Functional genomic characterization of diverse bat innate immune mechanisms

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PROJECT SUMMARY Human health can be inextricably linked to our understanding of bats, as bats are known or predicted reservoirs for the precursors of many zoonotic viruses. Unfortunately, we still know little about bat immunology, and we do not know what characteristics allow them to survive with the same viruses that can readily kill other hosts such as humans. This problem is exacerbated by the fact that bats are an incredibly diverse order of mammals, so what we learn about the immunological mechanisms of one bat species may not be true for another. We need new techniques and technologies to efficiently explore the vast unknown universe of immunological mechanisms that exist across diverse bats. In this application, we propose to develop key tools needed to functionally characterize the genetic underpinnings of bat immunology. In the first aim, we propose to pair modern advances in recombinant DNA synthesis and cell engineering to more comprehensively investigate the function of orthologous genes already proposed to be important in bat immune homeostasis. We will focus our investigations on STING, a key node in the cytoplasmic nucleic acid sensing pathway. We will characterize a dozen diverse STING orthologs across Chiroptera to determine whether the same regulatory mechanisms exist across the order, or if multiple distinct regulatory mechanisms exist. We will narrow down the genetic determinants underlying these mechanisms by developing and applying “chimeric mutational scanning”, where we will create libraries of human-bat STING chimeras to identify the key residues that confer bat-specific regulation to signaling. In the second aim, we will shift our focus to identifying previously unappreciated immune genes and pathways present in bats. We will perform this with an unbiased transposon mutagenesis approach, which will be applied to cell lines from diverse bat species. Selection and sequencing of mutagenized bat cells resistant to the cytopathic effects of Vesicular Stomatitis Virus (VSV) replication, a proxy for rabies virus, will reveal innate immune genes that allow bats to survive infection. The mutagenized cells will also be exposed to VSV-EboGP (VSV pseudotyped with the Ebola virus glycoprotein), mimicking the mechanism used by Ebola virus to enter cells. Thus, with little extra effort, the same library will be used to identify bat innate immune genes that confer resistance to two zoonotic viruses thought to use bats as an animal reservoir. This work will both create the technologies needed to explore and understand the unknown aspects of bat immunity, and generate biological findings directly improving our understanding of bat innate immune mechanisms. These complementary approaches span a wide field of scope, from the functional interrogation of the importances of individual amino acids within known innate immune genes, to the whole genome level to highlight additional genes that should be the subject of focused investigation in the future.