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

描述(申请人提供)：RNA裂解是控制植物和动物中病毒感染的一种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，核糖核酸的切割是由I型干扰素通过其效应器介导的，唯一受调控的内切核酸酶，核糖核酸酶，L是由病毒感染过程中产生的不寻常的2‘，5’-连接的寡腺苷(2-5A)激活的。2-5A激活核糖核酸酶L，导致宿主和病毒RNA在单链区域内被切割，主要发生在Uu和UA之后。由于其特异性，核糖核酸酶L产生小的，高度结构的核糖核酸切割产物。2007年，我们报道了核糖核酸酶L对自身核糖核酸的裂解产物激活了RIG-I样受体，导致I型干扰素合成的扩增。这些RNA裂解产物代表了一类新的小RNA分子，命名为病毒RNA抑制因子(SvRNA)。我们在这个项目中的目标是克隆、鉴定和探索从宿主RNA和病毒[丙型肝炎病毒]RNA产生的svRNAs的功能。我们的假设是，svRNA对于宿主防御对人类致病的广泛病毒是必不可少的。我们的具体目标是：(1)为了分离和鉴定核糖核酸酶L从宿主和病毒RNA中释放出来的svRNA，我们将用纯化的核糖核酸酶L切割丙型肝炎病毒RNA，克隆与RLR结合的小RNA，并在2-5A处理的完整细胞中切割细胞(自身)RNA，克隆与RLR结合的小RNA；(2)为了鉴定svRNAs激活RIG-I和MDA5的特性，我们将进行ATPase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，并建立激活RIG-I和MDA5的svRNA的序列和结构要求；以及(3)为了确定svRNA在抗病毒天然免疫中的作用，我们将在丙型肝炎病毒感染细胞中鉴定svRNA，并确定svRNAs在小鼠体内的抗病毒作用。我们最近的研究表明，svRNAs在高等脊椎动物的抗病毒状态中发挥着重要作用。在拟议的研究中，我们试图对这一重要途径有一个基本的了解，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和健康相关的理由。 与公共健康相关：我们已经确定了一种新型的小RNA，它可以放大并保持干扰素的产生，这是人体抵御广泛不同类型病毒的前线防御。在拟议的研究中，我们将识别和鉴定这些小RNA，并使用它们来抑制病毒感染。从拟议的研究中获得的知识可能有助于改进病毒感染的治疗方法。

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.