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反应和病毒对策的显著多样性 在宿主和病原体共同进化的过程中。在这个进化过程中识别和描述病毒与宿主的相互作用 “军备竞赛”对于理解这些相互作用的结果如何有利于任何一种高产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)，它们可能被利用来发现和探测它们的目标细胞机械。我们正在开发新的 识别以保守的真核机制为靶标并能够打破平衡的IEP的方法 在流产性和生产性病毒感染之间。虽然经典的方法采用了病毒-宿主模型 病毒可以有效地感染选定的宿主，我们开发了一种新的范例，利用 鳞翅目昆虫(蛾和蝴蝶)细胞中自然流产的虫媒病毒感染作为筛选工具 由哺乳动物病原体编码的新的IEP，通过以下方式将流产感染转化为生产性感染 对抗宿主免疫反应。通过识别由哺乳动物病原体编码的IEP 在昆虫细胞中的免疫抑制功能，我们可以选择针对保守的抗病毒机制的IEP 在无脊椎动物和脊椎动物宿主之间。使用这种方法，我们已经识别了几个由 以保守的宿主机制为目标的哺乳动物病原体，我们随后发现这些宿主机制执行 抗病毒功能。例如，当前关注的一个领域是痘病毒编码的A51R的特征 蛋白质作为一个新的哺乳动物IEP家族，靶向易染色质转录(FACT)复合体， 一种进化保守的组蛋白伴侣复合体，可抑制昆虫和 人类细胞。使用A51R蛋白来探索FACT功能，我们发现一个翻译后修饰的 在痘病毒过程中，A51R蛋白结合了FAX复合亚单位的形式并错误地定位到细胞质 感染。我们还发现了几种不相关的RNA病毒来编码IEP，以防止和/或逆转事实 亚基修饰。利用病毒学、遗传学和生化方法，我们的目标是揭示病毒IEP是如何 阻碍FACT复合体活性、FACT在决定宿主感染易感性中的作用以及其功能(S) 事实上，亚基在抗病毒和正常细胞过程中的翻译后修饰。最后，我们是 开发一条管道来利用我们的虫媒病毒-鳞翅目昆虫宿主系统来识别更多的靶向IEP 保守的宿主因子，作为细胞内病原体限制的关键决定因素。使用这条管道，我们 确定由感染哺乳动物的细菌病原体编码的~10个IEP，以解除昆虫中虫媒病毒的限制 细胞，这表明这些IEP针对的是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的IEP，并使用它们来提供 对他们操纵的保守宿主机制的抗病毒(和正常)功能的机械式洞察。

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.