Publications by authors named "Edward S Mocarski"

110 Publications

TNF Signaling Dictates Myeloid and Non-Myeloid Cell Crosstalk to Execute MCMV-Induced Extrinsic Apoptosis.

Viruses 2020 10 28;12(11). Epub 2020 Oct 28.

Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322, USA.

Cytomegaloviruses all encode the viral inhibitor of caspase-8-induced apoptosis (vICA). After binding to this initiator caspase, vICA blocks caspase-8 proteolytic activity and ability to activate caspase-3 and/or caspase-7. In this manner, vICA has long been known to prevent apoptosis triggered via tumor necrosis factor (TNF) family death receptor-dependent extrinsic signaling. Here, we employ fully wild-type murine cytomegalovirus (MCMV) and vICA-deficient MCMV (∆M36) to investigate the contribution of TNF signaling to apoptosis during infection of different cell types. ∆M36 shows the expected ability to kill mouse splenic hematopoietic cells, bone marrow-derived macrophages (BMDM), and dendritic cells (BMDC). Antibody blockade or genetic elimination of TNF protects myeloid cells from death, and caspase-8 activation accompanies cell death. Interferons, necroptosis, and pyroptotic gasdermin D (GSDMD) do not contribute to myeloid cell death. Human and murine fibroblasts or murine endothelial cells (SVEC4-10) normally insensitive to TNF become sensitized to ∆M36-induced apoptosis when treated with TNF or TNF-containing BMDM-conditioned medium. We demonstrate that myeloid cells are the natural source of TNF that triggers apoptosis in either myeloid (autocrine) or non-myeloid cells (paracrine) during ∆M36 infection of mice. Caspase-8 suppression by vICA emerges as key to subverting innate immune elimination of a wide variety of infected cell types.
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http://dx.doi.org/10.3390/v12111221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693317PMC
October 2020

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

Proc Natl Acad Sci U S A 2020 08 3;117(33):20109-20116. Epub 2020 Aug 3.

Department of Microbiology, Immunology, and Molecular Genetics, University of Texas Health at San Antonio, San Antonio, TX 78229;

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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http://dx.doi.org/10.1073/pnas.1921315117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443917PMC
August 2020

Squalene emulsion-based vaccine adjuvants stimulate CD8 T cell, but not antibody responses, through a RIPK3-dependent pathway.

Elife 2020 06 9;9. Epub 2020 Jun 9.

Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford University, Stanford, United States.

The squalene-based oil-in-water emulsion (SE) vaccine adjuvant MF59 has been administered to more than 100 million people in more than 30 countries, in both seasonal and pandemic influenza vaccines. Despite its wide use and efficacy, its mechanisms of action remain unclear. In this study we demonstrate that immunization of mice with MF59 or its mimetic AddaVax (AV) plus soluble antigen results in robust antigen-specific antibody and CD8 T cell responses in lymph nodes and non-lymphoid tissues. Immunization triggered rapid RIPK3-kinase dependent necroptosis in the lymph node which peaked at 6 hr, followed by a sequential wave of apoptosis. Immunization with alum plus antigen did not induce RIPK3-dependent signaling. RIPK3-dependent signaling induced by MF59 or AV was essential for cross-presentation of antigen to CD8 T cells by Batf3-dependent CD8 DCs. Consistent with this, RIPK3 deficient or Batf3 deficient mice were impaired in their ability to mount adjuvant-enhanced CD8 T cell responses. However, CD8 T cell responses were unaffected in mice deficient in MLKL, a downstream mediator of necroptosis. Surprisingly, antibody responses were unaffected in RIPK3-kinase or Batf3 deficient mice. In contrast, antibody responses were impaired by in vivo administration of the pan-caspase inhibitor Z-VAD-FMK, but normal in caspase-1 deficient mice, suggesting a contribution from apoptotic caspases, in the induction of antibody responses. These results demonstrate that squalene emulsion-based vaccine adjuvants induce antigen-specific CD8 T cell and antibody responses, through RIPK3-dependent and-independent pathways, respectively.
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http://dx.doi.org/10.7554/eLife.52687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314549PMC
June 2020

Cytomegalovirus inhibition of extrinsic apoptosis determines fitness and resistance to cytotoxic CD8 T cells.

Proc Natl Acad Sci U S A 2020 06 22;117(23):12961-12968. Epub 2020 May 22.

Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany;

Viral immune evasion is currently understood to focus on deflecting CD8 T cell recognition of infected cells by disrupting antigen presentation pathways. We evaluated viral interference with the ultimate step in cytotoxic T cell function, the death of infected cells. The viral inhibitor of caspase-8 activation (vICA) conserved in human cytomegalovirus (HCMV) and murine CMV (MCMV) prevents the activation of caspase-8 and proapoptotic signaling. We demonstrate the key role of vICA from either virus, in deflecting antigen-specific CD8 T cell-killing of infected cells. vICA-deficient mutants, lacking either UL36 or M36, exhibit greater susceptibility to CD8 T cell control than mutants lacking the set of immunoevasins known to disrupt antigen presentation via MHC class I. This difference is evident during infection in the natural mouse host infected with MCMV, in settings where virus-specific CD8 T cells are adoptively transferred. Finally, we identify the molecular mechanism through which vICA acts, demonstrating the central contribution of caspase-8 signaling at a point of convergence of death receptor-induced apoptosis and perforin/granzyme-dependent cytotoxicity.
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http://dx.doi.org/10.1073/pnas.1914667117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293702PMC
June 2020

Characterisation of genetic regulatory effects for osteoporosis risk variants in human osteoclasts.

