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

定义西尼罗河病毒-宿主蛋白相互作用在逃避抗病毒免疫中的作用

• 批准号: 10141739
• 负责人: Holly Ramage
• 金额: $ 7.22万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10141739
• 关键词: Defining; Role; West; Nile; Virus

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.

项目摘要 西尼罗河病毒（WNV）是一种新出现的黄病毒属嗜神经病毒，可传播给人类 被受感染的蚊子叮咬。黄病毒包括全球重要的病原体，如登革热 寨卡病毒（ZIKV）和登革病毒（DENV）每年感染数亿人。目前还没有特效的抗病毒药物 治疗任何黄病毒。给定 选项 的 相互作用 利用 弗拉维夫 互动 显著 功能上 的 的 三 黄病毒在地球仪上的持续传播以及 为了预防或治疗它们，我们必须更好地了解宿主过程 影响感染。我们使用亲和纯化/质谱法来鉴定 发生在西尼罗河病毒和宿主蛋白质之间。与加州大学旧金山分校的Nevan Krogan合作，我们 来自DENV和ZIKV的平行研究的数据集中于由多种病毒靶向的宿主蛋白， 病毒。我们发现了259个高可信度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 与DENV或ZIKV中的类似病毒蛋白质。分析显示，大多数 黄病毒-宿主相互作用之间的重叠是衣壳-和NS 5-相互作用蛋白。到 为了确定对感染最重要的共享相互作用因子，我们采用了RNAi筛选， 在WNV、DENV和ZIKV感染的背景下。这揭示了影响西尼罗河病毒感染的23个因素，12 受影响的西尼罗河病毒和至少一种额外的黄病毒，以及8种影响所有感染的宿主蛋白质 其中，我们鉴定了USP 15，一种与黄病毒相互作用的宿主去泛素化酶 NS 5蛋白，并参与响应病毒感染诱导I型干扰素。我们证明了 USP 15是感染所必需的，并作为I型干扰素的负调节剂。我们将建立 我们通过使用USP 15和WNV NS 5观察到的表型中USP 15和WNV NS 5之间相互作用的要求 诱变以鉴定USP 15中对NS 5相互作用至关重要的残基，并产生NS 5- 结合缺陷型USP 15突变体进行测试。鉴于USP 15是一种去泛素化酶， I型干扰素反应的步骤需要泛素化，我们认为USP 15促进感染 通过去泛素化和抑制I型干扰素信号传导。为了测试这一点，我们将监控激活， 干扰素信号传导途径中已知靶点在USP 15敲低后的泛素化，并确定 USP 15-NS 5相互作用影响这种反应。我们还确定了黄病毒衣壳相互作用的主机 蛋白质WIBG。WIBG是参与无义介导的RNA衰变（NMD）的RNA结合蛋白， 黄病毒感染的抗病毒药物。我们表明，NMD在黄病毒感染中受到抑制;此外， 典型NMD因子UPF 1表明NMD限制黄病毒感染。我们认为NMD是一个 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WNV感染，并确定WIBG-衣壳相互作用如何影响这一过程。这个目标 该提案旨在揭示黄病毒破坏宿主先天抗病毒机制的机制。