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

描述（由申请人提供）：RNA切割是植物和动物控制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，作为控制病毒的一种手段，RNA裂解是由I型干扰素（IFN）通过其效应物介导的，即独特调节的核糖核酸内切酶RNase L. RNase L被病毒感染过程中产生的不寻常的2‘,5’连接的低聚腺苷酸（2- 5a）激活。2-5A激活RNase L，导致单链区域内宿主和病毒rna的切割，主要发生在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，我们报道了由RNase L激活的rig -I样受体（RLR）消化自身RNA获得的RNA切割产物，导致I型IFN合成扩增。这些RNA裂解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和从病毒[丙型肝炎病毒（HCV）] RNA中产生的svRNAs的功能。我们的假设是，svrna对宿主防御多种人类致病性病毒至关重要。我们的具体目标是：(1)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RLRs结合的小RNA，并在完整细胞中用2-5A切割细胞（自身）RNA并克隆与RLRs结合的小RNA；(2)为了表征svRNA对RIG-I和MDA5的激活，我们将进行atp酶激活研究，通过表面等离子体共振确定svRNA与RIG-I和MDA5相互作用的动力学参数，测量RIG-I和MDA5的构象变化，并确定svRNA激活RIG-I和MDA5的序列和结构要求；(3)为了确定svRNA在抗病毒先天免疫中的作用，我们将在HCV感染细胞中鉴定svRNA，并在小鼠中确定svRNA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发挥了重要作用。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，因为它与宿主防御病毒有关。因此，这些研究有令人信服的与健康相关的理由。

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.