Efficacy of the antiviral state versus Sindbis and Venezuelan equine encephalitis

描述(申请人提供)：I型干扰素(干扰素-1/2)系统，如果在没有病毒拮抗的情况下被激活，在动物模型中可以非常有效地阻止多种病毒的复制，预防或显著减少疾病。此外，这个系统的一些组成部分已经进化到可以有效地区分宿主和病毒的结构和代谢过程，以对宿主的最小非靶标影响抑制病毒过程。这些属性为新的抗病毒药物的开发提供了一个极好的概念模型：一种病毒抑制系统，它将“自我”(宿主)与“非我”(病毒)区分开来，并有效地抑制非我，在某些情况下，对多种病毒具有广谱活性。虽然这个系统总体上是有效的，但我们发现，人类毒力的甲型病毒委内瑞拉马脑炎病毒(VEEV)比人类无毒的辛德比斯病毒(SINV)对抗病毒状态的活性更具抵抗力，并且在动物模型中这些病毒的毒力在很大程度上反映了这些差异。利用树突状细胞(一种与甲病毒在体内传播、扩增和疾病高度相关的细胞类型)的全球转录图谱，我们鉴定了两种具有强大抗病毒活性的干扰素-1/2诱导蛋白，ISG20外切核酸酶和MX2 GTP酶。为了发现这些蛋白的抗病毒活性的机制以及VEEV抵抗这些抗病毒状态的效应物的方式，我们建议表征每种蛋白相对于SINV和VEEV的抑制相对有效性和抗病毒活性的机制。为了实现这一目标，我们将使用过度表达和干扰RNA敲除技术来确定病毒复制周期中每个效应器的作用点，然后进行突变，以确定单个效应器中的负责功能域，并进行亚细胞定位和蛋白质相互作用研究，以确定针对抑制的病毒结构和/或过程。这些研究的结果将：i)确定MX2和ISG20针对甲型病毒的作用机制(S)，ii)确定每种病毒的脆弱性，作为抗病毒治疗药物设计的第一步，以及iii)为VEEV抵抗抗病毒状态活性的分子机制提供见解，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干扰素诱导的抗病毒效应蛋白的作用机制，这两种蛋白参与干扰素介导的抑制甲型病毒复制，以及良性和高毒力病毒对效应物的相对敏感性。这些信息可以用来设计人工模拟这些效应器活性或人工刺激这些效应器诱导的抗病毒药物。此外，这些研究将提供有关对特定效应物的耐药性对甲型病毒在小鼠和人类中的毒力的贡献的信息，这些信息可用于甲型病毒疫苗的设计。

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.