Exploiting Pathogen-Encoded Immune Evasion Proteins to Uncover Evolutionarily Conserved Antiviral Host Machinery

Project Summary The activation of innate antiviral responses after viral infection is critical for the restriction of viral pathogens by eukaryotic hosts. However, viral immune evasion proteins (IEPs) can subvert these host responses, allowing viral pathogens to replicate and cause disease. Thus, identifying and characterizing virus- host interactions within this evolutionary "arms race" is critical for understanding how hosts combat virus infection and the mechanisms viral pathogens use to counter host defenses. Moreover, viral IEPs can be used as tools to discover and probe the cellular machinery they target, thereby revealing fundamental aspects of host biology. We developed innovative approaches to identify viral IEPs that target conserved eukaryotic antiviral machinery and that are capable of tipping the balance between abortive and productive viral infection. We exploit naturally abortive arbovirus infections in lepidopteran (moth and butterfly) cells as a screening tool to identify novel IEPs encoded by mammalian viruses that convert these abortive infections to productive infections by countering host immune responses. By identifying IEPs encoded by mammalian viruses that are immunosuppressive in insect cells, we select for IEPs that target antiviral responses conserved between insect and mammalian hosts. Using this approach, we identified mammalian poxvirus-encoded A51R proteins as novel IEPs that target a previously unappreciated "FACT-ETS-1 Antiviral Response (FEAR)" pathway. This pathway requires the evolutionarily- conserved "FACT" histone chaperone complex and ETS-1 transcription factor, which activate antiviral gene expression programs in cells to block viral replication. Here, we seek to understand how this pathway is both activated and countered by viruses and to identify the FEAR pathway-induced factors required for virus restriction. Our preliminary data suggest that host sensors of pathogen-associated molecular patterns are required for FEAR pathway activation and, using transcriptomics, we identify a core set of FEAR pathway- induced host factors, some of which display antiviral properties. In addition, we identified new FEAR pathway antagonists from disparate mammalian viruses, suggesting this pathway is relevant to a wide array of viral pathogens. Our data suggest these antagonists inhibit the FEAR pathway through multiple, distinct mechanisms. Thus, we aim to characterize these antagonists and use them to interrogate FEAR pathway activation/regulation and to reveal new FEAR pathway factors. Finally, we will further exploit our arbovirus-lepidopteran host system to identify other viral IEPs targeting conserved host factors by screening a newly constructed mammalian virus ORFeome library. Our initial screens have already identified several putative viral IEPs that relieve arbovirus restriction in insect cells, suggesting they target defenses shared between insects and mammals. Our ultimate goal is to identify novel IEPs encoded by mammalian viruses in our unique system and use them to provide mechanistic insights into the antiviral (and normal) functions of the conserved host machinery they manipulate.