Z-DNA and Z-RNA are left-handed double helix nucleic acid structures with poorly understood biological function 1 – 3 . Z-DNA binding protein 1 (ZBP1, also known as DAI or DLM-1) is a nucleic acid sensor containing two Zα domains that bind Z-DNA 4 , 5 and Z-RNA 6 – 8 . ZBP1 mediates host-defence against certain viruses 6 , 7 , 9 – 14 by sensing viral nucleic acids 6 , 7 , 10 . RIPK1 deficiency or mutation of its RIP homotypic interaction motif (RHIM) triggers ZBP1-dependent necroptosis and inflammation in mice 15 , 16 , however, the mechanisms inducing ZBP1 activation in the absence of viral infection remain elusive. Here we show that Zα-dependent sensing of endogenous ligands induces ZBP1-mediated perinatal lethality in mice expressing RIPK1 with mutated RHIM ( Ripk1 mR/mR ) and skin inflammation in mice with epidermis-specific RIPK1 deficiency (RIPK1 E-KO ), as well as colitis in mice with intestinal epithelial-specific FADD deficiency (FADD IEC-KO ). Consistently, functional Zα domains were required for ZBP1-induced necroptosis in fibroblasts that express RIPK1 with mutated RHIM or were treated with caspase inhibitors. Moreover, inhibition of nuclear export triggered Zα-dependent activation of RIPK3 in the nucleus resulting in cell death, suggesting that ZBP1 may recognise Z-form nucleic acids (Z-NA) in the nucleus. We found that ZBP1 constitutively bound cellular double stranded RNA (dsRNA) in a Zα-dependent manner. Furthermore, endogenous retroelement (ERE)-derived complementary reads were detected in epidermal RNA, suggesting that ERE-derived dsRNA may act as Zα domain ligand triggering ZBP1 activation. Collectively, our results provide evidence that sensing of endogenous Z-NA by ZBP1 triggers RIPK3-dependent necroptosis and inflammation, which could underlie the development of chronic inflammatory conditions particularly in patients with mutations in the RIPK1 and CASPASE-8 genes 17 – 20 .

The Linear Ubiquitin chain Assembly Complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR11. Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, due to deregulation of TNFR1-mediated cell death2–8. In humans, deficiency in the third LUBAC component, HOIL-1, causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype9–11. By creating HOIL-1-deficient mice, we here show that HOIL-1 is, however, as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is LUBAC’s catalytically active component, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1-signalling complex (TNFR1-SC), thereby preventing aberrant cell death. Both, HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of Caspase-8 with RIPK3 or MLKL prevents cell death in Hoil-1-/- embryos, yet only combined loss of Caspase-8 with MLKL results in viable HOIL-1-deficient mice. Interestingly, Ripk3-/-Caspase-8-/-Hoil-1-/- embryos die at late-gestation due to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both, HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they unveil that, when LUBAC and Caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in foetal haematopoiesis.

Necroptosis complements apoptosis as a host defense pathway to stop virus infection. Herpes simplex virus shows a propensity to trigger necroptosis of mouse cells and mice even though cell death is blocked in human cells through UL39-encoded ICP6. This ribonucleotide reductase large subunit (R1) nucleates RHIM-dependent oligomerization of RIP3 kinase (RIPK3, also known as RIP3) in mouse cells but inhibits activation in cells from the natural human host. By interrogating the comparative behavior of ICP6-deficient viruses in mouse and human cells, here we unveil virus-induced necroptosis mediated by Z-DNA-binding protein 1 (ZBP1, also known as DAI). ZBP1 acts as a pathogen sensor to detect nascent RNA transcripts rather than input viral DNA or viral DNA generated through replication. Consistent with the implicated role of virus-induced necroptosis in restricting infection, viral pathogenesis is restored in Zbp1−/−, Ripk3−/− and Mlkl−/− mice. Thus, in addition to direct activation of RIPK3 via ICP6, HSV1 infection in mice and mouse cells triggers virus-induced necroptosis through ZBP1. Importantly, virus-induced necroptosis is also induced in human HT-29 cells by ICP6 mutant viruses; however, ZBP1 levels must be elevated for this pathway to be active. Thus, our studies reveal a common, species-independent role of this nucleic acid sensor to detect the presence of this virus. HSV1 ICP6 functions as a bona fide RHIM signaling inhibitor to block virus-induced necroptosis in its natural host. Altogether, ZBP1-dependent restriction of herpesvirus infection emerges as a potent antiviral armament of the innate immune system.

