Proteomics of Venezuelan Equine Encephalitis Virus nsP3 protein

委内瑞拉马脑炎病毒 nsP3 蛋白的蛋白质组学

• 批准号: 9302884
• 负责人: Aarthi Narayanan
• 金额: $ 45.6万
• 依托单位: GEORGE MASON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9302884
• 关键词: Acute; Adult; Aerosols; Alphavirus; Alphavirus Infections; Antiviral Agents; Asses; Attenuated; Brain; Categories; Cell Line; Cells; Child; Culicidae; DDX1 gene; Dependence; Development; Disease; Eastern Equine Encephalitis Virus; Encephalitis; Enzymes; Equus caballus; Evaluation; Event; FDA approved; Family; Human; In Vitro; Infection; Infectious Agent; Innate Immune Response; Insecta; Knowledge; Laboratories; Link; Manuals; Manuscripts; Mass Spectrum Analysis; Morbidity - disease rate; Mutate; Mutation; Nonstructural Protein; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Population; Prevention; Proteins; Proteomics; Publications; Publishing; Research; Role; Testing; Therapeutic; Tissues; Togaviridae; Treatment Efficacy; Trinidad; Vaccine Design; Vaccines; Validation; Venezuelan Equine Encephalitis Virus; Viral; Viral Load result; Viral Nonstructural Proteins; Viral Proteins; Virulence; Virulent; Virus; Virus Diseases; Virus Replication; Western Equine Encephalitis Virus; base; helicase; improved; in vivo; kinase inhibitor; mortality; mouse model; mutant; novel; pathogen; phosphoproteomics; protein protein interaction; prototype; small molecule inhibitor

Abstract Venezuelan Equine Encephalitis Virus (VEEV) is an alphavirus that is classified as a category B select agent and an emerging infectious agent. There are no FDA approved therapeutics or vaccines currently available for treatment of VEEV infection. Our research over the past two years have uncovered that host kinases play an important role in the establishment of a productive infection in VEEV infected cells. Inhibition of host kinase activity by small molecule inhibitors decreases viral load and improves host survival in vitro and in vivo. We have identified that the viral protein nsP3 interacted with a host kinase (IKKwhich prompted us to consider the phosphorylation status of nsP3 in infected cells. Our analysis revealed multiple phosphorylated residues on nsP3, many of which were susceptible to our small molecule inhibitor treatment. We have also determined that in addition to IKK nsP3 interacted with DDX-1 and DDX-3 in infected cells. An unmet need is a better understanding of the phosphorylation events that occur on nsP3 and the impact of those events on the protein:protein interactions involving nsP3 and host proteins. Understanding the importance of nsP3 phosphorylation to viral multiplication will pave the way for effective therapeutics and rationally designed vaccines. Our hypothesis is that nsP3 will be phosphorylated on multiple residues in infected cells and at least some of these phosphorylation events will be critical to viral multiplication. In addition, the phosphorylation status of nsP3 will impact its ability to interact with the host proteins in the infected cell. As a consequence, when target phosphorylation sites are mutated in the virus, the virulence of the virus will be reduced in vivo. We will test our hypothesis with the following specific aims: Aim 1. Phosphorylation of nsP3 in infected cells. Aim 2. Interaction of nsP3 with host proteins in infected cells.

摘要 委内瑞拉马脑炎病毒(VEEV)是一种甲型病毒，被归类为B类选择因子 以及一种新的感染性病原体。目前还没有FDA批准的疗法或疫苗可用于 VEEV感染的治疗。我们在过去两年的研究发现，宿主激酶在 在VEEV感染细胞中建立生产性感染的重要作用。对宿主激酶的抑制 小分子抑制剂的活性降低了病毒载量，提高了体内和体外宿主的存活率。我们 已经确定病毒蛋白NSP3与宿主激酶(IKK)相互作用，这促使我们考虑 感染细胞中NSP3的磷酸化状态。我们的分析揭示了多个磷酸化残基 NSP3，其中许多对我们的小分子抑制剂治疗敏感。我们还确定， 在感染细胞中，除IKK外，NSP3还与DDX-1和DDX-3相互作用。未得到满足的需求是更好的 了解NSP3上发生的磷酸化事件以及这些事件对 蛋白质：涉及NSP3和宿主蛋白的蛋白质相互作用。理解NSP3的重要性 对病毒增殖的磷酸化将为有效的治疗和合理的设计铺平道路 疫苗。我们的假设是，NSP3将在感染细胞中的多个残基上被磷酸化，至少 其中一些磷酸化事件将是病毒增殖的关键。此外，磷酸化 NSP3的状态将影响其与感染细胞中的宿主蛋白相互作用的能力。因此， 当病毒中的目标磷酸化位点发生突变时，病毒在体内的毒力将会降低。 我们将通过以下具体目标来验证我们的假设： 目的1.感染细胞中NSP3的磷酸化。 目的2.感染细胞中NSP3与宿主蛋白的相互作用。