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: 10434020
• 负责人: Holly Ramage
• 金额: $ 39万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10434020
• 关键词: Affect; Affinity Chromatography; Attenuated; Binding; Biology; Capsid; Cells; Co-Immunoprecipitations; Collaborations; Complex; Culicidae; Data; Degradation Pathway; Dengue; Dengue Virus; Development; Epidemic; Excision; Exons; Flavivirus; Flavivirus Infections; Future; Genes; Goals; Human; Immune Evasion; Immunity; Immunologic Surveillance; Infection; Integration Host Factors; Interferon Type I; Interferons; Invaded; Knockout Mice; Mass Spectrum Analysis; Mediating; Modification; Monitor; Nonsense-Mediated Decay; Pathway interactions; Phenotype; Physiological; Process; Production; Proteins; RNA; RNA Decay; RNA Degradation; RNA Interference; RNA Probes; RNA Stability; RNA interference screen; RNA-Binding Proteins; Role; Signal Pathway; Signal Transduction; Site; Testing; Therapeutic Intervention; Ubiquitin; United States; Viral; Viral Pathogenesis; Viral Proteins; Virus; Virus Replication; West Nile viral infection; West Nile virus; Work; ZIKA; ZIKV infection; Zika Virus; antiviral immunity; gene induction; knock-down; member; mutant; neurotropic; neurotropic virus; new therapeutic target; novel therapeutics; overexpression; pathogen; prevent; protein complex; protein function; response; sensor; viral RNA; virus host interaction

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.

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 通过受感染的蚊子。黄病毒包括全球重要的病原体，如登革热（DENV）和寨卡病毒（Zika）。 ZIKV病毒，每年感染数亿人。目前，没有特异性的抗病毒治疗方法， 任何黄病毒鉴于黄病毒在地球仪的传播以及缺乏预防或治疗的选择 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法来鉴定发生在 WNV和宿主蛋白。在与Nevan Krogan的合作中，我们利用了来自DENV平行研究的数据， 和ZIKV集中于多种黄病毒靶向的宿主蛋白。我们发现了259个高可信度的 WNV相互作用宿主蛋白;其中，49种宿主蛋白与WNV中的类似病毒蛋白相互作用。 DENV或ZIKV。该分析显示，最显著的重叠是衣壳-和NS 5-。 相互作用的蛋白质为了确定对感染重要的共享相互作用因子，我们采用了RNAi技术， 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，8个因素影响所有三个感染 黄病毒其中，我们鉴定了两个在抗病毒信号传导和细胞内分泌中起作用的宿主蛋白。 免疫力USP 15是促进WNV感染并与黄病毒NS 5相互作用的宿主去泛素化酶 proteins.我们发现USP 15是西尼罗河病毒感染所必需的，并在感染中作为I型干扰素的负调节剂。 这个背景。我们将通过鉴定WNV NS 5-USP 15相互作用在该表型中的作用， USP 15中对于与NS 5相互作用和生成NS 5结合缺陷型USP 15至关重要的残基 在我们的研究中进行测试I型干扰素应答的激活需要RIG-I的泛素化，并且 我们提出USP 15通过RIG-I的去泛素化抑制I型干扰素信号传导。为了验证这一点，我们 监测RIG-I在USP 15敲低后的活化和泛素化，并确定USP 15- NS 5在此响应中的相互作用。我们还鉴定了黄病毒衣壳相互作用宿主蛋白WIBG， 限制了西尼罗河病毒的感染WIBG参与RNA调控过程，包括无义介导的 RNA衰变（NMD）和与外显子连接复合物（EJC）蛋白Y14和MAGOH的相互作用。我们表明 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，因为典型的NMD因子UPF 1的缺失， EJC蛋白MAGOH导致黄病毒感染增加。我们认为NMD是一种抗病毒药物 被黄病毒拮抗的宿主过程。我们将确定NMD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。