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 通过受感染的蚊子。黄病毒包括全球重要病原体，如登革热(DENV)和寨卡 (ZIKV)病毒，每年感染数亿人。目前，还没有专门的抗病毒疗法来治疗 任何黄病毒。鉴于黄病毒在全球的传播，以及预防或治疗选择的匮乏 因此，我们必须更好地了解影响感染的宿主过程。我们 使用亲和纯化和质谱学来鉴定发生在 西尼罗河病毒和宿主蛋白。在与内万·克罗根的合作中，我们利用了DENV的平行研究数据 和ZIKV专注于多种黄病毒靶标的宿主蛋白。我们发现了259个高自信 与西尼罗河病毒相互作用的宿主蛋白；其中49种宿主蛋白与类似的病毒蛋白在 DENV或ZIKV。这一分析表明，最显著的重叠是衣壳蛋白-和NS5- 相互作用的蛋白质。为了定义对感染很重要的共享交互作用因子，我们使用了RNAi 在WNV、DENV和ZIKV感染的背景下进行筛查。我们发现了23个影响西尼罗河病毒感染的因素， 12个影响西尼罗河病毒和一个额外的黄病毒，以及8个影响这三个病毒感染的因素 黄病毒。其中，我们鉴定了两种宿主蛋白，它们分别在抗病毒信号和细胞内源性蛋白中发挥作用。 豁免权。USP15是一种宿主脱泛素酶，可促进西尼罗河病毒感染并与黄病毒NS5相互作用 蛋白质。我们发现USP15是西尼罗河病毒感染所必需的，并作为I型干扰素的负调控因子 这一背景。我们将通过鉴定WNV NS5-USP15相互作用在这种表型中的作用 USP15中对与NS5相互作用和产生NS5结合缺陷USP15至关重要的残基 在我们的研究中测试突变体。I型干扰素反应的激活需要RIG-I的泛素化，并且 我们认为USP15通过RIG-I的去泛素化抑制I型干扰素信号转导。为了测试这一点，我们将 监测RIG-I在USP15被敲除后的激活和泛素化，并确定USP15- NS5在这一反应中的相互作用。我们还鉴定了黄病毒衣壳相互作用的宿主蛋白WIBG， 从而限制西尼罗河病毒的感染。WIBG参与了RNA调控过程，包括无意义介导的过程 RNA衰变(NMD)以及与外显子连接复合体(EJC)蛋白Y14和MAGOH的相互作用。我们展示了 在黄病毒感染中，NMD被抑制，WIBG与Y14/MAGOH的相互作用被破坏。 此外，我们证明了NMD和EJC是抗病毒的，因为缺乏典型的NMD因子UPF1， 而一种名为MAGOH的EJC蛋白会导致黄病毒感染增加。我们认为NMD是一种抗病毒药物 被黄病毒对抗的宿主进程。我们将确定NMD抑制的机制 WNV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 via infected mosquitoes. 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 the spread of flaviviruses across the globe and the dearth of options to prevent or treat them, it is imperative that we develop a better understanding of host processes that impact infection. We used affinity purification and mass spectrometry to identify the physical interactions that occur between WNV and host proteins. In collaboration with Nevan Krogan, we utilized data from parallel studies of DENV and ZIKV to focus on host proteins targeted by multiple flaviviruses. We discovered 259 high-confidence WNV-interacting host proteins; of those, 49 host proteins interacted with the analogous viral protein in either DENV or ZIKV. This analysis revealed that the most significant overlap was for capsid- and NS5- interacting proteins. To define the shared interactors that are important for infection, we employed an RNAi screen in the context of WNV, DENV and ZIKV infection. We found 23 factors that impacted WNV infection, 12 that impacted WNV and one additional flavivirus, and 8 factors influencing infection of all three flaviviruses. Among these, we identified two host proteins with roles in antiviral signaling and cell-intrinsic immunity. USP15 is a host deubiquitylase that promotes WNV infection and interacts with flavivirus NS5 proteins. We show USP15 is required for WNV infection and acts as a negative regulator of Type I interferon in this context. We will establish the role of the WNV NS5-USP15 interaction in this phenotype by identifying the residues in USP15 that are critical for interaction with NS5 and generating NS5-binding deficient USP15 mutants to test in our studies. Activation of the Type I interferon response requires ubiquitylation of RIG-I, and we propose that USP15 inhibits Type I interferon signaling via deubiquitylation of RIG-I. To test this, we will monitor activation and ubiquitylation of RIG-I upon knockdown of USP15 and determine the role of the USP15- NS5 interaction in this response. We have also identified the flavivirus capsid-interacting host protein WIBG, which restricts WNV infection. WIBG is involved in RNA regulatory processes, including nonsense-mediated RNA decay (NMD) and interaction with the exon-junction complex (EJC) proteins, Y14 and MAGOH. We show that NMD is inhibited and that the interaction of WIBG with Y14/MAGOH is disrupted in flaviviral infection. Moreover, we demonstrate that NMD and the EJC are antiviral, as depletion of a canonical NMD factor, UPF1, and an EJC protein, MAGOH, results in increased 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.