Genome Biol 2020 03 26;21(1):80. Epub 2020 Mar 26.

Department of Endocrinology & Diabetes, Sir Charles Gairdner Hospital, Nedlands, WA, Australia.

Background: Osteoporosis is a complex disease with a strong genetic contribution. A recently published genome-wide association study (GWAS) for estimated bone mineral density (eBMD) identified 1103 independent genome-wide significant association signals. Most of these variants are non-coding, suggesting that regulatory effects may drive many of the associations. To identify genes with a role in osteoporosis, we integrate the eBMD GWAS association results with those from our previous osteoclast expression quantitative trait locus (eQTL) dataset.

Results: We identify sixty-nine significant cis-eQTL effects for eBMD GWAS variants after correction for multiple testing. We detect co-localisation of eBMD GWAS and osteoclast eQTL association signals for 21 of the 69 loci, implicating a number of genes including CCR5, ZBTB38, CPE, GNA12, RIPK3, IQGAP1 and FLCN. Summary-data-based Mendelian Randomisation analysis of the eBMD GWAS and osteoclast eQTL datasets identifies significant associations for 53 genes, with TULP4 presenting as a strong candidate for pleiotropic effects on eBMD and gene expression in osteoclasts. By performing analysis using the GARFIELD software, we demonstrate significant enrichment of osteoporosis risk variants among high-confidence osteoclast eQTL across multiple GWAS P value thresholds. Mice lacking one of the genes of interest, the apoptosis/necroptosis gene RIPK3, show disturbed bone micro-architecture and increased osteoclast number, highlighting a new biological pathway relevant to osteoporosis.

Conclusion: We utilise a unique osteoclast eQTL dataset to identify a number of potential effector genes for osteoporosis risk variants, which will help focus functional studies in this area.
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http://dx.doi.org/10.1186/s13059-020-01997-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098081PMC
March 2020

Recognizing limits of Z-nucleic acid binding protein (ZBP1/DAI/DLM1) function.

FEBS J 2020 10 24;287(20):4362-4369. Epub 2020 Feb 24.

Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, USA.

Z-nucleic acid binding protein (ZBP)1 (also known as DAI and DLM1) is a pathogen sensor activated by double-strand character RNA to recruit receptor-interacting protein (RIP) kinase via a RIP homotypic interaction motif. The activation of receptor-interacting protein kinase (RIPK)3 and initiation of virus-induced necroptosis were initially reported in a landmark publication Upton et al. (Cell Host Microbe 11: 290, 2012) employing the DNA virus murine cytomegalovirus (MCMV). M45-encoded viral inhibitor of RIP activation prevents virus-induced necroptosis. Additional virus-encoded suppressors of necroptosis were then identified, including herpes simplex virus ICP6 and vaccinia virus E3L. Caspase-8 suppressors encoded by these DNA viruses block apoptosis, unleashing necroptosis mediated through Z-nucleic acid binding protein 1 (ZBP1) recruitment of RIPK3. These studies all utilized ZBP1-deficient mice generated by the Akira Lab (Zbp1 ) to bring the significance of virus-induced necroptosis to light. C57BL/6 mice were chosen as controls based on the assumption that mutant mice were congenic; however, these mice were recently found to display an unexpected innate immune deficit, lacking C57BL/6-specific NK1.1 and Ly49H natural killer cell subpopulations important in the early control of MCMV infection. Short nucleotide polymorphism analysis of Zbp1 breeders revealed a mixed genetic background (~ 71% C57BL/6 DNA and ~ 29% 129). Even though this level of 129 strain background does not alter ZBP1 cell-autonomous function as a sensor and mediator of necroptosis, it confounds innate immune response characteristics. In the future, genetic background must be carefully controlled before implicating ZBP1 function in response characteristics that shape immunity, inflammation, metabolism, and pathogenesis.
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http://dx.doi.org/10.1111/febs.15242DOI Listing
October 2020

HIF1α Regulates Early Metabolic Changes due to Activation of Innate Immunity in Nuclear Reprogramming.

Stem Cell Reports 2020 02 11;14(2):192-200. Epub 2020 Feb 11.

Stanford Cardiovascular Institute, Stanford University School of Medicine, 265 Campus Drive, Stanford, CA 94305-5454, USA; Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

Innate immune signaling has recently been shown to play an important role in nuclear reprogramming, by altering the epigenetic landscape and thereby facilitating transcription. However, the mechanisms that link innate immune activation and metabolic regulation in pluripotent stem cells remain poorly defined, particularly with regard to key molecular components. In this study, we show that hypoxia-inducible factor 1α (HIF1α), a central regulator of adaptation to limiting oxygen tension, is an unexpected but crucial regulator of innate immune-mediated nuclear reprogramming. HIF1α is dramatically upregulated as a consequence of Toll-like receptor 3 (TLR3) signaling and is necessary for efficient induction of pluripotency and transdifferentiation. Bioenergetics studies reveal that HIF1α regulates the reconfiguration of innate immune-mediated reprogramming through its well-established role in throwing a glycolytic switch. We believe that results from these studies can help us better understand the influence of immune signaling in tissue regeneration and lead to new therapeutic strategies.
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http://dx.doi.org/10.1016/j.stemcr.2020.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7013248PMC
February 2020

Caspase-8 restricts antiviral CD8 T cell hyperaccumulation.

