Patch repair protects cells from the small pore-forming toxin aerolysin.

Aerolysin family pore-forming toxins damage the membrane, but membrane repair responses used to resist them, if any, remain controversial. Four proposed membrane repair mechanisms include toxin removal by caveolar endocytosis, clogging by annexins, microvesicle shedding catalyzed by MEK, and patch repair. Which repair mechanism aerolysin triggers is unknown. Membrane repair requires Ca2+, but it is controversial if Ca2+ flux is triggered by aerolysin. Here, we determined Ca2+ influx and repair mechanisms activated by aerolysin. In contrast to what is seen with cholesterol-dependent cytolysins (CDCs), removal of extracellular Ca2+ protected cells from aerolysin. Aerolysin triggered sustained Ca2+ influx. Intracellular Ca2+ chelation increased cell death, indicating that Ca2+-dependent repair pathways were triggered. Caveolar endocytosis failed to protect cells from aerolysin or CDCs. MEK-dependent repair did not protect against aerolysin. Aerolysin triggered slower annexin A6 membrane recruitment compared to CDCs. In contrast to what is seen with CDCs, expression of the patch repair protein dysferlin protected cells from aerolysin. We propose aerolysin triggers a Ca2+-dependent death mechanism that obscures repair, and the primary repair mechanism used to resist aerolysin is patch repair. We conclude that different classes of bacterial toxins trigger distinct repair mechanisms.

ZBP1/DAI Drives RIPK3-Mediated Cell Death Induced by IFNs in the Absence of RIPK1.

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.

RIP1 suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition.

The pronecrotic kinase, receptor interacting protein (RIP1, also called RIPK1) mediates programmed necrosis and, together with its partner, RIP3 (RIPK3), drives midgestational death of caspase 8 (Casp8)-deficient embryos. RIP1 controls a second vital step in mammalian development immediately after birth, the mechanism of which remains unresolved. Rip1(-/-) mice display perinatal lethality, accompanied by gross immune system abnormalities. Here we show that RIP1 K45A (kinase dead) knockin mice develop normally into adulthood, indicating that development does not require RIP1 kinase activity. In the face of complete RIP1 deficiency, cells develop sensitivity to RIP3-mixed lineage kinase domain-like-mediated necroptosis as well as to Casp8-mediated apoptosis activated by diverse innate immune stimuli (e.g., TNF, IFN, double-stranded RNA). When either RIP3 or Casp8 is disrupted in combination with RIP1, the resulting double knockout mice exhibit slightly prolonged survival over RIP1-deficient animals. Surprisingly, triple knockout mice with combined RIP1, RIP3, and Casp8 deficiency develop into viable and fertile adults, with the capacity to produce normal levels of myeloid and lymphoid lineage cells. Despite the combined deficiency, these mice sustain a functional immune system that responds robustly to viral challenge. A single allele of Rip3 is tolerated in Rip1(-/-)Casp8(-/-)Rip3(+/-) mice, contrasting the need to eliminate both alleles of either Rip1 or Rip3 to rescue midgestational death of Casp8-deficient mice. These observations reveal a vital kinase-independent role for RIP1 in preventing pronecrotic as well as proapoptotic signaling events associated with life-threatening innate immune activation at the time of mammalian parturition.

Interferon-induced RIP1/RIP3-mediated necrosis requires PKR and is licensed by FADD and caspases.

Interferons (IFNs) are cytokines with powerful immunomodulatory and antiviral properties, but less is known about how they induce cell death. Here, we show that both type I (α/ß) and type II (γ) IFNs induce precipitous receptor-interacting protein (RIP)1/RIP3 kinase-mediated necrosis when the adaptor protein Fas-associated death domain (FADD) is lost or disabled by phosphorylation, or when caspases (e.g., caspase 8) are inactivated. IFN-induced necrosis proceeds via progressive assembly of a RIP1-RIP3 "necrosome" complex that requires Jak1/STAT1-dependent transcription, but does not need the kinase activity of RIP1. Instead, IFNs transcriptionally activate the RNA-responsive protein kinase PKR, which then interacts with RIP1 to initiate necrosome formation and trigger necrosis. Although IFNs are powerful activators of necrosis when FADD is absent, these cytokines are likely not the dominant inducers of RIP kinase-driven embryonic lethality in FADD-deficient mice. We also identify phosphorylation on serine 191 as a mechanism that disables FADD and collaborates with caspase inactivation to allow IFN-activated necrosis. Collectively, these findings outline a mechanism of IFN-induced RIP kinase-dependent necrotic cell death and identify FADD and caspases as negative regulators of this process.

