Apoptosis and necroptosis are complementary pathways controlled by common signaling adaptors, kinases and proteases; among these, caspase-8 (Casp8) is critical for death receptor (DR)-induced apoptosis. This caspase has also been implicated in nonapoptotic pathways that regulate Fas-associated via death domain (FADD)-dependent signaling and other less defined biological processes as diverse as innate immune signaling and myeloid or lymphoid differentiation patterns 1. Casp8 suppresses RIP3/RIP1 kinase complex-dependent 2–4 necroptosis 5 that follows DR-activation as well as a RIP3-dependent, RIP1-independent necrotic pathway that has emerged as a host defense mechanism against murine cytomegalovirus (MCMV) 6. Disruption of Casp8 expression leads to embryonic lethality in mice between E10.5 and E11.5 7. Thus, Casp8 may naturally hold alternative RIP3-dependent death pathways in check in addition to its role promoting apoptosis. We find that RIP3 is responsible for the midgestational death of Casp8-deficient embryos. Remarkably, Casp8−/−Rip3−/− double mutant mice are viable and mature into fertile adults with a full immune complement of myeloid and lymphoid cell types. These mice appear immunocompetent but develop lymphadenopathy by four months of age marked by accumulation of abnormal T cells in the periphery, a phenotype reminiscent of mice with Fas-deficiency (lpr/lpr). Casp8 contributes to homeostatic control in the adult immune system; however, RIP3 and Casp8 are together completely dispensable for mammalian development.

Background: RIP3-dependent programmed necrosis is an alternative to apoptosis. Results: When caspase-8 is compromised, TRIF-dependent TLRs directly activate RIP3 kinase through RHIM-dependent interactions. Conclusion: TRIF mediates direct RHIM-dependent signaling, triggering necrosis via RIP3 and MLKL. Significance: Programmed necrosis eliminates cells following stimulation of either MyD88 or TRIF signaling pathways that converge on RIP3.

Summary Viral pathogenesis relies upon modulation of host cytokine activation as well as cell death pathways. Infection by murine cytomegalovirus induces a novel receptor-interacting protein (RIP)3-dependent necrosis. RIP3 kinase activity and homotypic interaction motif (RHIM)-dependent interactions control virus-associated necrosis as occurs in TNFα-induced necroptosis; however, the virus-induced death pathway proceeds independent of RIP1, and is therefore distinct from the TNFα-dependent death pathway. The viral inhibitor of RIP activation (vIRA, encoded by the viral M45 gene) suppresses either pathway, disrupting RHIM-dependent RIP3-RIP1 interaction that is critical for TNFα-induced necroptosis as well as a RIP3 RHIM-dependent step in virus-induced necrosis. Importantly, the attenuation of vIRA-deficient virus in wild type mice is completely normalized in a RIP3-deficient genetic background. Thus, vIRA function validates necrosis as central to the elimination of infected cells in host defense and highlights the benefit of multiple virus-encoded cell death suppressors that subvert not only apoptotic, but also necrotic mechanisms of virus clearance.

Summary Programmed necrosis, like apoptosis, eliminates pathogen infected cells as a component of host defense. Receptor interacting protein kinase (RIP) 3 (also called RIPK3) mediates programmed necrosis via RIP homotypic interaction motif (RHIM)-dependent interactions, which is induced by murine cytomegalovirus (MCMV) infection or death receptor activation and is suppressed by the MCMV-encoded viral inhibitor of RIP activation (vIRA). We find that interferon-independent expression of DNA-dependent activator of interferon regulatory factors (DAI; also known as ZBP1 or DLM-1), sensitizes cells to virus-induced necrosis and DAI knockdown or knockout cells are resistant to this death pathway. Importantly, as with RIP3−/− mice, vIRA mutant MCMV pathogenesis is restored in DAI−/− mice, consistent with a DAI-RIP3 complex being the natural target of vIRA. Thus, DAI interacts with RIP3 to mediate virus-induced necrosis analogous to the RIP1-RIP3 complex controlling death receptor-induced necroptosis. These studies unveil a role for DAI as the RIP3 partner mediating virus-induced necrosis.

Summary Receptor interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pro-necrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound; whereas, D161G, D143N, and K51A mutants only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3K51A/K51A) are viable and fertile, in stark contrast to the perinatal lethality of Rip3D161N/D161N mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.

Summary Influenza A virus (IAV) is an RNA virus that is cytotoxic to most cell types in which it replicates. IAV activates the host kinase RIPK3, which induces cell death via parallel pathways of necroptosis, driven by the pseudokinase MLKL, and apoptosis, dependent on the adaptor proteins RIPK1 and FADD. How IAV activates RIPK3 remains unknown. We report that DAI (ZBP-1/DLM-1), previously implicated as a cytoplasmic DNA sensor, is essential for RIPK3 activation by IAV. Upon infection, DAI recognizes IAV genomic RNA, associates with RIPK3, and is required for recruitment of MLKL and RIPK1 to RIPK3. Cells lacking DAI or containing DAI mutants deficient in nucleic acid binding are resistant to IAV-triggered necroptosis and apoptosis. DAI-deficient mice fail to control IAV replication and succumb to lethal respiratory infection. These results identify DAI as a link between IAV replication and RIPK3 activation, and implicate DAI as a sensor of RNA viruses.

Highlights d Replicating influenza A virus (IAV) produces Z-RNAs d IAV Z-RNAs are sensed by host ZBP1 in the nucleus d ZBP1 activates MLKL in the nucleus, triggering nuclear envelope rupture d MLKL-induced nuclear rupture and necroptosis drive IAV disease severity Authors

Pathogens specifically target both the caspase 8-dependent apoptotic cell death pathway and the necrotic cell death pathway that is dependent on receptor-interacting protein 1 (RIP1; also known as RIPK1) and RIP3 (also known as RIPK3). The fundamental co-regulation of these two cell death pathways emerged when the midgestational death of mice deficient in FAS-associated death domain protein (FADD) or caspase 8 was reversed by elimination of RIP1 or RIP3, indicating a far more entwined relationship than previously appreciated. Thus, mammals require caspase 8 activity during embryogenesis to suppress the kinases RIP1 and RIP3 as part of the dialogue between two distinct cell death processes that together fulfil reinforcing roles in the host defence against intracellular pathogens such as herpesviruses.