of the small molecules to control viral infections, and investigate effects of the RNase L inhibitors on cell death caused by ADAR1 deficiency. (2) To investigate how the ATP-binding cassette member, ABCE1 (RLI), limits RNase L activity, we will determine precisely how ABCE1 interacts with RNase L to inhibit its activity, establish how the host cells ramps up RNase L activity during viral infections by caspase-mediated cleavage of ABCE1, and determine the ability of ABCE1 to counteract cellular toxicity of double stranded RNA by inhibiting RNase L. (3) To probe the mechanism of NLRP3 inflammasome activation by RNase L, we will identify and characterize RNase L-cleaved viral RNA activators of the NLRP3 inflammasome, determine effects of the small RNA cleavage products on inflammasome activation, and determine the impact of RNase L inhibitor drugs on viral mediated inflammation in vivo. Viral infections remain a serious threat to human health worldwide. Despite viral countermeasures, the OAS-RNase L system has a major impact against many types of pathogenic viruses. These studies emphasize how central and important it is to regulate RNase L in order to mediate the balance between antiviral activity and cell death. It is believed that a better understanding of the viral and host factors that regulate RNase L activity may eventually lead to novel and improved therapeutic approaches for treating established and emerging viral infections in the human population.