Identification of STAT2 serine 287 as a novel regulatory phosphorylation site in type I interferon-induced cellular responses.

STAT2 is a positive modulator of the transcriptional response to type I interferons (IFNs). STAT2 acquires transcriptional function by becoming tyrosine phosphorylated and imported to the nucleus following type I IFN receptor activation. Although most STAT proteins become dually phosphorylated on specific tyrosine and serine residues to acquire full transcriptional activity, no serine phosphorylation site in STAT2 has been reported. To find novel phosphorylation sites, mass spectrometry of immunoprecipitated STAT2 was used to identify several phosphorylated residues. Of these, substitution of serine 287 with alanine (S287A) generated a gain-of-function mutant that enhanced the biological effects of IFN-α. S287A-STAT2 increased cell growth inhibition, prolonged protection against vesicular stomatitis virus infection and enhanced transcriptional responses following exposure of cells to IFN-α. In contrast, a phosphomimetic STAT2 mutant (S287D) produced a loss-of-function protein that weakly activated IFN-induced ISGs. Our mechanistic studies suggest that S287A-STAT2 likely mediates its gain-of-function effects by prolonging STAT2/STAT1 dimer activation and retaining it in transcriptionally active complexes with chromatin. Altogether, we have uncovered that in response to type I IFN, STAT2 is serine phosphorylated in the coiled-coil domain that when phosphorylated can negatively regulate the biological activities of type I IFNs.

Inactivation of ribosomal protein L22 promotes transformation by induction of the stemness factor, Lin28B.

Ribosomal protein (RP) mutations in diseases such as 5q- syndrome both disrupt hematopoiesis and increase the risk of developing hematologic malignancy. However, the mechanism by which RP mutations increase cancer risk has remained an important unanswered question. We show here that monoallelic, germline inactivation of the ribosomal protein L22 (Rpl22) predisposes T-lineage progenitors to transformation. Indeed, RPL22 was found to be inactivated in ∼ 10% of human T-acute lymphoblastic leukemias. Moreover, monoallelic loss of Rpl22 accelerates development of thymic lymphoma in both a mouse model of T-cell malignancy and in acute transformation assays in vitro. We show that Rpl22 inactivation enhances transformation potential through induction of the stemness factor, Lin28B. Our finding that Rpl22 inactivation promotes transformation by inducing expression of Lin28B provides the first insight into the mechanistic basis by which mutations in Rpl22, and perhaps some other RP genes, increases cancer risk.

Translational implications of targeting annexin A2: From membrane repair to muscular dystrophy, cardiovascular disease and cancer.

Annexin A2 (A2) contributes to several key cellular functions and processes, including membrane repair. Effective repair prevents cell death and degeneration, especially in skeletal or cardiac muscle, epithelia, and endothelial cells. To maintain cell integrity after damage, mammalian cells activate multiple membrane repair mechanisms. One protein family that facilitates membrane repair processes are the Ca2+-regulated phospholipid-binding annexins. Annexin A2 facilitates repair in association with S100A10 and related S100 proteins by forming a plug and linking repair to other physiologic functions. Deficiency of annexin A2 enhances cellular degeneration, exacerbating muscular dystrophy and degeneration. Downstream of repair, annexin A2 links membrane with the cytoskeleton, calcium-dependent endocytosis, exocytosis, cell proliferation, transcription, and apoptosis to extracellular roles, including vascular fibrinolysis, and angiogenesis. These roles regulate cardiovascular disease progression. Finally, annexin A2 protects cancer cells from membrane damage due to immune cells or chemotherapy. Since these functions are regulated by post-translational modifications, they represent a therapeutic target for reducing the negative consequences of annexin A2 expression. Thus, connecting the roles of annexin A2 in repair to its other physiologic functions represents a new translational approach to treating muscular dystrophy and cardiovascular diseases without enhancing its pro-tumorigenic activities.

