Necroptosis in SARS-CoV-2 pathogenesis, evolution, and therapy

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PROJECT SUMMARY/ABSTRACT Our laboratory has recently implicated necroptosis as a pathogenic and targetable host pathway during pulmonary influenza A virus (IAV) infections. In this proposal, we seek to extend these findings to SARS-CoV-2 because we have strong reason to believe that SARS-CoV-2, like IAV, activates necroptosis. We have identified a mechanism by which SARS-CoV-2 may trigger necroptosis, and propose that such necroptosis underlies the alveolar cell death and inflammatory ‘cytokine storm’ observed in severe COVID-19 disease. Importantly, necroptosis can be targeted by dedicated RIPK3 kinase inhibitors, opening up a new and unanticipated therapeutic entry-point for COVID-19. Specifically, we have discovered that a SARS-CoV-2 nonstructural protein contains a functional RHIM motif that is essential for propagating necroptosis signaling. In all known cell types, necroptosis is initiated when the kinase RIPK3 engages in RHIM-RHIM interactions with other RHIM-containing proteins. For example, during IAV infection, the RHIM in RIPK3 interacts with the RHIM in the IAV sensor protein ZBP1 to trigger necroptosis. We thus hypothesized that the RHIM in the CoV-2 protein allows it to interact with RIPK3 to activate necroptosis. Indeed, we found that the SARS-CoV-2 protein engages RIPK3 and activates necroptosis in human cells. The precise mechanism responsible remains unknown. We have also found that all three pathogenic CoVs (SARS-CoV, MERS-CoV, and SARS-CoV-2) have a RHIM in this protein, whereas none of the human-adapted strains (HKU-1, CO43, NL63, and 229E) possess one. Finally, we have found that bats, the likely natural hosts of SARS-CoV-2 and other pathogenic CoVs, encode a variant of RIPK3 which contains a single amino acid change from non-bat RIPK3. This change significantly dampens necroptosis signaling, suggesting that the necroptosis machinery is defective or non-functional in bats. Based on these and other observations, we hypothesize that SARS-CoV-2 and allied pathogenic CoVs activate necroptosis in human pulmonary epithelia, via a RHIM-RHIM interaction involving the CoV-2 RHIM-containing protein and RIPK3, and that such necroptosis initiates and amplifies the lung injury and inflammation seen in severe cases of COVID- 19. We further propose that dampened necroptosis signaling in bats allows them to harbor pathogenic (to humans) CoVs without apparent hyper-inflammatory consequences. In this proposal, we will examine how SARS-CoV activates necroptosis in human cells, and if such necroptosis is a new therapeutic opportunity in vivo by evaluating FDA-approved and new, high potency RIPK3 inhibitors in a mouse model of SARS-CoV-2 infection. We have also developed a knock-in mouse harboring the bat RIPK3 polymorphism, and will test if SARS-CoV-2-initiated lung pathology is dampened in this mouse, compared to controls. The successful completion of these studies will provide pioneering insight into the mechanism and evolutionary biology of necroptosis signaling in SARS-CoV-2 pathogenesis and stand to have important ramifications for the treatment of severe COVID-19.