ANTIVIRAL MECHANISMS OF 2-5A-DEPENDENT RNASE L

2-5A依赖性RNA酶L的抗病毒机制

• 批准号: 10005110
• 负责人: Robert H. Silverman
• 金额: $ 40.25万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-25 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10005110
• 关键词: 2-5A Synthetase; ABCE1 gene; ADAR1; Antiviral Agents; Antiviral Response; Apoptotic; Autophagocytosis; Binding; Biological Assay; Caspase; Cause of Death; Cell Death; Cells; Cleaved cell; Complex; Crystallization; DNA Viruses; Double-Stranded RNA; Enhancers; Enzymes; Equilibrium; Family; Health; Host Defense; Human; Immune system; Inflammasome; Inflammation; Inflammatory; Integration Host Factors; Interferons; Knockout Mice; Knowledge; Lead; Mediating; Mus; Oncolytic viruses; Pathogenicity; Pathway interactions; Pharmaceutical Preparations; Philadelphia; Population; Protein Kinase; Protocols documentation; RNA Viruses; Ramp; Reporting; Ribonucleases; Roentgen Rays; Role; Signal Transduction; Small RNA; Structure; System; Therapeutic; Toxic effect; Vertebrates; Viral; Virus; Virus Diseases; dimer; improved; in vivo; inhibitor/antagonist; knockout gene; member; novel; oligoadenylate; pathogenic virus; phosphodiesterase V; small molecule; small molecule inhibitor; viral RNA

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.

2',5'-寡腺苷酸 (2-5A) 合成酶 (OAS)-RNase L 系统是一种强大的干扰素 (IFN) 诱导型 抗病毒途径有助于高等脊椎动物抵抗 RNA 和 DNA 病毒的生存。 因此，病毒已经获得并进化出多种拮抗 OAS-RNase L 的机制。 在过去 5 年中，我们确定了 RNase L 在 IFN 信号传导、炎性体激活和 自噬。然而，关于 RNase 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 RNase L 激活中恢复。特别是，我们对如何 RNase L 通过其蛋白激酶样结构域进行调节，RNase L 如何介导 ADAR1 导致的细胞死亡 RNase L 抑制剂（ABCE1 又名 RLI）如何发挥作用，以及 RNase L 如何激活 NLRP3 炎性小体仍然是碎片。因此，建议继续该项目将系统地 调查有关这一至关重要的宿主抗病毒途径的主要悬而未决的问题。统一的 本申请的主题涉及 RNase L 功能如何在 2-5A 激活之外进行调节， 特别是通过其蛋白激酶样（PKL）结构域来控制病毒感染和炎症。我们的发现 导致这样的假设：OAS-RNase L 系统是宿主免疫系统的一个组成部分，必须 受到严格监管以消除病毒，同时限制其有害的促炎作用。我们的具体 目标是：（1）研究 RNase L PKL 结构域对细胞和病毒的影响，我们最近将进行评估 鉴定了小分子抑制剂、增强剂及其衍生物对 RNase L 活性的影响，确定了 控制病毒感染的小分子，并研究 RNase L 抑制剂对细胞死亡的影响 由 ADAR1 缺陷引起。 (2) 研究 ATP 结合盒成员 ABCE1 (RLI) 如何限制 RNase L 活性，我们将精确确定 ABCE1 如何与 RNase L 相互作用以抑制其活性，建立 病毒感染期间宿主细胞如何通过 caspase 介导的 ABCE1 裂解来增强 RNase L 活性， 并确定 ABCE1 通过抑制 RNase L 来抵消双链 RNA 细胞毒性的能力。 (3) 为了探究 RNase L 激活 NLRP3 炎症小体的机制，我们将鉴定和表征 NLRP3 炎症小体的 RNase L 切割病毒 RNA 激活剂，确定小 RNA 的作用 裂解产物对炎症小体激活的影响，并确定 RNase L 抑制剂药物对病毒的影响 介导体内炎症。病毒感染仍然对全世界人类健康构成严重威胁。尽管病毒式传播 对抗措施中，OAS-RNase L系统对多种病原病毒具有重大影响。 这些研究强调了调节 RNase L 以调节平衡的核心和重要性 抗病毒活性和细胞死亡之间。据信，更好地了解病毒和宿主因素 调节 RNase L 活性可能最终会带来新的、改进的治疗方法 人群中已建立和新出现的病毒感染。