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

RNA病毒通常存在于野生动物物种中，如甲型流感（鸭子、滨鸟）、埃博拉病毒（果蝠）、寨卡病毒（非人灵长类动物）和sars相关冠状病毒（食虫蝙蝠），这些病毒引起的意外疾病流行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。尽管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名为杰里米角病毒（JPTV）的新发现的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（病毒捕获的趋化因子；ORF6），其特征是与白细胞介素-8 （IL-8）相似的ELR+ CXC趋化因子同源物，具有完全的功能，并将免疫细胞招募到感染部位。值得注意的是，ORF6在复制过程中也被复制，现在正在进化成一个具有替代功能的新基因（ORF5）。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引起疾病。在Specific Aim 1中，我们将通过反向遗传学、高级免疫细胞微流体和交联质谱（XL-MS）研究JPTV病毒因子和ORF5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，证明其具有趋化活性。使用这种纯化蛋白，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，以确定它是否可以作为激动剂（即与免疫细胞受体结合并触发迁移）和/或拮抗剂（即与受体结合但不触发迁移，从而阻断其他趋化因子激动剂的作用）作用于来自不同宿主的各种免疫细胞，以及执行xml - ms来识别病毒因子的推定结合伙伴。最后，我们最近构建了一个JPTV的反向遗传系统，我们将使用该系统来研究携带或不携带宿主捕获基因的突变病毒的生物学特性。在Specific Aim 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，如有必要，我们也可以使用核磁共振波谱等替代结构方法。重要的是，JPTV病毒因子（ORF6）的结构测定也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，我们也将以蛋白质结晶为目标。据我们所知，盗取的宿主基因与其副本（已进化成不同的蛋白质）之间的这种结构比较尚未进行，因此将为RNA病毒进化提供以前从未观察到的新见解。

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.