Proc Natl Acad Sci U S A 2019 07 8;116(30):15170-15177. Epub 2019 Jul 8.

Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30033;

The magnitude of CD8 T cell responses against viruses is checked by the balance of proliferation and death. Caspase-8 (CASP8) has the potential to influence response characteristics through initiation of apoptosis, suppression of necroptosis, and modulation of cell death-independent signal transduction. Mice deficient in CASP8 and RIPK3 ( ) mount enhanced peak CD8 T cell levels against the natural mouse pathogen murine cytomegalovirus (MCMV) or the human pathogen herpes simplex virus-1 compared with littermate control RIPK3-deficient or WT C57BL/6 mice, suggesting an impact of CASP8 on the magnitude of antiviral CD8 T cell expansion and not on contraction. The higher peak response to MCMV in mice resulted from accumulation of greater numbers of terminally differentiated KLRG1 effector CD8 T cell subsets. Antiviral T cells exhibited enhanced proliferation when splenocytes were transferred into WT recipient mice. Thus, cell-autonomous CASP8 normally restricts CD8 T cell proliferation following T cell receptor activation in response to foreign antigen. Memory inflation is a hallmark quality of the T cell response to cytomegalovirus infection. Surprisingly, MCMV-specific memory inflation was not sustained long-term in mice even though these mice retained immunity to secondary challenge. In addition, the accumulation of abnormal B220CD3 T cells in these viable CASP8-deficient mice was reduced by chronic MCMV infection. Combined, these data brings to light the cell death-independent role of CASP8 during CD8 T cell expansion in mice lacking the confounding impact of RIPK3-mediated necroptosis.
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http://dx.doi.org/10.1073/pnas.1904319116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6660791PMC
July 2019

Caspase-8 restricts natural killer cell accumulation during MCMV Infection.

Med Microbiol Immunol 2019 Aug 21;208(3-4):543-554. Epub 2019 May 21.

Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, 1462 Clifton Rd. N.E, Atlanta, GA, 30322, USA.

Natural killer (NK) cells provide important host defense against herpesvirus infections and influence subsequent T cell control of replication and maintenance of latency. NK cells exhibit phases of expansion, contraction and memory formation in response to the natural mouse pathogen murine cytomegalovirus (MCMV). Innate and adaptive immune responses are tightly regulated in mammals to avoid excess tissue damage while preventing acute and chronic viral disease and assuring resistance to reinfection. Caspase (CASP)8 is an autoactivating aspartate-specific cysteine protease that initiates extrinsic apoptosis and prevents receptor interacting protein (RIP) kinase (RIPK)1-RIPK3-driven necroptosis. CASP8 also promotes death-independent signal transduction. All of these activities make contributions to inflammation. Here, we demonstrate that CASP8 restricts NK cell expansion during MCMV infection but does not influence NK memory. Casp8Ripk3 mice mount higher NK response levels than Casp8Ripk3 littermate controls or WT C57BL/6 J mice, indicating that RIPK3 deficiency alone does not contribute to NK response patterns. MCMV m157-responsive Ly49H NK cells support increased expansion of both Ly49H NK cells and CD8 T cells in Casp8Ripk3 mice. Surprisingly, hyperaccumulation of NK cells depends on the pronecrotic kinase RIPK1. Ripk1Casp8Ripk3 mice fail to show the enhanced expansion of lymphocytes observed in Casp8Ripk3 mice even though development and homeostasis are preserved in uninfected Ripk1Casp8Ripk3 mice. Thus, CASP8 naturally regulates the magnitude of NK cell responses in response to infection where strong activation signals depend on another key regulator of death signaling, RIPK1. In addition, the strong NK cell response promotes survival of effector CD8 T cells during their expansion. Thus, hyperaccumulation of NK cells and crosstalk with T cells becomes amplified in the absence of extrinsic cell death machinery.
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http://dx.doi.org/10.1007/s00430-019-00617-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635065PMC
August 2019

Caspase-8-dependent control of NK- and T cell responses during cytomegalovirus infection.

Med Microbiol Immunol 2019 Aug 16;208(3-4):555-571. Epub 2019 May 16.

Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, 1462 Clifton Rd. N.E, Atlanta, GA, 30322, USA.

