Efficacy of the antiviral state versus Sindbis and Venezuelan equine encephalitis

描述（由申请人提供）：I型干扰素（IFN-1/2）系统，如果在没有病毒拮抗的情况下被激活，在阻断多种病毒的复制方面非常有效，在动物模型中可以预防或大大减少疾病。此外，该系统的一些成分已经进化到可以有效区分宿主和病毒的结构和代谢过程，以最小的非靶标效应抑制病毒过程。这些特性为开发新的抗病毒药物提供了一个极好的概念模型：一个区分“自我”（宿主）和“非自我”（病毒）并有效抑制“非自我”的病毒抑制系统，在某些情况下具有广谱活性，而不是多种病毒。虽然这一系统通常是有效的，但我们发现，人类毒性的甲病毒委内瑞拉马脑炎病毒（VEEV）比人类毒性的辛德比斯病毒（SINV）更能抵抗抗病毒状态的活性，动物模型中病毒的毒性在很大程度上反映了这些差异。树突状细胞是一种与甲病毒传播、扩增和体内疾病高度相关的细胞类型，通过树突状细胞的全局转录谱分析，我们鉴定出两种ifn -1/2诱导蛋白，ISG20外核糖核酸酶和Mx2 GTPase对SINV具有有效的抗病毒活性。为了发现这些蛋白的抗病毒活性机制以及VEEV抵抗这些抗病毒状态效应物的手段，我们建议表征每种蛋白对SINV和VEEV的相对抑制效果和抗病毒活性机制。为了实现这一目标，我们将使用过表达和干扰rna敲低技术来确定病毒复制周期中每个效应物起作用的点，然后进行诱变，以确定单个效应物的相关功能域，并进行亚细胞定位和蛋白质-蛋白质相互作用研究，以确定抑制的病毒结构和/或过程。这些研究的结果将：i)确定对Mx2和ISG20 α病毒的作用机制，ii)确定每种病毒的易感性点，作为设计抗病毒疗法的第一步，以及iii)深入了解VEEV对抗病毒状态活性的抗性的分子机制，用于设计疫苗。

DESCRIPTION (provided by applicant): The type I interferon (IFN-1/2) system, if activated in the absence of virus antagonism, is very effective at blocking the replication of multiple viruses, either preventing or substantially reducing disease in animal models. Furthermore, some components of this system have evolved such that they can effectively distinguish between host and viral structural and metabolic processes, suppressing the viral processes with minimal non- target effects upon the host. These attributes provide an excellent conceptual model for development of new antiviral drugs: a virus suppression system that distinguishes "self" (host) from "nonself" (virus) and effectively suppresses nonself, in some cases with broad spectrum activity versus multiple viruses. While this system is generally effective, we have found that the human-virulent alphavirus, Venezuelan equine encephalitis virus (VEEV), is much more resistant to the activity of the antiviral state than the human-avirulent Sindbis virus (SINV) and that the virulence of the viruses in animal models is largely reflective of these differences. Using global transcription profiling of dendritic cells, a cell-type highly relevant to alphavirus dissemination, amplification and disease in vivo, we have identified two IFN-1/2-inducible proteins with potent antiviral activity versus SINV, the ISG20 exoribonuclease and the Mx2 GTPase. To discover the mechanisms of antiviral activity employed by these proteins and the means by which VEEV resists these effectors of the antiviral state, we propose to characterize the relative efficacy of inhibition and mechanisms of antiviral activity exerted by each protein versus SINV and VEEV. To achieve this, we will use over-expression and interfering-RNA knockdown techniques to identify the point in the virus replication cycle at which each effector acts followed by mutagenesis to identify the responsible functional domains in the individual effectors and subcellular localization and protein-protein interaction studies to identify the viral structures and/or processes targeted for inhibition. The results of these studies will: i) determine the mechanism(s) of action versus alphaviruses of Mx2 and ISG20, ii) identify points of vulnerability of each virus as a first step in design of antiviral therapeutics, and iii) provide insights into molecular mechanisms underlying the resistance of VEEV to the activities of the antiviral state that be utilized in design of vaccines. PUBLIC HEALTH RELEVANCE: The results of these studies will identify the mechanisms of action of two highly active IFN-induced antiviral effector proteins that are involved in interferon-mediated suppression of alphavirus replication and the relative sensitivity of benign and highly virulent viruses to the effectors. This information can be used in the design of antiviral drugs that artificially mimic the activity or artificially stimulate the induction of these effectors. Furthermore, these studies will provide information regarding the contribution of resistance to particular effectors to the virulence of alphaviruses in mice and humans that can be used in design of alphavirus vaccines.