Exploiting Pathogen-Encoded Immune Evasion Proteins to Uncover Evolutionarily Conserved Antiviral Host Machinery

项目摘要 病毒-宿主相互作用驱动宿主免疫应答和病毒对策的显著多样性 在宿主-病原体共同进化过程中。识别和表征这种进化过程中的病毒-宿主相互作用 “军备竞赛”对于理解这些相互作用的结果如何有利于生产性病原体是至关重要的。 复制或流产感染。此外，如果能够识别病原体编码的免疫逃避蛋白， （IEP），它们可能被用来发现和探测它们所针对的细胞机制。我们正在开发新 鉴定靶向保守真核生物机制并能够打破平衡的IEPs的方法 流产和生产性病毒感染之间的关系。虽然经典方法采用病毒宿主模型， 其中病毒可以有效地感染所选择的宿主，我们已经开发了一种新的范例， 自然流产虫媒病毒感染鳞翅目（蛾和蝴蝶）细胞作为筛选工具，以确定 由哺乳动物病原体编码的新IEPs，其通过以下途径将流产感染转化为生产性感染： 对抗宿主免疫反应通过鉴定哺乳动物病原体编码的IEPs， 在昆虫细胞中的免疫抑制功能，我们可以选择靶向保守的抗病毒机制的IEPs 在无脊椎动物和脊椎动物宿主之间。使用这种方法，我们已经确定了几个IEP编码， 哺乳动物病原体的目标是保守的宿主机制，我们随后发现， 抗病毒功能。例如，一个当前关注的领域是痘病毒编码的A51 R的表征 蛋白质作为哺乳动物IEP的新家族，靶向促进染色质转录（FACT）复合物， 一种进化上保守的组蛋白伴侣复合物，可抑制昆虫细胞质病毒复制， 人体细胞使用A51 R蛋白来探测FACT功能，我们发现一种后修饰的FACT蛋白， 一种FACT复合物亚基的形式与A51 R蛋白结合，并在痘病毒感染期间错误定位于细胞质。 感染我们还发现了几种不相关的RNA病毒编码的IEPs，可以阻止和/或逆转FACT 亚基修饰使用病毒学，遗传学和生物化学方法，我们的目标是揭示病毒IEPs如何 阻碍FACT复合物活性，FACT在确定宿主感染易感性中的作用，以及FACT的功能 FACT亚基在抗病毒和正常细胞过程中的翻译后修饰。最后，我们 开发一个管道，利用我们的虫媒病毒-鳞翅目宿主系统，以确定更多的IEPs靶向 作为细胞内病原体限制的关键决定因素的保守宿主因子。通过这条管道，我们 鉴定了约10种由感染哺乳动物的细菌病原体编码的IEPs，它们可以解除虫媒病毒对昆虫的限制 这表明这些IEPs针对广泛限制病毒和细菌的宿主防御。我们的总体使命 是在我们独特的系统中识别由哺乳动物病原体编码的新型IEPs， 对它们操纵的保守宿主机制的抗病毒（和正常）功能的机械见解。

Project Summary Virus-host interactions drive a remarkable diversity of host immune responses and viral countermeasures during host-pathogen coevolution. Identifying and characterizing virus-host interactions within this evolutionary “arms race” is critical for understanding how the outcome of these interactions favor either productive pathogen replication or abortive infection. Furthermore, if one can identify pathogen-encoded immune evasion proteins (IEPs), they might be exploited to discover and probe the cellular machinery they target. We are developing new approaches to identify IEPs that target conserved eukaryotic machinery and are capable of tipping the balance between abortive and productive viral infection. While classical approaches have employed virus-host models wherein the virus can productively infect the chosen host, we have developed a new paradigm that exploits naturally abortive arbovirus infections in lepidopteran (moth and butterfly) cells as a screening tool to identify novel IEPs encoded by mammalian pathogens that convert abortive infections to productive infections by countering host immune responses. By identifying IEPs encoded by mammalian pathogens that retain immunosuppressive function in insect cells, we can select for IEPs that target antiviral machinery conserved between invertebrate and vertebrate hosts. Using this approach, we have identified several IEPs encoded by mammalian pathogens that target conserved host machinery that we have subsequently found to perform antiviral functions. For example, one current area of focus is the characterization of poxvirus-encoded A51R proteins as a new family of mammalian IEPs that target the facilitates chromatin transcription (FACT) complex, an evolutionarily-conserved histone chaperone complex, that inhibits cytoplasmic virus replication in insect and human cells. Using A51R proteins to probe FACT functions, we discovered that a post-translationally modified form of a FACT complex subunit is bound by A51R proteins and mislocalized to the cytoplasm during poxvirus infection. We also found several, unrelated RNA viruses to encode IEPs that prevent and/or reverse FACT subunit modification. Using virological, genetic, and biochemical approaches, we aim to reveal how viral IEPs impede FACT complex activity, the role of FACT in determining host susceptibility to infection, and the function(s) of FACT subunit post-translational modification in both antiviral and normal cellular processes. Finally, we are developing a pipeline to exploit our arbovirus-lepidopteran host system to identify additional IEPs targeting conserved host factors that act as key determinants of intracellular pathogen restriction. Using this pipeline, we identified ~10 IEPs encoded by bacterial pathogens infecting mammals that relieve arbovirus restriction in insect cells, suggesting these IEPs target host defenses that broadly restrict viruses and bacteria. Our overall mission is to identify novel IEPs encoded by mammalian pathogens in our unique system and use them to provide mechanistic insights into the antiviral (and normal) functions of the conserved host machinery they manipulate.