Antiviral Mechanisms of 2-5A Dependent RNase L

描述（由申请人提供）：RNase L是先天抗病毒反应的主要介质，因此对人类健康至关重要。高等脊椎动物中的病毒复制受到干扰素（IFN）的抑制，干扰素导致细胞转录编码抗病毒蛋白的基因，包括2‘-5’寡聚腺苷酸合成酶（OAS）。病毒病原体相关分子模式（PAMP），双链RNA (dsRNA)，激活OAS产生5'-磷酸化，2',5'-连接的低聚腺苷酸（2- 5a），其功能是刺激RNase L. OAS-RNase L系统因此构成经典的先天免疫途径，快速响应PAMPs（这里，dsRNA）产生广泛活性的抗病毒反应。在我们之前根据该方案进行的研究中，我们克隆了RNase L，在小鼠中敲除它，在体内建立了它的抗病毒和凋亡活性，并确定了它诱导抗病毒基因的转录信号通路。我们未来的目标是探索影响病毒生命周期的RNase L周围的基本事件和生物学终点。我们的假设是RNase L通过切割病毒和细胞RNA来抑制病毒复制，启动应激反应途径并信号表达抗病毒基因。我们的具体目标是：(1)优化激活RNase L的新型小分子2-5A模拟物，并确定其对细胞活力和病毒生长的影响；(2)确定2- 5a激活RNase L如何信号抗病毒和免疫调节基因的转录，包括研究dsRNA信号因子和MAP激酶的参与；(3)通过检测病毒对I型IFN的诱导作用，确定RNase L在野生型和无RNase L小鼠体内抗病毒活性的根本依据，确定RNase L诱导的IFN对病毒复制的影响以及2-5A和2-5A模拟物对IFN诱导和病毒复制的影响。本提案中描述的实验将研究一种普遍存在的强效抗病毒酶（RNase L）的小分子激活剂如何刺激主要的抗病毒途径。由此获得的知识可能导致控制重要人类病毒病原体的策略。因此，这些研究有令人信服的与健康相关的理由。

DESCRIPTION (provided by applicant): RNase L is a principal mediator of the innate antiviral response and is thus critically important for human health. Virus replication in higher vertebrates is restrained by interferons (IFN) that cause cells to transcribe genes encoding antiviral proteins, including the 2'-5' oligoadenylate synthetases (OAS). The viral pathogen associated molecular pattern (PAMP), double-stranded RNA (dsRNA), activates OAS to produce 5'- phosphorylated, 2', 5'-linked oligoadenylates (2-5A) whose function is to stimulate RNase L. The OAS-RNase L system thus constitutes a classical innate immune pathway that rapidly responds to PAMPs (here, dsRNA) to produce a broadly active antiviral response. In our prior studies performed under this protocol, we cloned RNase L, knocked it out in mice, established its antiviral and apoptotic activities in vivo, and determined that it induces transcriptional signaling pathways to antiviral genes. Our future GOALS are to probe fundamental events and biologic endpoints surrounding RNase L that impact on viral lifecycles. Our HYPOTHESIS is that RNase L inhibits viral replication by cleaving viral and cellular RNA, initiating a stress-response pathway and signaling expression of antiviral genes. Our Specific Aims are to: (1) optimize novel small molecular 2-5A mimics that activate RNase L and determine their effects on cell viability and viral growth; (2) determine how 2-5A activation of RNase L signals transcription of antiviral and immunoregulatory genes, including investigations into the involvement of dsRNA signaling factors and MAP kinases; and (3) determine the fundamental basis for the antiviral activity of RNase L in vivo in wild type and RNase L-null mice by measuring viral induction of type I IFNs, determining the impact of RNase L-induced IFNs on viral replication and the effects of 2-5A and 2-5A mimics on IFN induction and viral replication. The experiments described in this PROPOSAL will investigate how a major antiviral pathway is stimulated by small molecule activators of a ubiquitous and potent antiviral enzyme (RNase L). The knowledge to be gained may thus lead to strategies for controlling important human viral pathogens. Therefore, there are cogent and health-related justifications for these studies.