AMPLIFICATION OF ANTIVIRAL INNATE IMMUNITY BY SUPPRESSOR OF VIRUS RNA (svRNA)

描述（由申请人提供）：RNA切割是控制植物和动物中病毒感染的基本和古老的宿主反应。在包括人类在内的高等脊椎动物中，RNA切割作为控制病毒的一种手段是由I型干扰素（IFN）通过其效应物（独特调节的核糖核酸内切酶RNase L）介导的。RNA酶L被病毒感染过程中产生的不寻常的2 '，5'-连接的寡腺苷酸（2-5A）激活。2-5A激活RNA酶L，导致在单链区域内切割宿主和病毒RNA，主要在UU和UA之后。由于其特异性，RNase L产生小的、高度结构化的RNA切割产物。在2007年，我们报道了通过RNase L消化自身RNA获得的RNA切割产物激活RIG-I样受体（RLR），导致I型IFN合成的扩增。这些RNA切割产物代表了一类新的小RNA分子，称为“病毒RNA抑制因子”（svRNA）。我们在这个项目中的目标是克隆，鉴定和探测从宿主RNA和病毒[丙型肝炎病毒（HCV）] RNA产生的svRNA的功能。我们的假设是svRNA对于宿主防御对人类致病的各种病毒是必不可少的。我们的具体目标是：（1）为了从宿主和病毒RNA中分离和鉴定由RNase L释放的svRNA，我们将用纯化的RNase L切割HCV RNA并克隆与RLR结合的小RNA，并在用2-5A处理的完整细胞中切割细胞（自身）RNA并克隆与RLR结合的小RNA;（2）为了表征svRNA对RIG-I和MDA 5的活化，我们将进行ATP酶活化研究，通过表面等离子体共振确定svRNA与RIG-I和MDA 5相互作用的动力学参数，测量RIG-I和MDA 5的构象变化，并建立激活RIG-I和MDA 5所需的svRNA的序列和结构;（3）为了确定svRNA在抗病毒天然免疫中的作用，我们将鉴定HCV感染细胞中的svRNA，并确定svRNA在小鼠中的抗病毒作用。我们最近的研究表明svRNA在高等脊椎动物的抗病毒状态中起重要作用。在拟议的研究中，我们试图获得对这一重要途径的基本理解，因为它与宿主对病毒的防御有关。因此，这些研究有令人信服的健康相关理由。 公共卫生相关性：我们已经发现了一种新型的小RNA，它可以放大和维持干扰素的产生，干扰素是人体对抗各种不同类型病毒的前线防御。在拟议的研究中，我们将鉴定和表征这些小RNA，并将它们用于抑制病毒感染。从拟议的研究中获得的知识可能有助于改善病毒感染的治疗。

DESCRIPTION (provided by applicant): RNA cleavage is a fundamental and ancient host response for controlling viral infections in both plants and animals. In higher vertebrates, including humans, RNA cleavage as a means of controlling viruses is mediated by the type I interferons (IFN) through its effector, the uniquely regulated endoribonuclease, RNase L. RNase L is activated by unusual 2',5'-linked oligoadenylates (2-5A) produced during viral infections. 2-5A activates RNase L resulting in cleavage of host and viral RNAs within single stranded regions, predominantly after UU and UA. As a result of its specificity, RNase L produces small, highly structured RNA cleavage products. In 2007 we reported that RNA cleavage products obtained from digestion of self RNA by RNase L activated RIG-I-like receptors (RLR) resulting in amplification of type I IFN synthesis. These RNA cleavage products represent a novel class of small RNA molecules named "Suppressor of Virus RNA" (svRNA). Our GOALS in this project are to clone, identify and probe the functions of svRNAs generated from both host RNA and from viral [hepatitis C virus (HCV)] RNA. Our HYPOTHESIS is that svRNAs are essential to host defense against a wide range of viruses that are pathogenic for humans. Our Specific Aims are: (1) to isolate and identify svRNA liberated by RNase L from host and viral RNA, we will cleave HCV RNA with purified RNase L and clone small RNAs that bind to RLRs, and cleave cellular (self) RNA in intact cells treated with 2-5A and clone small RNAs that bind to RLRs; (2) To characterize activation of RIG-I and MDA5 by svRNAs we will perform ATPase activation studies, determine the kinetic parameters for svRNA interactions with RIG-I and MDA5 by surface plasmon resonance, measure conformational changes in RIG-I and MDA5, and establish the sequence and structural requirement of svRNA for activation of RIG-I and MDA5; and (3) to determine the role of svRNA in antiviral innate immunity we will identify svRNAs in HCV infected cells, and determine the antiviral effects of svRNAs in mice. Our recent studies suggest an essential role of svRNAs in the antiviral state in higher vertebrates. In the proposed studies we seek to obtain a fundamental understanding of this important pathway as it relates to host defense against viruses. Therefore, there are cogent and health-related justifications for these studies. PUBLIC HEALTH RELEVANCE: We have identified a novel type of small RNA that amplifies and perpetuates interferon production, the body's frontline defense against a broad-range of different types of viruses. In the proposed studies, we will identify and characterize these small RNAs and we will use them to inhibit virus infections. The knowledge to be gained from the proposed studies could contribute to improved treatments for viral infections.