Antiviral Mechanisms of 2-5A Dependent RNase L

描述(申请人提供)：核糖核酸酶L是先天抗病毒反应的主要介体，因此对人类健康至关重要。在高等脊椎动物中，病毒复制受到干扰素的抑制，干扰素使细胞转录编码抗病毒蛋白的基因，包括2‘-5’寡腺苷合成酶(OAS)。病毒病原体相关分子模式，双链RNA(DsRNA)，激活OAS产生5‘-磷酸化，2’，5‘-连接的寡腺苷(2-5A)，其功能是刺激核糖核酸酶L。因此，OAS-RNaseL系统构成了一种经典的先天免疫途径，对PAMPs(这里指双链RNA)快速反应，产生广泛活性的抗病毒反应。在我们以前的研究中，我们克隆了核糖核酸酶L，在小鼠中将其敲除，在体内建立了它的抗病毒和凋亡活性，并确定了它诱导了抗病毒基因的转录信号通路。我们未来的目标是探索围绕核糖核酸酶L影响病毒生命周期的基本事件和生物终点。我们的假设是，核糖核酸酶L通过裂解病毒和细胞核糖核酸，启动应激反应途径和抗病毒基因的信号表达来抑制病毒复制。我们的具体目标是：(1)优化激活核糖核酸酶L的新型小分子2-5A模拟物，并确定它们对细胞存活和病毒生长的影响：(2)确定2-5A激活核糖核酸酶L如何信号转导抗病毒和免疫调节基因的转录，包括研究双链RNA信号转导因子和MAP激酶的参与；(3)通过检测I型干扰素的病毒诱导作用、核糖核酸酶L诱导的干扰素对病毒复制的影响以及2-5A和2-5A类似物对干扰素诱导和病毒复制的影响，确定核糖核酸酶L在野生型和L缺失小鼠体内抗病毒活性的基本依据。这项建议中描述的实验将研究普遍存在的有效抗病毒酶(核糖核酸酶L)的小分子激活剂如何刺激主要的抗病毒途径。因此，将获得的知识可能会导致控制重要的人类病毒病原体的策略。因此，这些研究有令人信服的和健康相关的理由。

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.