Pathogenic Mutations in the C2A Domain of Dysferlin form Amyloid that Activates the Inflammasome.

Limb-Girdle Muscular Dystrophy Type-2B/2R is caused by mutations in the dysferlin gene ( DYSF ). This disease has two known pathogenic missense mutations that occur within dysferlin's C2A domain, namely C2A W52R and C2A V67D . Yet, the etiological rationale to explain the disease linkage for these two mutations is still unclear. In this study, we have presented evidence from biophysical, computational, and immunological experiments which suggest that these missense mutations interfere with dysferlin's ability to repair cells. The failure of C2A W52R and C2A V67D to initiate membrane repair arises from their propensity to form stable amyloid. The misfolding of the C2A domain caused by either mutation exposes ß-strands, which are predicted to nucleate classical amyloid structures. When dysferlin C2A amyloid is formed, it triggers the NLRP3 inflammasome, leading to the secretion of inflammatory cytokines, including IL-1ß. The present study suggests that the muscle dysfunction and inflammation evident in Limb-Girdle Muscular Dystrophy types-2B/2R, specifically in cases involving C2A W52R and C2A V67D , as well as other C2 domain mutations with considerable hydrophobic core involvement, may be attributed to this mechanism.

Distinct roles for the NF-kappa B RelA subunit during antiviral innate immune responses.

Production of type I interferons (IFNs; prominently, IFN-α/ß) following virus infection is a pivotal antiviral innate immune response in higher vertebrates. The synthesis of IFN-ß proceeds via the virus-induced assembly of the transcription factors IRF-3/7, ATF-2/c-Jun, and NF-κB on the ifnß promoter. Surprisingly, recent data indicate that the NF-κB subunit RelA is not essential for virus-stimulated ifnß expression. Here, we show that RelA instead sustains autocrine IFN-ß signaling prior to infection. In the absence of RelA, virus infection results in significantly delayed ifnß induction and consequently defective secondary antiviral gene expression. While RelA is not required for ifnß expression after infection, it is nonetheless essential for fully one-fourth of double-stranded RNA (dsRNA)-activated genes, including several mediators of inflammation and immune cell recruitment. Further, RelA directly regulates a small subset of interferon-stimulated genes (ISGs). Finally, RelA also protects cells from dsRNA-triggered RIP1-dependent programmed necrosis. Taken together, our findings suggest distinct roles for RelA in antiviral innate immunity: RelA maintains autocrine IFN-ß signaling in uninfected cells, facilitates inflammatory and adaptive immune responses following infection, and promotes infected-cell survival during this process.

A harmful religio-cultural practice (Chhaupadi) during menstruation among adolescent girls in Nepal: Prevalence and policies for eradication.

BACKGROUND: Chhaupadi is a deeply rooted tradition and a centuries-old harmful religio-cultural practice. Chhaupadi is common in some parts of Karnali and Sudurpaschim Provinces of western Nepal, where women and girls are considered impure, unclean, and untouchable in the menstrual period or immediately following childbirth. In Chhaupadi practice, women and girls are isolated from a range of daily household chores, social events and forbidden from touching other people and objects. Chhaupadi tradition banishes women and girls into menstruation huts', or Chhau huts or livestock sheds to live and sleep. These practices are guided by existing harmful beliefs and practices in western Nepal, resulting in poor menstrual hygiene and poor physical and mental health outcomes. This study examined the magnitude of Chhaupadi practice and reviewed the existing policies for Chhaupadi eradication in Nepal. METHODS: We used both quantitative survey and qualitative content analysis of the available policies. First, a quantitative cross-sectional survey assessed the prevalence of Chhaupadi among 221 adolescent girls in Mangalsen Municipality of Achham district. Second, the contents of prevailing policies on Chhaupadi eradication were analysed qualitatively using the policy cube framework. RESULTS: The current survey revealed that most adolescent girls (84%) practised Chhaupadi in their most recent menstruation. The Chhaupadi practice was high if the girls were aged 15-17 years, born to an illiterate mother, and belonged to a nuclear family. Out of the girls practising Chhaupadi, most (86%) reported social and household activities restrictions. The policy content analysis of identified higher-level policy documents (constitution, acts, and regulations) have provisioned financial resources, ensured independent monitoring mechanisms, and had judiciary remedial measures. However, middle (policies and plans) and lower-level (directives) documents lacked adequate budgetary commitment and independent monitoring mechanisms. CONCLUSION: Chhaupadi remains prevalent in western Nepal and has several impacts to the health of adolescent girls. Existing policy mechanisms lack multilevel (individual, family, community, subnational and national) interventions, including financial and monitoring systems for Chhaupadi eradication. Eradicating Chhaupadi practice requires a robust multilevel implementation mechanism at the national and sub-national levels, including adequate financing and accountable systems up to the community level.

