Defining the Role of West Nile Virus-Host Protein Interactions in Evading Antiviral Immunity

项目摘要 西尼罗河病毒(West Nile Virus，WNV)是一种新出现的嗜神经性黄病毒属病毒，可传播给人类 通过被感染的蚊子叮咬。黄病毒包括全球重要的病原体，如登革热 每年感染数亿人的登革热(DENV)和寨卡(ZIKV)病毒。目前，还没有特定的抗病毒药物 对任何黄病毒的治疗。vt.给出 选项 那 互动 利用 弗拉维夫 互动 显着性 在功能上 这个 那 三 黄病毒在全球范围内的持续传播以及缺乏 为了预防或治疗它们，我们必须更好地了解宿主过程 冲击性感染。我们使用亲和纯化/质谱学方法来鉴定物理 发生在西尼罗河病毒和宿主蛋白之间。在加州大学旧金山分校的内万·克罗根的合作下，我们 来自DENV和ZIKV的平行研究的数据集中在多个靶向的宿主蛋白上 艾瑞斯。我们发现了259个高度可信的与西尼罗河病毒相互作用的宿主蛋白，其中49个宿主蛋白 在DENV或ZIKV中使用类似的病毒蛋白。这一分析显示，最 黄病毒-宿主相互作用之间的重叠是衣壳和NS5相互作用的蛋白质。至 定义对感染最重要的共享交互作用，我们在 西尼罗河病毒、登革热病毒和寨卡病毒感染的背景。这揭示了23个影响西尼罗病毒感染的因素，12 影响西尼罗河病毒和至少一个额外的黄病毒，以及影响ALL感染的8个宿主蛋白 在这些病毒中，我们鉴定了USP15，一种与黄病毒相互作用的宿主脱泛素酶 NS5蛋白，并与病毒感染时I型干扰素的诱导有关。我们证明了 USP15是感染所必需的，是I型干扰素的负调节因子。我们将建立 USP15与西尼罗河病毒NS5相互作用的表型要求 突变以确定USP15中对NS5相互作用至关重要并将产生NS5-的残基 结合缺陷的USP15突变体在我们的研究中进行测试。鉴于USP15是一种脱泛素酶，而且几个 I型干扰素反应中的步骤需要泛素化，我们认为USP15促进感染 通过去泛素化和抑制I型干扰素信号。为了测试这一点，我们将监控激活和 敲除USP15后干扰素信号通路中已知靶点的泛素化，并确定 USP15-NS5相互作用影响这种反应。我们还鉴定了与黄病毒衣壳相互作用的宿主 蛋白质WIBG。WIBG是一种参与无义介导的RNA衰退(NMD)的RNA结合蛋白，它是 治疗黄病毒感染的抗病毒药物。我们发现NMD在黄病毒感染中受到抑制；此外，NMD的耗竭 典型的NMD因子UPF1表明NMD限制了黄病毒的感染。我们认为，NMD是一种 被黄病毒拮抗的抗病毒宿主过程。我们将确定NMD的机制 抑制西尼罗河病毒感染，并确定WIBG-衣壳相互作用如何影响这一过程。这样做的目的是 建议是揭示黄病毒颠覆宿主固有的抗病毒机制的机制。

Project Summary West Nile virus (WNV) is an emerging, neurotropic virus of the Flavivirus genus that is transmitted to humans through the bite of an infected mosquito. Flaviviruses include globally important pathogens, such as dengue (DENV) and Zika (ZIKV) virus which infect hundreds of millions yearly. Currently, there are no specific antiviral treatments for any flavivirus. Given options that interactions utilized flaviv interacted significant functionally the that three the continuing spread of flaviviruses across the globe and the dearth of to prevent or treat them, it is imperative that we develop a better understanding the host processes impact infection. W e used an affinity purification/mass spectrometry approach to identify the physical that occur between WNV and host proteins. In collaboration with Nevan Krogan at UCSF, we the data from parallel studies of DENV and ZIKV to focus on host proteins targeted by multiple iruses. We discovered 259 high- confidence WNV -interacting host proteins; of those, 49 host proteins with the analogous viral protein in either DENV or ZIKV. This analysis revealed that the most overlap between flavivirus-host interactions was for capsid- and NS5-interacting proteins. To define shared interactors that are most important for infection, we employed an RNAi screen in context of WNV, DENV and ZIKV infection. This revealed 23 factors that impacted WNV infection , 12 impacted WNV and at least one additional flavivirus, and 8 host proteins influencing infection for all flaviviruses.Among these, we identified USP15, a host deubiquitylase that interacts with the flavivirus NS5 proteins and is implicated in the induction of Type I interferons in response to viral infection. We show that USP15 is required for infection and acts as a negative regulator of Type I interferon. We will establish the requirement for the interaction between USP15 and WNV NS5 in the phenotypes we observe by using mutagenesis to identify the residues in USP15 that are critical for the NS5 interaction and will generate NS5- binding deficient USP15 mutants to test in our studies. Given that USP15 is a deubiquitylase and that several steps in the Type I interferon response require ubiquitylation, we propose that USP15 promotes infection through deubiquitylation and inhibition of Type I interferon signaling. To test this, we will monitor activation and ubiquitylation of known targets in the interferon signaling pathway upon knockdown of USP15 and determine if the USP15-NS5 interaction affects this response. We have also identified the flavivirus capsid-interacting host protein WIBG. WIBG is an RNA-binding protein involved in nonsense-mediated RNA decay (NMD) and is antiviral in flavivirus infection. We show that NMD is inhibited in flavivirus infection; moreover, depletion of the canonical NMD factor UPF1 indicates that NMD restricts flavivirus infection. We propose that NMD is an antiviral host process that is antagonized by flaviviruses. We will determine the mechanism by which NMD inhibits WNV infection and determine how the WIBG-capsid interaction influences this process. The goal of this proposal is to uncover the mechanisms by which flaviviruses subvert host innate antiviral mechanisms.