Caspase-8 (CASP8) impacts antiviral immunity in expected as well as unexpected ways. Mice with combined deficiency in CASP8 and RIPK3 cannot support extrinsic apoptosis or RIPK3-dependent programmed necrosis, enabling studies of CASP8 function without complications of unleashed necroptosis. These extrinsic cell death pathways are naturally targeted by murine cytomegalovirus (MCMV)-encoded cell death suppressors, showing they are key to cell-autonomous host defense. Remarkably, Casp8Ripk3, Ripk1Casp8Ripk3 and Casp8Ripk3 mice mount robust antiviral T cell responses to control MCMV infection. Studies in Casp8Ripk3 mice show that CASP8 restrains expansion of MCMV-specific natural killer (NK) and CD8 T cells without compromising contraction or immune memory. Infected Casp8Ripk3 or Casp8Ripk3 mice have higher levels of virus-specific NK cells and CD8 T cells compared to matched RIPK3-deficient littermates or WT mice. CASP8, likely acting downstream of Fas death receptor, dampens proliferation of CD8 T cells during expansion. Importantly, contraction proceeds unimpaired in the absence of extrinsic death pathways owing to intact Bim-dependent (intrinsic) apoptosis. CD8 T cell memory develops in Casp8Ripk3 mice, but memory inflation characteristic of MCMV infection is not sustained in the absence of CASP8 function. Despite this, Casp8Ripk3 mice are immune to secondary challenge. Interferon (IFN)γ is recognized as a key cytokine for adaptive immune control of MCMV. IfngrCasp8Ripk3 mice exhibit increased lifelong persistence in salivary glands as well as lungs compared to Ifngr and Casp8Ripk3 mice. Thus, mice deficient in CASP8 and RIPK3 are more dependent on IFNγ mechanisms for sustained T cell immune control of MCMV. Overall, appropriate NK- and T cell immunity to MCMV is dependent on host CASP8 function independent of RIPK3-regulated pathways.
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http://dx.doi.org/10.1007/s00430-019-00616-7DOI Listing
August 2019

Herpes simplex virus 1 ICP6 impedes TNF receptor 1-induced necrosome assembly during compartmentalization to detergent-resistant membrane vesicles.

J Biol Chem 2019 01 30;294(3):991-1004. Epub 2018 Nov 30.

From the Department of Microbiology & Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia 30322

Receptor-interacting protein (RIP) kinase 3 (RIPK3)-dependent necroptosis directs inflammation and tissue injury, as well as anti-viral host defense. In human cells, herpes simplex virus 1 (HSV1) UL39-encoded ICP6 blocks RIP homotypic interacting motif (RHIM) signal transduction, preventing this leakage form of cell death and sustaining viral infection. TNF receptor 1 (TNFR1)-induced necroptosis is known to require the formation of a RIPK1-RIPK3-mixed lineage kinase domain-like pseudokinase (MLKL) signaling complex (necrosome) that we find compartmentalizes exclusively to caveolin-1-associated detergent-resistant membrane (DRM) vesicles in HT-29 cells. Translocation proceeds in the presence of RIPK3 kinase inhibitor GSK'840 or MLKL inhibitor necrosulfonomide but requires the kinase activity, as well as RHIM signaling of RIPK1. ICP6 impedes the translocation of RIPK1, RIPK3, and MLKL to caveolin-1-containing DRM vesicles without fully blocking the activation of RIPK3 or phosphorylation of MLKL. Consistent with the important contribution of RIPK1 RHIM-dependent recruitment of RIPK3, overexpression of RHIM-deficient RIPK3 results in phosphorylation of MLKL, but this does not lead to either translocation or necroptosis. Combined, these data reveal a critical role of RHIM signaling in the recruitment of the MLKL-containing necrosome to membrane vesicle-associated sites of aggregation. A similar mechanism is predicted for other RHIM-containing signaling adaptors, Z-nucleic acid-binding protein 1 (ZBP1) (also called DAI and DLM1), and TIR domain-containing adapter-inducing interferon-β (TRIF).
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http://dx.doi.org/10.1074/jbc.RA118.004651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6341382PMC
January 2019

Remarkably Robust Antiviral Immune Response despite Combined Deficiency in Caspase-8 and RIPK3.

J Immunol 2018 10 7;201(8):2244-2255. Epub 2018 Sep 7.

Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322

Caspase-8 (Casp8)-mediated signaling triggers extrinsic apoptosis while suppressing receptor-interacting protein kinase (RIPK) 3-dependent necroptosis. Although Casp8 is dispensable for the development of innate and adaptive immune compartments in mice, the importance of this proapoptotic protease in the orchestration of immune response to pathogens remains to be fully explored. In this study, C57BL/6 mice show robust innate and adaptive immune responses to the natural mouse pathogen, murine CMV. When young, these mice lack -like lymphoid hyperplasia and accumulation of either B220 CD3 or B220CD3CD4 and CD8 T cells with increased numbers of immature myeloid cells that are evident in older mice. Dendritic cell activation and cytokine production drive both NK and T cell responses to control viral infection in these mice, suggesting that Casp8 is dispensable to the generation of antiviral host defense. Curiously, NK and T cell expansion is amplified, with greater numbers observed by 7 d postinfection compared with either or wild type ( ) littermate controls. Casp8 and RIPK3 are natural targets of virus-encoded cell death suppressors that prevent infected cell apoptosis and necroptosis, respectively. It is clear from the current studies that the initiation of innate immunity and the execution of cytotoxic lymphocyte functions are all preserved despite the absence of Casp8 in responding cells. Thus, Casp8 and RIPK3 signaling is completely dispensable to the generation of immunity against this natural herpesvirus infection, although the pathways driven by these initiators serve as a crucial first line for host defense within virus-infected cells.
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http://dx.doi.org/10.4049/jimmunol.1800110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6211196PMC
October 2018

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

Cell Death Dis 2018 07 26;9(8):816. Epub 2018 Jul 26.

