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作用的情况下被激活，在阻断多种病毒复制方面非常有效，在动物模型中预防或显著减少疾病。此外，该系统的一些组分已经进化，使得它们可以有效地区分宿主和病毒的结构和代谢过程，抑制病毒过程，对宿主的非靶效应最小。这些属性为开发新的抗病毒药物提供了一个很好的概念模型：一种病毒抑制系统，可以区分“自我”（宿主）和“非自我”（病毒），并有效抑制非自我，在某些情况下对多种病毒具有广谱活性。虽然这种系统通常是有效的，但我们发现，人毒性甲病毒，委内瑞拉马脑炎病毒（VEEV），比人无毒力辛德毕斯病毒（SINV）对抗病毒状态的活性更具抗性，并且动物模型中病毒的毒力在很大程度上反映了这些差异。使用树突状细胞（一种与体内甲病毒传播、扩增和疾病高度相关的细胞类型）的全局转录谱，我们已经鉴定了两种IFN-1/2诱导的蛋白质，其对SINV具有有效的抗病毒活性，ISG 20核糖核酸外切酶和Mx2 GT3。为了发现这些蛋白质的抗病毒活性的机制和VEEV抵抗这些抗病毒状态的效应物的手段，我们建议表征每种蛋白质与SINV和VEEV的相对抑制效力和抗病毒活性的机制。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子作用的点，然后进行诱变以确定单个效应子中负责的功能结构域，并进行亚细胞定位和蛋白质-蛋白质相互作用研究以确定靶向抑制的病毒结构和/或过程。这些研究的结果将：i）确定Mx2和ISG 20对抗甲病毒的作用机制，ii）鉴定每种病毒的脆弱点，作为设计抗病毒治疗剂的第一步，和iii）提供对VEEV对抗病毒状态活性的抗性的分子机制的见解，所述分子机制用于疫苗设计。 公共卫生相关性：这些研究的结果将确定两种高活性IFN诱导的抗病毒效应蛋白的作用机制，这些蛋白参与干扰素介导的甲病毒复制抑制以及良性和高毒力病毒对效应物的相对敏感性。这些信息可用于设计人工模拟活性或人工刺激这些效应物的诱导的抗病毒药物。此外，这些研究将提供关于对特定效应物的抗性对小鼠和人类中甲病毒毒力的贡献的信息，这些信息可用于甲病毒疫苗的设计。

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.