A RIPK1-specific PROTAC degrader achieves potent antitumor activity by enhancing immunogenic cell death.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a critical stress sentinel that coordinates cell survival, inflammation, and immunogenic cell death (ICD). Although the catalytic function of RIPK1 is required to trigger cell death, its non-catalytic scaffold function mediates strong pro-survival signaling. Accordingly, cancer cells can hijack RIPK1 to block necroptosis and evade immune detection. We generated a small-molecule proteolysis-targeting chimera (PROTAC) that selectively degraded human and murine RIPK1. PROTAC-mediated depletion of RIPK1 deregulated TNFR1 and TLR3/4 signaling hubs, accentuating the output of NF-κB, MAPK, and IFN signaling. Additionally, RIPK1 degradation simultaneously promoted RIPK3 activation and necroptosis induction. We further demonstrated that RIPK1 degradation enhanced the immunostimulatory effects of radio- and immunotherapy by sensitizing cancer cells to treatment-induced TNF and interferons. This promoted ICD, antitumor immunity, and durable treatment responses. Consequently, targeting RIPK1 by PROTACs emerges as a promising approach to overcome radio- or immunotherapy resistance and enhance anticancer therapies.

Cooperative sensing of mitochondrial DNA by ZBP1 and cGAS promotes cardiotoxicity.

Mitochondrial DNA (mtDNA) is a potent agonist of the innate immune system; however, the exact immunostimulatory features of mtDNA and the kinetics of detection by cytosolic nucleic acid sensors remain poorly defined. Here, we show that mitochondrial genome instability promotes Z-form DNA accumulation. Z-DNA binding protein 1 (ZBP1) stabilizes Z-form mtDNA and nucleates a cytosolic complex containing cGAS, RIPK1, and RIPK3 to sustain STAT1 phosphorylation and type I interferon (IFN-I) signaling. Elevated Z-form mtDNA, ZBP1 expression, and IFN-I signaling are observed in cardiomyocytes after exposure to Doxorubicin, a first-line chemotherapeutic agent that induces frequent cardiotoxicity in cancer patients. Strikingly, mice lacking ZBP1 or IFN-I signaling are protected from Doxorubicin-induced cardiotoxicity. Our findings reveal ZBP1 as a cooperative partner for cGAS that sustains IFN-I responses to mitochondrial genome instability and highlight ZBP1 as a potential target in heart failure and other disorders where mtDNA stress contributes to interferon-related pathology.

Differential effects of CMV infection on the viability of cardiac cells.

Cytomegalovirus (CMV) is a widely prevalent herpesvirus that reaches seroprevalence rates of up to 95% in several parts of the world. The majority of CMV infections are asymptomatic, albeit they have severe detrimental effects on immunocompromised individuals. Congenital CMV infection is a leading cause of developmental abnormalities in the USA. CMV infection is a significant risk factor for cardiovascular diseases in individuals of all ages. Like other herpesviruses, CMV regulates cell death for its replication and establishes and maintains a latent state in the host. Although CMV-mediated regulation of cell death is reported by several groups, it is unknown how CMV infection affects necroptosis and apoptosis in cardiac cells. Here, we infected primary cardiomyocytes, the contractile cells in the heart, and primary cardiac fibroblasts with wild-type and cell-death suppressor deficient mutant CMVs to determine how CMV regulates necroptosis and apoptosis in cardiac cells. Our results reveal that CMV infection prevents TNF-induced necroptosis in cardiomyocytes; however, the opposite phenotype is observed in cardiac fibroblasts. CMV infection also suppresses inflammation, reactive oxygen species (ROS) generation, and apoptosis in cardiomyocytes. Furthermore, CMV infection improves mitochondrial biogenesis and viability in cardiomyocytes. We conclude that CMV infection differentially affects the viability of cardiac cells.

Ubiquitylation of MLKL at lysine 219 positively regulates necroptosis-induced tissue injury and pathogen clearance.

Necroptosis is a lytic, inflammatory form of cell death that not only contributes to pathogen clearance but can also lead to disease pathogenesis. Necroptosis is triggered by RIPK3-mediated phosphorylation of MLKL, which is thought to initiate MLKL oligomerisation, membrane translocation and membrane rupture, although the precise mechanism is incompletely understood. Here, we show that K63-linked ubiquitin chains are attached to MLKL during necroptosis and that ubiquitylation of MLKL at K219 significantly contributes to the cytotoxic potential of phosphorylated MLKL. The K219R MLKL mutation protects animals from necroptosis-induced skin damage and renders cells resistant to pathogen-induced necroptosis. Mechanistically, we show that ubiquitylation of MLKL at K219 is required for higher-order assembly of MLKL at membranes, facilitating its rupture and necroptosis. We demonstrate that K219 ubiquitylation licenses MLKL activity to induce lytic cell death, suggesting that necroptotic clearance of pathogens as well as MLKL-dependent pathologies are influenced by the ubiquitin-signalling system.