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Receptor-interacting protein kinase 1 (RIPK1) regulates cell fate and proinflammatory signaling downstream of multiple innate immune pathways, including those initiated by TNF-α, TLR ligands, and IFNs. Genetic ablation of Ripk1 results in perinatal lethality arising from both RIPK3-mediated necroptosis and FADD/caspase-8–driven apoptosis. IFNs are thought to contribute to the lethality of Ripk1-deficient mice by activating inopportune cell death during parturition, but how IFNs activate cell death in the absence of RIPK1 is not understood. In this study, we show that Z-form nucleic acid binding protein 1 (ZBP1; also known as DAI) drives IFN-stimulated cell death in settings of RIPK1 deficiency. IFN-activated Jak/STAT signaling induces robust expression of ZBP1, which complexes with RIPK3 in the absence of RIPK1 to trigger RIPK3-driven pathways of caspase-8–mediated apoptosis and MLKL-driven necroptosis. In vivo, deletion of either Zbp1 or core IFN signaling components prolong viability of Ripk1−/− mice for up to 3 mo beyond parturition. Together, these studies implicate ZBP1 as the dominant activator of IFN-driven RIPK3 activation and perinatal lethality in the absence of RIPK1.

Herpesviruses are ubiquitous human pathogens that cause a wide range of health complications. Currently, there is an incomplete understanding of cellular factors that contribute to herpesvirus infection. Here, we report an antiviral necroptosis-based genetic screen to identify novel host cell factors required for infection with the β-herpesvirus murine cytomegalovirus (MCMV). Our genome-wide CRISPR-based screen harnessed the capacity of herpesvirus mutants that trigger antiviral necroptotic cell death upon early viral gene expression. Vascular endothelial growth factor (VEGF) and semaphorin-binding receptor Neuropilin-1 (Nrp-1) emerge as crucial determinants of MCMV infection. We find that elimination of Nrp-1 impairs early viral gene expression and reduces infection rates in endothelial cells, fibroblasts, and macrophages. Furthermore, preincubation of virus with soluble Nrp-1 dramatically inhibits infection by reducing virus attachment. Thus, Nrp-1 is a key determinant of the initial phase of MCMV infection.

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A progressive decline in islet function is a major obstacle to the success of islet transplantation. The cause of this decline in islet function is unclear, but immunosuppressive agents may contribute. Insulin-like growth factor-I (IGF-I) and betacellulin are important for islet cell survival and/or proliferation. In the present study, we performed studies in INS-1 cells and murine islets to define the effect of IGF-I and betacellulin on progression of islet cells through the cell cycle and the impact of immunosuppressive agents. Treatment of INS-1 cells for 24 hrs with 20 ng/ml betacellulin or 50 ng/ml IGF-I increased cells in S phase by ~2-fold. Treatment of INS-1 cells with IGF-I or betacellulin also increased cyclin D1 expression and nuclear exclusion of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1. In INS-1 cells and islets, betacellulin- and IGF-I increased Akt, ERK, and p70S6 kinase phosphorylation. Rapamycin, an immunosuppressant which inhibits mTOR, inhibited the increase in p70S6 kinase phosphorylation stimulated by betacellulin- and IGF-I in INS-1 cells. Rapamycin also inhibited betacellulin- and IGF-I-induced entry of cells into S phase and BrdU incorporation as well as the effect of betacellulin and IGF-I on cyclin D1 expression and nuclear exclusion of p21Cip1 and p27Kip1. Together, these data suggest that the effect of betacellulin and IGF-I on islet cell growth and proliferation is mediated, in part, via signaling through mTOR. As rapamycin is used to treat islet transplant recipients, these results suggest that rapamycin could have deleterious effects on islet proliferation and function over time.