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

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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.