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.