Reovirus Z-RNAs activate ZBP1 dependent necroptosis

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ABSTRACT Reovirus (ReoV) is an enteric dsRNA virus which can infect a wide range of mammals and induce intestinal and autoimmune diseases due to the cell death elicited during infection. The mechanisms by which ReoV induces such cell death are unclear. For decades ReoV was thought to cause mainly apoptosis in infected cells. Recently, the Danthi lab has shown that ReoV can also activate necroptosis, a caspase-independent form of inflammatory programmed cell death reliant on the kinase RIPK3 and its substrate MLKL. Necroptosis is highly immunogenic because it results in the release of damage-associated molecular patterns (DAMPs), pathogen-associated molecular patterns (PAMPs), and intracellular cytokines, whereas apoptosis is more immunologically ‘silent’. The mechanisms by which both necroptosis and apoptosis are activated during ReoV infection, as well as the immunological consequences of this activation, are largely unknown. I have found that the innate immune sensor Z-DNA Binding Protein 1 (ZBP1) initiates both apoptosis and necroptosis during ReoV infections. I have also discovered that ReoV generates Z-RNA, the left-handed conformation of double-helical (ds)RNA, in infected cells. Additionally, I have found that ZBP1 is modified by K63-mediated polyubiquitin linkages during ReoV infections. Together, these new findings allow us to hypothesize that (1) ReoV produces cytoplasmic Z-RNAs, which are activating ligands for ZBP1; (2) ZBP1 ubiquitination is essential for ZBP1 activation, engagement of RIPK3, and execution of both apoptosis and necroptosis downstream of RIPK3; and (3) ZBP1-triggered necroptosis drives virus pathogenesis by both promoting virus release and inducing detrimental inflammation in the gut. In this proposal, we will test these hypotheses by identifying the ReoV-generated Z-RNA ligands for ZBP1 (Aim 1.1), determining the mechanism and contribution of K63 ubiquitination to ZBP1 activation (Aim 1.2), and investigating the role ZBP1-activated to host defense and pathogenesis during ReoV infections in vivo, distinguishing between the contributions of apoptosis and necroptosis to these outcomes (Aim 2). We will also delete ZBP1 and its downstream pathway effectors in intestinal epithelial cells, the primary ReoV targets in the gut, to determine the selective role of ZBP1-triggered cell death to ReoV spread and pathogenesis.