Research Project 3: Role of Posttranslational Protein Modifications in the Pathogenesis of Ebola Virus Disease

RESEARCH PROJECT 3 (RP3): PROJECT SUMMARY/ABSTRACT The epidemic of Ebola virus (EBOV) in Africa in 2013-2016 and its reappearance in subsequent years have reached alarming numbers of infections and deaths, prompting extensive efforts to develop antiviral strategies; however, none have thus far been licensed for use in humans. Pathogenesis of EBOV infection derives from a combination of the viral suppression of the host's antiviral protective immune responses and induction of hyper- inflammation, including a "cytokine storm". Cytokine production and inflammatory responses require activation of multiple signaling pathways that are extensively regulated at the posttranslational level to maintain a balance between efficient antiviral responses and excessive inflammation. Posttranslational modifications (PTMs) of pro- teins serve as molecular regulatory mechanisms, a process which requires the activity of specific enzymes (i.e., kinases, ubiquitin ligases, etc.). Thus, the expression and activation patterns of these enzymes in specific cell types determines the complex regulation that balances an effective immune physiological response. The goal of Research Project 3 (RP3) is to elucidate how EBOV transforms the PTM cell-type specific landscape leading to immune dysregulation and disease, with the long-term goal of applying this knowledge in the development of effective approaches to treat the disease caused by EBOV. Our preliminary data indicate that interferon antagonist domains of EBOV proteins can hijack the host ubiquitin system to increase virus replication while causing cell-type specific dysregulation of signaling pathways that control hyperinflammation. However, it is not known which PTM enzymes or what changes in host protein mod- ifications drive immune dysregulation and disease. Our hypothesis is that EBOV targets specific enzymes that regulate PTM in a cell-type specific manner resulting in uncontrolled inflammation, while also blocking protective innate immune signaling. We will use novel approaches to assess how EBOV infection dysregulates immune signaling by targeting unconventional PTMs, in vitro and in vivo. With information obtained from the BSL-4 Core (Core B), Proteogenomics Core (Core C), and Bioinformatics and Modeling Core (Core D), and from RP1 and RP2 we will build a complete model of inflammatory pathways activated during EBOV infection. In Aim 1 we will characterize the PTM landscape during EBOV infection in primary human immune and nonimmune cells, and in Aim 2 in cells isolated from infected nonhuman primates, using mass spectrometry analysis and validation assays. In Aim 3, we will validate PTM enzymes and their modifications, and elucidate cell-type specific PTM- mediated mechanisms that result in dysregulated immune responses to EBOV. The outcome of these studies is significant because it will provide fundamental knowledge on the function of specific PTMs during infection and uncover molecular targets to treat pathogenic inflammation. Our work on posttranslational regulation together with information derived from changes in the transcriptional (RP1) and post- transcriptional (RP2) landscapes will provide a comprehensive model of immune dysregulation.