Netrin G1 Promotes Pancreatic Tumorigenesis through Cancer-Associated Fibroblast-Driven Nutritional Support and Immunosuppression.

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1+ cancer-associated fibroblasts (CAF) support PDAC survival, through a NetG1-mediated effect on glutamate/glutamine metabolism. Also, NetG1+ CAFs are intrinsically immunosuppressive and inhibit natural killer cell-mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC. SIGNIFICANCE: This study demonstrates the feasibility of targeting a fibroblastic protein, NetG1, which can limit PDAC tumorigenesis in vivo by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function.See related commentary by Sherman, p. 230.This article is highlighted in the In This Issue feature, p. 211.

Interaction of Macrophages and Cholesterol-Dependent Cytolysins: The Impact on Immune Response and Cellular Survival.

Cholesterol-dependent cytolysins (CDCs) are key virulence factors involved in many lethal bacterial infections, including pneumonia, necrotizing soft tissue infections, bacterial meningitis, and miscarriage. Host responses to these diseases involve myeloid cells, especially macrophages. Macrophages use several systems to detect and respond to cholesterol-dependent cytolysins, including membrane repair, mitogen-activated protein (MAP) kinase signaling, phagocytosis, cytokine production, and activation of the adaptive immune system. However, CDCs also promote immune evasion by silencing and/or destroying myeloid cells. While there are many common themes between the various CDCs, each CDC also possesses specific features to optimally benefit the pathogen producing it. This review highlights host responses to CDC pathogenesis with a focus on macrophages. Due to their robust plasticity, macrophages play key roles in the outcome of bacterial infections. Understanding the unique features and differences within the common theme of CDCs bolsters new tools for research and therapy.

Implementing a quality improvement initiative for the management of chronic obstructive pulmonary disease in rural Nepal.

Background: Chronic obstructive pulmonary disease accounts for a significant portion of the world's morbidity and mortality, and disproportionately affects low/middle-income countries. Chronic obstructive pulmonary disease management in low-resource settings is suboptimal with diagnostics, medications and high-quality, evidence-based care largely unavailable or unaffordable for most people. In early 2016, we aimed to improve the quality of chronic obstructive pulmonary disease management at Bayalpata Hospital in rural Achham, Nepal. Given that quality improvement infrastructure is limited in our setting, we also aimed to model the use of an electronic health record system for quality improvement, and to build local quality improvement capacity. Design: Using international chronic obstructive pulmonary disease guidelines, the quality improvement team designed a locally adapted chronic obstructive pulmonary disease protocol which was subsequently converted into an electronic health record template. Over several Plan-Do-Study-Act cycles, the team rolled out a multifaceted intervention including educational sessions, reminders, as well as audits and feedback. Results: The rate of oral corticosteroid prescriptions for acute exacerbations of chronic obstructive pulmonary disease increased from 14% at baseline to >60% by month 7, with the mean monthly rate maintained above this level for the remainder of the initiative. The process measure of chronic obstructive pulmonary disease template completion rate increased from 44% at baseline to >60% by month 2 and remained between 50% and 70% for the remainder of the initiative. Conclusion: This case study demonstrates the feasibility of robust quality improvement programmes in rural settings and the essential role of capacity building in ensuring sustainability. It also highlights how individual quality improvement initiatives can catalyse systems-level improvements, which in turn create a stronger foundation for continuous quality improvement and healthcare system strengthening.

