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

• 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
• 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

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.

通常在野生动物物种中存在的RNA病毒引起的意外疾病流行，如甲型流感（鸭，滨鸟），埃博拉病毒（果蝠），寨卡病毒（非人类灵长类动物）和SARS相关的冠状病毒（食虫蝙蝠）已成为日益严重的全球健康威胁。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。虽然RNA病毒已被证明利用许多不同的免疫操纵机制，但获得宿主基因（如趋化因子）-正如我们最近在一种新发现的名为杰里米点病毒（JPTV）的RNA病毒中所证明的那样-是一种极其罕见的事件。我们的中心假设是，这种病毒因子（病毒捕获趋化因子; ORF 6）-特征为ELR+ CXC趋化因子同系物类似于白细胞介素-8（IL-8）-是完全功能性的，并招募免疫细胞到感染部位。值得注意的是，ORF 6在复制过程中也被复制，现在正在进化成具有替代功能的新基因（ORF 5）。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的免疫系统并导致疾病。在具体目标1中，我们将通过反向遗传学，先进的免疫细胞微流体技术和交联质谱（XL-MS）研究JPTV病毒因子和ORF 5的功能。我们已经克隆、表达并通过亲和层析纯化了JPTV病毒因子，并证明其具有趋化活性。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化学引诱能力，以确定它是否可以作为激动剂（即，结合免疫细胞受体并触发迁移）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的作用）至来自不同宿主的各种免疫细胞，以及进行XL-MS以鉴定病毒因子的推定结合配偶体。最后，我们最近构建了一个反向遗传学系统的JPTV，我们将使用它来研究突变病毒的生物学特性与或没有宿主捕获的基因。在具体目标2中，我们将通过X射线晶体学确定JPTV病毒因子与脊椎动物ELR+ CXC趋化因子的结构关系，如果需要，我们还可以使用替代结构方法，如核磁共振光谱。重要的是，JPTV病毒因子（ORF 6）的结构测定也将提供我们与其随后的复制事件（ORF 5）直接比较的目标的第一步，我们也将靶向蛋白质结晶。据我们所知，这种被盗的宿主基因和它的复制品（已经进化成不同的蛋白质）之间的结构比较还没有进行，因此将提供一个新的见解RNA病毒进化，以前从未观察到。