Department of Microbiology, Immunology, and Molecular Genetics, University of Texas Heath San Antonio, San Antonio, TX, 78229, USA.

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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http://dx.doi.org/10.1038/s41419-018-0868-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6062522PMC
July 2018

Caspase-8 Collaborates with Caspase-11 to Drive Tissue Damage and Execution of Endotoxic Shock.

Immunity 2018 07;49(1):42-55.e6

Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta GA 30322, USA. Electronic address:

The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from caspase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-β-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.
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http://dx.doi.org/10.1016/j.immuni.2018.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6064639PMC
July 2018

MLKL Requires the Inositol Phosphate Code to Execute Necroptosis.

Mol Cell 2018 06 7;70(5):936-948.e7. Epub 2018 Jun 7.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

Necroptosis is an important form of lytic cell death triggered by injury and infection, but whether mixed lineage kinase domain-like (MLKL) is sufficient to execute this pathway is unknown. In a genetic selection for human cell mutants defective for MLKL-dependent necroptosis, we identified mutations in IPMK and ITPK1, which encode inositol phosphate (IP) kinases that regulate the IP code of soluble molecules. We show that IP kinases are essential for necroptosis triggered by death receptor activation, herpesvirus infection, or a pro-necrotic MLKL mutant. In IP kinase mutant cells, MLKL failed to oligomerize and localize to membranes despite proper receptor-interacting protein kinase-3 (RIPK3)-dependent phosphorylation. We demonstrate that necroptosis requires IP-specific kinase activity and that a highly phosphorylated product, but not a lowly phosphorylated precursor, potently displaces the MLKL auto-inhibitory brace region. These observations reveal control of MLKL-mediated necroptosis by a metabolite and identify a key molecular mechanism underlying regulated cell death.
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http://dx.doi.org/10.1016/j.molcel.2018.05.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5994928PMC
June 2018

Proteasome inhibition blocks necroptosis by attenuating death complex aggregation.

Cell Death Dis 2018 03 1;9(3):346. Epub 2018 Mar 1.

Department of Microbiology & Immunology, Emory Vaccine Center, Emory University School of Medicine, 1462 Clifton Rd., Atlanta, GA, 30322, USA.

Proteasome inhibitors have achieved clinical success because they trigger intrinsic and extrinsic cell death to eliminate susceptible human cancers. The ubiquitin-proteasome protein degradation system regulates signaling pathways by controlling levels of components such as cellular inhibitor of apoptosis (cIAP)1 and cIAP2 in TNF-mediated cell death. Here, we sought to evaluate the contribution of necroptosis to the cell death pattern induced by the specific proteasome inhibitor Carfilzomib (Cf). Proteasome inhibitor-sensitive multiple myeloma cell lines die in response to Cf by apoptosis in combination with serine protease-dependent death, without any contribution of RIPK3-dependent necroptosis. Proteasome inhibition leads to the induction of apoptotic markers such as activated caspase-3 rather than necroptotic markers such as phosphorylated-MLKL in all cell lines tested. In HT-29 cells, Cf attenuates the late RIPK1 interaction with TNFR1 during TNF-induced necroptosis without altering the sensitivity of cIAP antagonists. Cf treatment results in decreased translocation of death signaling components RIPK1, FADD, caspase-8, cFLIP, and RIPK3 to detergent insoluble fractions. Our results show that proteasome inhibition with Cf impairs necroptosis and favors apoptosis even in cells with intact necroptotic machinery. Following the induction of TNFR1-mediated necroptosis, proteasome activity stabilizes effective aggregation and activation of ripoptosome/necrosome complexes.
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http://dx.doi.org/10.1038/s41419-018-0371-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5832869PMC
March 2018

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

Proc Natl Acad Sci U S A 2017 10 9;114(43):11506-11511. Epub 2017 Oct 9.

Center for Immunotherapy, Vaccines, and Virotherapy, Biodesign Institute, Arizona State University, Tempe, AZ 85287;

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.
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http://dx.doi.org/10.1073/pnas.1700999114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664489PMC
October 2017

Genomic analysis of chimeric human cytomegalovirus vaccine candidates derived from strains Towne and Toledo.

Virus Genes 2017 Aug 8;53(4):650-655. Epub 2017 Apr 8.

MRC-University of Glasgow Centre for Virus Research, Glasgow, UK.