Designing and implementing an integrated non-communicable disease primary care intervention in rural Nepal.

Low-income and middle-income countries are struggling with a growing epidemic of non-communicable diseases. To achieve the Sustainable Development Goals, their healthcare systems need to be strengthened and redesigned. The Starfield 4Cs of primary care-first-contact access, care coordination, comprehensiveness and continuity-offer practical, high-quality design options for non-communicable disease care in low-income and middle-income countries. We describe an integrated non-communicable disease intervention in rural Nepal using the 4C principles. We present 18 months of retrospective assessment of implementation for patients with type II diabetes, hypertension and chronic obstructive pulmonary disease. We assessed feasibility using facility and community follow-up as proxy measures, and assessed effectiveness using singular 'at-goal' metrics for each condition. The median follow-up for diabetes, hypertension and chronic obstructive pulmonary disease was 6, 6 and 7 facility visits, and 10, 10 and 11 community visits, respectively (0.9 monthly patient touch-points). Loss-to-follow-up rates were 16%, 19% and 22%, respectively. The median time between visits was approximately 2 months for facility visits and 1 month for community visits. 'At-goal' status for patients with chronic obstructive pulmonary disease improved from baseline to endline (p=0.01), but not for diabetes or hypertension. This is the first integrated non-communicable disease intervention, based on the 4C principles, in Nepal. Our experience demonstrates high rates of facility and community follow-up, with comparatively low lost-to-follow-up rates. The mixed effectiveness results suggest that while this intervention may be valuable, it may not be sufficient to impact outcomes. To achieve the Sustainable Development Goals, further implementation research is urgently needed to determine how to optimise non-communicable disease interventions.

Multiple Parameters Beyond Lipid Binding Affinity Drive Cytotoxicity of Cholesterol-Dependent Cytolysins.

The largest superfamily of bacterial virulence factors is pore-forming toxins (PFTs). PFTs are secreted by both pathogenic and non-pathogenic bacteria. PFTs sometimes kill or induce pro-pathogen signaling in mammalian cells, all primarily through plasma membrane perforation, though the parameters that determine these outcomes are unclear. Membrane binding, calcium influx, pore size, and membrane repair are factors that influence PFT cytotoxicity. To test the contribution of membrane binding to cytotoxicity and repair, we compared the closely related, similarly-sized PFTs Perfringolysin O (PFO) from Clostridium perfringens and Streptolysin O (SLO) from Streptococcus pyogenes. Cell death kinetics for PFO and SLO were different because PFO increased in cytotoxicity over time. We introduced known L3 loop mutations that swap binding affinity between toxins and measured hemolytic activity, nucleated cell death kinetics and membrane repair using viability assays, and live cell imaging. Altered hemolytic activity was directly proportional to toxin binding affinity. In contrast, L3 loop alterations reduced nucleated cell death, and they had limited effects on cytotoxicity kinetics and membrane repair. This suggests other toxin structural features, like oligomerization, drives these parameters. Overall, these findings suggest that repair mechanisms and toxin oligomerization add constraints beyond membrane binding on toxin evolution and activity against nucleated cells.

Analysis of Cytokine- and Influenza A Virus-Driven RIPK3 Necrosome Formation.

In multicellular organisms, regulated cell death plays a vital role in development, adult tissue homeostasis, and clearance of damaged or infected cells. Necroptosis is one such form of regulated cell death, characterized by its reliance on receptor-interacting protein kinase 3 (RIPK3). Once activated, RIPK3 nucleates a protein complex, termed the "necrosome," which includes the adaptors RIPK1 and FADD, and the effector protein MLKL. From the necrosome, RIPK3 phosphorylates MLKL to drive necroptosis, and can also induce RIPK1/FADD-mediated apoptosis, via caspase-8. Assembly of the necrosome thus serves as a useful readout of RIPK3 activation. In this chapter, we describe molecular methods for examining necrosome activation in response to the cytokines TNF-α, IFN-ß, and IFN-γ, and upon infection with influenza A virus (IAV).