Vaccinia virus E3 prevents sensing of Z-RNA to block ZBP1-dependent necroptosis.

Necroptosis mediated by Z-nucleic-acid-binding protein (ZBP)1 (also called DAI or DLM1) contributes to innate host defense against viruses by triggering cell death to eliminate infected cells. During infection, vaccinia virus (VACV) protein E3 prevents death signaling by competing for Z-form RNA through an N-terminal Zα domain. In the absence of this E3 domain, Z-form RNA accumulates during the early phase of VACV infection, triggering ZBP1 to recruit receptor interacting protein kinase (RIPK)3 and execute necroptosis. The C-terminal E3 double-strand RNA-binding domain must be retained to observe accumulation of Z-form RNA and induction of necroptosis. Substitutions of Zα from either ZBP1 or the RNA-editing enzyme double-stranded RNA adenosine deaminase (ADAR)1 yields fully functional E3 capable of suppressing virus-induced necroptosis. Overall, our evidence reveals the importance of Z-form RNA generated during VACV infection as a pathogen-associated molecular pattern (PAMP) unleashing ZBP1/RIPK3/MLKL-dependent necroptosis unless suppressed by viral E3.

Necroptosis-based CRISPR knockout screen reveals Neuropilin-1 as a critical host factor for early stages of murine cytomegalovirus infection.

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 ß-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.

Influenza Virus Z-RNAs Induce ZBP1-Mediated Necroptosis.

Influenza A virus (IAV) is a lytic RNA virus that triggers receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated pathways of apoptosis and mixed lineage kinase domain-like pseudokinase (MLKL)-dependent necroptosis in infected cells. ZBP1 initiates RIPK3-driven cell death by sensing IAV RNA and activating RIPK3. Here, we show that replicating IAV generates Z-RNAs, which activate ZBP1 in the nucleus of infected cells. ZBP1 then initiates RIPK3-mediated MLKL activation in the nucleus, resulting in nuclear envelope disruption, leakage of DNA into the cytosol, and eventual necroptosis. Cell death induced by nuclear MLKL was a potent activator of neutrophils, a cell type known to drive inflammatory pathology in virulent IAV disease. Consequently, MLKL-deficient mice manifest reduced nuclear disruption of lung epithelia, decreased neutrophil recruitment into infected lungs, and increased survival following a lethal dose of IAV. These results implicate Z-RNA as a new pathogen-associated molecular pattern and describe a ZBP1-initiated nucleus-to-plasma membrane "inside-out" death pathway with potentially pathogenic consequences in severe cases of influenza.

Murine cytomegalovirus M72 promotes acute virus replication in vivo and is a substrate of the TRiC/CCT complex.

Betaherpesvirus dUTPase homologs are core herpesvirus proteins, but little is known about their role during infection. Human cytomegalovirus (HCMV) UL72 and murine cytomegalovirus (MCMV) M72 have been designated dUTPase homologs, and previous studies indicate UL72 is dispensable for replication and enzymatically inactive. Here, we report the initial characterization of MCMV M72. M72 does not possess dUTPase activity, and is expressed as a leaky-late gene product with multiple protein isoforms. Importantly, M72 augments MCMV replication in vitro and during the early stage of acute infection in vivo. We identify and confirm interaction of M72 with the eukaryotic chaperonin tailless complex protein -1 (TCP-1) ring complex (TRiC) or chaperonin containing tailless complex polypeptide 1 (CCT). Accumulating biochemical evidence indicates M72 forms homo-oligomers and is a substrate of TRiC/CCT. Taken together, we provide the first evidence of M72's contribution to viral pathogenesis, and identify a novel interaction with the TRiC/CCT complex.

Species-independent contribution of ZBP1/DAI/DLM-1-triggered necroptosis in host defense against HSV1.

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.

Inhibition of DAI-dependent necroptosis by the Z-DNA binding domain of the vaccinia virus innate immune evasion protein, E3.