Human cytomegalovirus (HCMV) is an important opportunistic pathogen in immunocompromised patients and a major cause of congenital birth defects when acquired in utero. In the 1990s, four chimeric viruses were constructed by replacing genome segments of the high passage Towne strain with segments of the low passage Toledo strain, with the goal of obtaining live attenuated vaccine candidates that remained safe but were more immunogenic than the overly attenuated Towne vaccine. The chimeras were found to be safe when administered to HCMV-seronegative human volunteers, but to differ significantly in their ability to induce seroconversion. This suggests that chimera-specific genetic differences impacted the ability to replicate or persist in vivo and the consequent ability to induce an antibody response. To identify specific genomic breakpoints between Towne and Toledo sequences and establish whether spontaneous mutations or rearrangements had occurred during construction of the chimeras, complete genome sequences were determined. No major deletions or rearrangements were observed, although a number of unanticipated mutations were identified. However, no clear association emerged between the genetic content of the chimeras and the reported levels of vaccine-induced HCMV-specific humoral or cellular immune responses, suggesting that multiple genetic determinants are likely to impact immunogenicity. In addition to revealing the genome organization of the four vaccine candidates, this study provided an opportunity to probe the genetics of HCMV attenuation in humans. The results may be valuable in the future design of safe live or replication-defective vaccines that optimize immunogenicity and efficacy.
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http://dx.doi.org/10.1007/s11262-017-1452-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5527331PMC
August 2017

Mouse cytomegalovirus M36 and M45 death suppressors cooperate to prevent inflammation resulting from antiviral programmed cell death pathways.

Proc Natl Acad Sci U S A 2017 03 14;114(13):E2786-E2795. Epub 2017 Mar 14.

Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322

The complex interplay between caspase-8 and receptor-interacting protein (RIP) kinase RIP 3 (RIPK3) driving extrinsic apoptosis and necroptosis is not fully understood. Murine cytomegalovirus triggers both apoptosis and necroptosis in infected cells; however, encoded inhibitors of caspase-8 activity (M36) and RIP3 signaling (M45) suppress these antiviral responses. Here, we report that this virus activates caspase-8 in macrophages to trigger apoptosis that gives rise to secondary necroptosis. Infection with double-mutant ΔM36/M45mutRHIM virus reveals a signaling pattern in which caspase-8 activates caspase-3 to drive apoptosis with subsequent RIP3-dependent activation of mixed lineage kinase domain-like (MLKL) leading to necroptosis. This combined cell death signaling is highly inflammatory, greater than either apoptosis induced by ΔM36 or necroptosis induced by M45mutRHIM virus. IL-6 production by macrophages is dramatically increased during double-mutant virus infection and correlates with faster antiviral responses in the host. Collaboratively, M36 and M45 target caspase-8 and RIP3 pathways together to suppress this proinflammatory cell death. This study reveals the effect of antiviral programmed cell death pathways on inflammation, shows that caspase-8 activation may go hand-in-hand with necroptosis in macrophages, and revises current understanding of independent and collaborative functions of M36 and M45 in blocking apoptotic and necroptotic cell death responses.
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http://dx.doi.org/10.1073/pnas.1616829114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5380087PMC
March 2017

Retinoic Acid Inducible Gene 1 Protein (RIG1)-Like Receptor Pathway Is Required for Efficient Nuclear Reprogramming.

Stem Cells 2017 05 27;35(5):1197-1207. Epub 2017 Mar 27.

Department of Cardiovascular Sciences, Houston Methodist Research Institute, Houston, Texas, 77030, USA.

We have revealed a critical role for innate immune signaling in nuclear reprogramming to pluripotency, and in the nuclear reprogramming required for somatic cell transdifferentiation. Activation of innate immune signaling causes global changes in the expression and activity of epigenetic modifiers to promote epigenetic plasticity. In our previous articles, we focused on the role of toll-like receptor 3 (TLR3) in this signaling pathway. Here, we define the role of another innate immunity pathway known to participate in response to viral RNA, the retinoic acid-inducible gene 1 receptor (RIG-1)-like receptor (RLR) pathway. This pathway is represented by the sensors of viral RNA, RIG-1, LGP2, and melanoma differentiation-associated protein 5 (MDA5). We first found that TLR3 deficiency only causes a partial inhibition of nuclear reprogramming to pluripotency in mouse tail-tip fibroblasts, which motivated us to determine the contribution of RLR. We found that knockdown of interferon beta promoter stimulator 1, the common adaptor protein for the RLR family, substantially reduced nuclear reprogramming induced by retroviral or by modified messenger RNA expression of Oct 4, Sox2, KLF4, and c-MYC (OSKM). Importantly, a double knockdown of both RLR and TLR3 pathway led to a further decrease in induced pluripotent stem cell (iPSC) colonies suggesting an additive effect of both these pathways on nuclear reprogramming. Furthermore, in murine embryonic fibroblasts expressing a doxycycline (dox)-inducible cassette of the genes encoding OSKM, an RLR agonist increased the yield of iPSCs. Similarly, the RLR agonist enhanced nuclear reprogramming by cell permeant peptides of the Yamanaka factors. Finally, in the dox-inducible system, RLR activation promotes activating histone marks in the promoter region of pluripotency genes. To conclude, innate immune signaling mediated by RLR plays a critical role in nuclear reprogramming. Manipulation of innate immune signaling may facilitate nuclear reprogramming to achieve pluripotency. Stem Cells 2017;35:1197-1207.
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http://dx.doi.org/10.1002/stem.2607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5426114PMC
May 2017

RIPK3 Activates Parallel Pathways of MLKL-Driven Necroptosis and FADD-Mediated Apoptosis to Protect against Influenza A Virus.