Host functions used by hepatitis B virus to complete its life cycle: Implications for developing host-targeting agents to treat chronic hepatitis B.

Similar to other mammalian viruses, the life cycle of hepatitis B virus (HBV) is heavily dependent upon and regulated by cellular (host) functions. These cellular functions can be generally placed in to two categories: (a) intrinsic host restriction factors and innate defenses, which must be evaded or repressed by the virus; and (b) gene products that provide functions necessary for the virus to complete its life cycle. Some of these functions may apply to all viruses, but some may be specific to HBV. In certain cases, the virus may depend upon the host function much more than does the host itself. Knowing which host functions regulate the different steps of a virus' life cycle, can lead to new antiviral targets and help in developing novel treatment strategies, in addition to improving a fundamental understanding of viral pathogenesis. Therefore, in this review we will discuss known host factors which influence key steps of HBV life cycle, and further elucidate therapeutic interventions targeting host-HBV interactions.

Isolation of Multidrug Resistant Bacteria from Patients Medical Charts.

BACKGROUND: Patient's medical charts in hospitals are potentially contaminated by pathogenic bacteria and might act as vehicles for transmission of bacterial infections.This study was aimed to determine the rate of contamination of medical charts by multidrug resistant bacteria. METHODS: Sampling of total 250 patient's medical charts from different wards was done with the help of cotton swabs soaked in sterile normal saline. The swabs thus collected were cultured using standard microbiological procedures.The colonies grown were then identified with the help of colony morphology, Gram's stain and biochemical tests. Antimicrobial susceptibility testing was performed by using Kirby-Bauer disc diffusion technique. RESULTS: Of the total 250 charts sampled, 98.8% grew bacteria; Bacillus spp. in 40.7%, followed by Staphylococcus aureus (17%), coagulase-negative Staphylococcus spp.(CoNS) (17%), Citrobacter freundii (9.6%) and Acinetobacter spp. (4.5%). Rate of multidrug resistance was highest in Acinetobacter spp. (50%). Among 83 isolates of S. aureus, methicillin resistance was found in 29 isolates. Similarly, two out of total 9 isolates of Enterococcus spp. were vancomycin resistant. CONCLUSIONS: This study showed that patient's medical charts were contaminated with multidrug resistant bacteria including methicillin resistant S. aureus and vancomycin resistant Enterococcus spp. Strict hand washing before and after handling medical charts is recommended.

RIPK3 Is Largely Dispensable for RIG-I-Like Receptor- and Type I Interferon-Driven Transcriptional Responses to Influenza A Virus in Murine Fibroblasts.

The kinase RIPK3 is a key regulator of cell death responses to a growing number of viral and microbial agents. We have found that influenza A virus (IAV)-mediated cell death is largely reliant on RIPK3 and that RIPK3-deficient mice are notably more susceptible to lethal infection by IAV than their wild-type counterparts. Recent studies demonstrate that RIPK3 also participates in regulating gene transcription programs during host pro-inflammatory and innate-immune responses, indicating that this kinase is not solely an inducer of cell death and that RIPK3-driven transcriptional responses may collaborate with cell death in promoting clearance of IAV. Here, we carried out DNA microarray analyses to determine the contribution of RIPK3 to the IAV-elicited host transcriptional response. We report that RIPK3 does not contribute significantly to the RLR-activated transcriptome or to the induction of type I IFN genes, although, interestingly, IFN-ß production at a post-transcriptional step was modestly attenuated in IAV-infected ripk3-/- fibroblasts. Overall, RIPK3 regulated the expression of <5% of the IAV-induced transcriptome, and no genes were found to be obligate RIPK3 targets. IFN-ß signaling was also found to be largely normal in the absence of RIPK3. Together, these results indicate that RIPK3 is not essential for the host antiviral transcriptional response to IAV in murine fibroblasts.

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.