Vaccinia virus (VACV) encodes an innate immune evasion protein, E3, which contains an N-terminal Z-nucleic acid binding (Zα) domain that is critical for pathogenicity in mice. Here we demonstrate that the N terminus of E3 is necessary to inhibit an IFN-primed virus-induced necroptosis. VACV deleted of the Zα domain of E3 (VACV-E3LΔ83N) induced rapid RIPK3-dependent cell death in IFN-treated L929 cells. Cell death was inhibited by the RIPK3 inhibitor, GSK872, and infection with this mutant virus led to phosphorylation and aggregation of MLKL, the executioner of necroptosis. In 293T cells, induction of necroptosis depended on expression of RIPK3 as well as the host-encoded Zα domain-containing DNA sensor, DAI. VACV-E3LΔ83N is attenuated in vivo, and pathogenicity was restored in either RIPK3- or DAI-deficient mice. These data demonstrate that the N terminus of the VACV E3 protein prevents DAI-mediated induction of necroptosis.

Sensing of viral and endogenous RNA by ZBP1/DAI induces necroptosis.

Nucleic acids are potent triggers for innate immunity. Double-stranded DNA and RNA adopt different helical conformations, including the unusual Z-conformation. Z-DNA/RNA is recognised by Z-binding domains (ZBDs), which are present in proteins implicated in antiviral immunity. These include ZBP1 (also known as DAI or DLM-1), which induces necroptosis, an inflammatory form of cell death. Using reconstitution and knock-in models, we report that mutation of key amino acids involved in Z-DNA/RNA binding in ZBP1's ZBDs prevented necroptosis upon infection with mouse cytomegalovirus. Induction of cell death was cell autonomous and required RNA synthesis but not viral DNA replication. Accordingly, ZBP1 directly bound to RNA via its ZBDs. Intact ZBP1-ZBDs were also required for necroptosis triggered by ectopic expression of ZBP1 and caspase blockade, and ZBP1 cross-linked to endogenous RNA These observations show that Z-RNA may constitute a molecular pattern that induces inflammatory cell death upon sensing by ZBP1.

Murine cytomegalovirus IE3-dependent transcription is required for DAI/ZBP1-mediated necroptosis.

DNA-dependent activator of interferon regulatory factors/Z-DNA binding protein 1 (DAI/ZBP1) is a crucial sensor of necroptotic cell death induced by murine cytomegalovirus (MCMV) in its natural host. Here, we show that viral capsid transport to the nucleus and subsequent viral IE3-dependent early transcription are required for necroptosis. Necroptosis induction does not depend on input virion DNA or newly synthesized viral DNA A putative RNA-binding domain of DAI/ZBP1, Zα2, is required to sense virus and trigger necroptosis. Thus, MCMV IE3-dependent transcription from the viral genome plays a crucial role in activating DAI/ZBP1-dependent necroptosis. This implicates RNA transcripts generated by a large double-stranded DNA virus as a biologically relevant ligand for DAI/ZBP1 during natural viral infection.

RIPK3-driven cell death during virus infections.

The programmed self-destruction of infected cells is a powerful antimicrobial strategy in metazoans. For decades, apoptosis represented the dominant mechanism by which the virus-infected cell was thought to undergo programmed cell death. More recently, however, new mechanisms of cell death have been described that are also key to host defense. One such mechanism in vertebrates is programmed necrosis, or "necroptosis", driven by receptor-interacting protein kinase 3 (RIPK3). Once activated by innate immune stimuli, including virus infections, RIPK3 phosphorylates the mixed lineage kinase domain-like protein (MLKL), which then disrupts cellular membranes to effect necroptosis. Emerging evidence demonstrates that RIPK3 can also mediate apoptosis and regulate inflammasomes. Here, we review studies on the mechanisms by which viruses activate RIPK3 and the pathways engaged by RIPK3 that drive cell death.

DAI Another Way: Necroptotic Control of Viral Infection.

Interrogation of murine cytomegalovirus (MCMV)-encoded cell-death suppressors revealed that necroptosis functions as a trap door to eliminate virally infected cells. This crucial host defense pathway is orchestrated by the sensing of infection by DAI/ZBP-1, engagement of the kinase RIPK3, and subsequent membrane permeablization by the pseudokinase MLKL.

Murine Cytomegalovirus Deubiquitinase Regulates Viral Chemokine Levels To Control Inflammation and Pathogenesis.

