RNA virus capture of host chemokines: Understanding novel viral mechanisms of immune manipulation

RNA病毒捕获宿主趋化因子：了解免疫操纵的新病毒机制

基本信息

批准号：
10452788
负责人：
Andrew Brownell Allison
金额：
$ 24.1万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-14 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10452788
关键词：
2019-nCoV Affect Affinity Chromatography Agonist Animals Antigenic Variation Binding Biological Biological Models Biological Process Birds COVID-19 pandemic CXC Chemokines Cell Culture System Cell Death Cells Cessation of life Chemotactic Factors Chemotaxis Chiroptera Chromatography Coupled Crystallization DNA-Directed RNA Polymerase Data Disease Double Stranded DNA Virus Ducks Ebola Economics Epidemic Event Evolution Family Filovirus Fright Fruit GTP-Binding Proteins Gene Duplication Genes Genetic Recombination Genome Goals HIV Herpesviridae Homologous Gene IL8RA gene Immune Immune response Immune system Immunity Infection Inflammation Influenza A virus Interleukin-8 Interleukin-8B Receptor Leukocytes Lymphocyte Mammalian Cell Mass Spectrum Analysis Membrane Proteins Methods Microfluidics Mononegavirales Mutation NMR Spectroscopy Names Panic Pathogenesis Pathogenicity Play Population Poxviridae Property Proteins Psychological Impact Public Health RNA Viruses Receptor Cell Research Respiratory syncytial virus Risk Role SARS coronavirus Site Social Impacts Structure Structure-Activity Relationship Suggestion System Time Viral Viral Pathogenesis Virus Virus Diseases X-Ray Crystallography ZIKA Zoonoses antagonist base chemokine cost crosslink cytokine experimental study global health insight migration mutant neutralizing antibody nonhuman primate novel overexpression receptor recruit reverse genetics tat Protein transcriptome sequencing

项目摘要

Unanticipated epidemics of disease caused by RNA viruses that are normally maintained in wildlife species, such as influenza A (ducks, shorebirds), Ebola (fruit bats), Zika (non-human primates), and SARS-related coronaviruses (insectivorous bats) have become an increasing global health threat. One important property of all RNA viruses is their ability to subvert the host immune response. Although RNA viruses have been shown to utilize many divergent mechanisms of immune manipulation, the acquisition of host genes such as chemokines – as we have recently demonstrated in a newly discovered RNA virus named Jeremy Point virus (JPTV) – is an exceedingly rare event. Our central hypothesis is that this virokine (virus-captured chemokine; ORF6) – characterized as an ELR+ CXC chemokine homologue similar to interleukin-8 (IL-8) – is fully functional and recruits immune cells to sites of infection. Remarkably, ORF6 was also duplicated during replication and is now evolving into a new gene (ORF5) with an alternate function. The primary goal of this project is to better understand how RNA viruses use gene capture to subvert the vertebrate immune system and cause disease. In Specific Aim 1, we will investigate the function of the JPTV virokine and ORF5 through reverse genetics, advanced immune cell microfluidics, and cross-linking mass spectrometry (XL-MS). We have already cloned, expressed and purified the JPTV virokine by affinity chromatography and demonstrated it has chemotactic activity. Using this purified protein, we will further assess its chemoattractant capabilities by immune cell-based microfluidics to determine if it may act either as an agonist (i.e., bind to immune cell receptors and trigger migration) and/or antagonist (i.e., bind to receptors without triggering migration and thus block the effects of other chemokine agonists) to various immune cells from different hosts, as well as perform XL-MS to identify putative binding partners of the virokine. Lastly, we have recently constructed a reverse genetics system for JPTV, which we will use to investigate the biological properties of mutant viruses with or without the host captured genes. In Specific Aim 2, we will determine the structural relationships of the JPTV virokine to vertebrate ELR+ CXC chemokines by X-ray crystallography and, if necessary, we can also use alternative structural methods such as nuclear magnetic resonance spectroscopy. Importantly, structural determination of the JPTV virokine (ORF6) will also provide the first step in our goal of direct comparison with its subsequent duplication event (ORF5), which we will also target for protein crystallization. To our knowledge, such structural comparisons between a stolen host gene and its duplicate (that has evolved into a different protein) has not been performed and thus would provide a new insight into RNA virus evolution that has never been previously observed.

由野生动物物种中通常存在的 RNA 病毒引起的疾病意外流行，例如甲型流感（鸭子、滨鸟）、埃博拉病毒（果蝠）、寨卡病毒（非人类灵长类动物）和 SARS 相关冠状病毒（食虫蝙蝠），所有RNA病毒的一个重要特性是它们破坏宿主免疫反应的能力，成为日益严重的全球健康威胁，尽管RNA病毒已被证明利用许多不同的免疫操纵机制，例如获得宿主基因。正如我们最近在一种新发现的名为 Jeremy Point 病毒 (JPTV) 的 RNA 病毒中所证明的，趋化因子是一种极其罕见的事件，我们的中心假设是这种病毒因子（病毒捕获的趋化因子；ORF6）——被定性为 ELR+ CXC 趋化因子同源物。与白细胞介素 8 (IL-8) 类似，功能齐全，可将免疫细胞募集到感染部位。值得注意的是，ORF6 在复制过程中也被复制，现在正在进化为一种新基因。该项目的主要目标是更好地了解 RNA 病毒如何利用基因捕获来破坏脊椎动物免疫系统并引起疾病。在具体目标 1 中，我们将研究 JPTV 病毒因子和 ORF5 的功能。通过反向遗传学、先进的免疫细胞微流体和交联质谱（XL-MS），我们已经通过亲和层析克隆、表达和纯化了JPTV病毒因子并进行了证明。使用这种纯化的蛋白质，我们将通过基于免疫细胞的微流体进一步评估其趋化能力，以确定它是否可以充当激动剂（即与免疫细胞受体结合并触发迁移）和/或拮抗剂（即与受体结合而不触发迁移，从而阻断其他趋化因子激动剂对来自不同宿主的各种免疫细胞的影响，以及进行 XL-MS 来识别假定的结合伙伴最后，我们最近构建了 JPTV 的反向遗传学系统，我们将用它来研究有或没有宿主捕获基因的突变病毒的生物学特性，在具体目标 2 中，我们将确定 JPTV 病毒因子的结构关系。通过 X 射线晶体学分析脊椎动物 ELR+ CXC 趋化因子，如有必要，我们还可以使用替代结构方法，例如核磁共振波谱法。重要的是，JPTV 的结构测定。 virokine (ORF6) 还将为我们与其后续复制事件 (ORF5) 进行直接比较的目标提供第一步，我们也将针对该复制事件进行蛋白质结晶，据我们所知，被盗宿主基因与其复制品之间的这种结构比较（已经进化成不同的蛋白质）尚未被执行，因此将为 RNA 病毒进化提供以前从未观察到的新见解。