Maintaining control over inflammatory processes represents a paradox for viral pathogens. Although many viruses induce host inflammatory responses to facilitate infection, control is necessary to avoid overactivation. One way is through the manipulation of proinflammatory chemokine levels, both host and viral. Murine cytomegalovirus (MCMV), a model betaherpesvirus, encodes a viral C-C chemokine, MCK2, which promotes host inflammatory responses and incorporates into virions to facilitate viral dissemination. Here, we show that the activity of M48, the conserved MCMV deubiquitinating enzyme (DUB), regulates MCK2 levels during infection. Inactivation of M48 DUB activity results in viral attenuation and exacerbates virally induced, MCK2-dependent inflammatory responses. M48 DUB activity also influences MCK2 incorporation into virions. Importantly, attenuation of DUB-mutant virus acute replication in vitro and in vivo is largely ameliorated by targeted deletion of MCK2. Thus, uncontrolled MCK2 levels appear to mediate DUB-mutant virus attenuation in specific tissues or cell types. This demonstrates that MCMV M48 DUB activity plays a previously unappreciated role in controlling MCK2 levels, thereby managing MCK2-dependent processes. These findings reveal a novel intrinsic control mechanism of virally induced inflammation and support the identification of betaherpesvirus DUBs as possible new targets for antiviral therapies. IMPORTANCE: Human cytomegalovirus infections represent a tremendous burden not only to those afflicted but also to health care systems worldwide. As cytomegalovirus infections are a leading cause of nongenetic sensory loss and neurodevelopmental delay, it is imperative that valuable model systems exist in order that we might understand what viral factors contribute to replication and pathogenesis. Currently, the only approved drug treatments against CMV infection are nucleoside analogues, to which some strains have become resistant. Understanding unique viral enzymatic contributions to infections will allow the development of novel pharmacological therapies. Here, we show that M48, the conserved MCMV deubiquitinase, is critical for MCMV replication in mice and demonstrate that attenuation is due to deregulated production of a viral proinflammatory chemokine. The deubiquitinases of both human and murine CMV represent structurally unique DUBs and are therefore attractive targets for pharmacological intervention. Continued research into the substrates of these DUBs will lend additional insight into their potential as targets.

Viral RNA at Two Stages of Reovirus Infection Is Required for the Induction of Necroptosis.

Necroptosis, a regulated form of necrotic cell death, requires the activation of the RIP3 kinase. Here, we identify that infection of host cells with reovirus can result in necroptosis. We find that necroptosis requires sensing of the genomic RNA within incoming virus particles via cytoplasmic RNA sensors to produce type I interferon (IFN). While these events that occur prior to the de novo synthesis of viral RNA are required for the induction of necroptosis, they are not sufficient. The induction of necroptosis also requires late stages of reovirus infection. Specifically, efficient synthesis of double-stranded RNA (dsRNA) within infected cells is required for necroptosis. These data indicate that viral RNA interfaces with host components at two different stages of infection to induce necroptosis. This work provides new molecular details about events in the viral replication cycle that contribute to the induction of necroptosis following infection with an RNA virus.IMPORTANCE An appreciation of how cell death pathways are regulated following viral infection may reveal strategies to limit tissue destruction and prevent the onset of disease. Cell death following virus infection can occur by apoptosis or a regulated form of necrosis known as necroptosis. Apoptotic cells are typically disposed of without activating the immune system. In contrast, necroptotic cells alert the immune system, resulting in inflammation and tissue damage. While apoptosis following virus infection has been extensively investigated, how necroptosis is unleashed following virus infection is understood for only a small group of viruses. Here, using mammalian reovirus, we highlight the molecular mechanism by which infection with a dsRNA virus results in necroptosis.

DAI Senses Influenza A Virus Genomic RNA and Activates RIPK3-Dependent Cell Death.

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 (ZBP1/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.

Enzymatically enhanced collisions on ultramicroelectrodes for specific and rapid detection of individual viruses.

We report the specific collision of a single murine cytomegalovirus (MCMV) on a platinum ultramicroelectrode (UME, radius of 1 µm). Antibody directed against the viral surface protein glycoprotein B functionalized with glucose oxidase (GOx) allowed for specific detection of the virus in solution and a biological sample (urine). The oxidation of ferrocene methanol to ferrocenium methanol was carried out at the electrode surface, and the ferrocenium methanol acted as the cosubstrate to GOx to catalyze the oxidation of glucose to gluconolactone. In the presence of glucose, the incident collision of a GOx-covered virus onto the UME while ferrocene methanol was being oxidized produced stepwise increases in current as observed by amperometry. These current increases were observed due to the feedback loop of ferrocene methanol to the surface of the electrode after GOx reduces ferrocenium methanol back to ferrocene. Negative controls (i) without glucose, (ii) with an irrelevant virus (murine gammaherpesvirus 68), and (iii) without either virus do not display these current increases. Stepwise current decreases were observed for the prior two negative controls and no discrete events were observed for the latter. We further apply this method to the detection of MCMV in urine of infected mice. The method provides for a selective, rapid, and sensitive detection technique based on electrochemical collisions.