Cell Host Microbe 2016 Jul 16;20(1):13-24. Epub 2016 Jun 16.

Blood Cell Development and Function Program, Fox Chase Cancer Center, Philadelphia, PA 19111, USA. Electronic address:

Influenza A virus (IAV) is a lytic virus in primary cultures of many cell types and in vivo. We report that the kinase RIPK3 is essential for IAV-induced lysis of mammalian fibroblasts and lung epithelial cells. Replicating IAV drives assembly of a RIPK3-containing complex that includes the kinase RIPK1, the pseudokinase MLKL, and the adaptor protein FADD, and forms independently of signaling by RNA-sensing innate immune receptors (RLRs, TLRs, PKR), or the cytokines type I interferons and TNF-α. Downstream of RIPK3, IAV activates parallel pathways of MLKL-driven necroptosis and FADD-mediated apoptosis, with the former reliant on RIPK3 kinase activity and neither on RIPK1 activity. Mice deficient in RIPK3 or doubly deficient in MLKL and FADD, but not MLKL alone, are more susceptible to IAV than their wild-type counterparts, revealing an important role for RIPK3-mediated apoptosis in antiviral immunity. Collectively, these results outline RIPK3-activated cytolytic mechanisms essential for controlling respiratory IAV infection.
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http://dx.doi.org/10.1016/j.chom.2016.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5026823PMC
July 2016

T cell-intrinsic ASC critically promotes T(H)17-mediated experimental autoimmune encephalomyelitis.

Nat Immunol 2016 May 21;17(5):583-92. Epub 2016 Mar 21.

Department of Immunology, Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, USA.

Interleukin 1β (IL-1β) is critical for the in vivo survival, expansion and effector function of IL-17-producing helper T (T(H)17) cells during autoimmune responses, including experimental autoimmune encephalomyelitis (EAE). However, the spatiotemporal role and cellular source of IL-1β during EAE pathogenesis are poorly defined. In the present study, we uncovered a T cell-intrinsic inflammasome that drives IL-1β production during T(H)17-mediated EAE pathogenesis. Activation of T cell antigen receptors induced expression of pro-IL-1β, whereas ATP stimulation triggered T cell production of IL-1β via ASC-NLRP3-dependent caspase-8 activation. IL-1R was detected on T(H)17 cells but not on type 1 helper T (T(H)1) cells, and ATP-treated T(H)17 cells showed enhanced survival compared with ATP-treated T(H)1 cells, suggesting autocrine action of T(H)17-derived IL-1β. Together these data reveal a critical role for IL-1β produced by a T(H)17 cell-intrinsic ASC-NLRP3-caspase-8 inflammasome during inflammation of the central nervous system.
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http://dx.doi.org/10.1038/ni.3389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5385929PMC
May 2016

Caspase-8 scaffolding function and MLKL regulate NLRP3 inflammasome activation downstream of TLR3.

Nat Commun 2015 Jun 24;6:7515. Epub 2015 Jun 24.

Department of Biochemistry and Molecular Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

TLR2 promotes NLRP3 inflammasome activation via an early MyD88-IRAK1-dependent pathway that provides a priming signal (signal 1) necessary for activation of the inflammasome by a second potassium-depleting signal (signal 2). Here we show that TLR3 binding to dsRNA promotes post-translational inflammasome activation through intermediate and late TRIF/RIPK1/FADD-dependent pathways. Both pathways require the scaffolding but not the catalytic function of caspase-8 or RIPK1. Only the late pathway requires kinase competent RIPK3 and MLKL function. Mechanistically, FADD/caspase-8 scaffolding function provides a post-translational signal 1 in the intermediate pathway, whereas in the late pathway it helps the oligomerization of RIPK3, which together with MLKL provides both signal 1 and 2 for inflammasome assembly. Cytoplasmic dsRNA activates NLRP3 independent of TRIF, RIPK1, RIPK3 or mitochondrial DRP1, but requires FADD/caspase-8 in wildtype macrophages to remove RIPK3 inhibition. Our study provides a comprehensive analysis of pathways that lead to NLRP3 inflammasome activation in response to dsRNA.
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http://dx.doi.org/10.1038/ncomms8515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480782PMC
June 2015

Caspase-8 as an Effector and Regulator of NLRP3 Inflammasome Signaling.

J Biol Chem 2015 Aug 22;290(33):20167-84. Epub 2015 Jun 22.

Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, From the Departments of Physiology and Biophysics and

We recently described the induction of noncanonical IL-1β processing via caspase-8 recruited to ripoptosome signaling platforms in myeloid leukocytes. Here, we demonstrate that activated NLRP3·ASC inflammasomes recruit caspase-8 to drive IL-1β processing in murine bone marrow-derived dendritic cells (BMDC) independent of caspase-1 and -11. Sustained stimulation (>2 h) of LPS-primed caspase-1-deficient (Casp1/11(-/-)) BMDC with the canonical NLRP3 inflammasome agonist nigericin results in release of bioactive IL-1β in conjunction with robust caspase-8 activation. This IL-1β processing and caspase-8 activation do not proceed in Nlrp3(-/-) or Asc(-/-) BMDC and are suppressed by pharmacological inhibition of caspase-8, indicating that caspase-8 can act as a direct IL-1β-converting enzyme during NLRP3 inflammasome activation. In contrast to the rapid caspase-1-mediated death of wild type (WT) BMDC via NLRP3-dependent pyroptosis, nigericin-stimulated Casp1/11(-/-) BMDC exhibit markedly delayed cell death via NLRP3-dependent apoptosis. Biochemical analyses of WT and Casp1/11(-/-) BMDC indicated that caspase-8 is proteolytically processed within detergent-insoluble ASC-enriched protein complexes prior to extracellular export during nigericin treatment. Although nigericin-stimulated caspase-1 activation and activity are only modestly attenuated in caspase-8-deficient (Casp8(-/-)Rip3(-/-)) BMDC, these cells do not exhibit the rapid loss of viability of WT cells. These results support a contribution of caspase-8 to both IL-1β production and regulated death signaling via NLRP3 inflammasomes. In the absence of caspase-1, NLRP3 inflammasomes directly utilize caspase-8 as both a pro-apoptotic initiator and major IL-1β-converting protease. In the presence of caspase-1, caspase-8 acts as a positive modulator of the NLRP3-dependent caspase-1 signaling cascades that drive both IL-1β production and pyroptotic death.
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http://dx.doi.org/10.1074/jbc.M115.652321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536427PMC
August 2015

Manipulation of apoptosis and necroptosis signaling by herpesviruses.

Med Microbiol Immunol 2015 Jun 1;204(3):439-48. Epub 2015 Apr 1.

Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, 30322, USA.

Like apoptosis, necroptosis is an innate immune mechanism that eliminates pathogen-infected cells. Receptor-interacting protein kinase (RIP)3 (also called RIPK3) mediates necrotic death by phosphorylating an executioner protein, MLKL, leading to plasma membrane leakage. The pathway is triggered against viruses that block caspase 8. In murine CMV, the viral inhibitor of caspase 8 activation prevents extrinsic apoptosis but also has the potential to unleash necroptosis. This virus encodes the viral inhibitor of RIP activation to prevent RIP homotypic interaction motif (RHIM)-dependent signal transduction and necroptosis. Recent investigations reveal a similar mechanism at play in the human alpha-herpesviruses, herpes simplex virus (HSV)1 and HSV2, where RHIM competitor function and caspase 8 suppression are carried out by the virus-encoded large subunit of ribonucleotide reductase (R1). In human cells, R1 inhibition of caspase 8 prevents TNF-induced apoptosis, but sensitizes to TNF-induced necroptosis. The RHIM and caspase 8 interaction domains of R1 collaborate to prevent RIP3-dependent steps and enable both herpesviruses to deflect host cell death machinery that would cut short infection. In mouse cells, HSV1 infection by itself triggers necroptosis by driving RIP3 protein kinase activity. HSV1 R1 contributes to the activation of RIP3 adaptor function in mice, a popular host animal for experimental infection. Based on these studies, infection of RIP3-kinase inactive mice should be explored in models of pathogenesis and latency. The necrotic death pathway that is suppressed during infection in the natural host becomes a cross-species barrier to infection in a non-natural host.
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http://dx.doi.org/10.1007/s00430-015-0410-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4520828PMC
June 2015

Necroptosis: The Trojan horse in cell autonomous antiviral host defense.

Virology 2015 May 24;479-480:160-6. Epub 2015 Mar 24.

Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322, USA.

Herpesviruses suppress cell death to assure sustained infection in their natural hosts. Murine cytomegalovirus (MCMV) encodes suppressors of apoptosis as well as M45-encoded viral inhibitor of RIP activation (vIRA) to block RIP homotypic interaction motif (RHIM)-signaling and recruitment of RIP3 (also called RIPK3), to prevent necroptosis. MCMV and human cytomegalovirus encode a viral inhibitor of caspase (Casp)8 activation to block apoptosis, an activity that unleashes necroptosis. Herpes simplex virus (HSV)1 and HSV2 incorporate both RHIM and Casp8 suppression strategies within UL39-encoded ICP6 and ICP10, respectively, which are herpesvirus-conserved homologs of MCMV M45. Both HSV proteins sensitize human cells to necroptosis by blocking Casp8 activity while preventing RHIM-dependent RIP3 activation and death. In mouse cells, HSV1 ICP6 interacts with RIP3 and, surprisingly, drives necroptosis. Thus, herpesviruses have illuminated the contribution of necoptosis to host defense in the natural host as well as its potential to restrict cross-species infections in nonnatural hosts.
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http://dx.doi.org/10.1016/j.virol.2015.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5115625PMC
May 2015

Stanley Plotkin: the bright spark of cytomegalovirus vaccines.

Med Microbiol Immunol 2015 Jun 20;204(3):243-5. Epub 2015 Mar 20.

Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, 30322, USA,

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http://dx.doi.org/10.1007/s00430-015-0392-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5115627PMC
June 2015

Suppression of RIP3-dependent necroptosis by human cytomegalovirus.

J Biol Chem 2015 May 16;290(18):11635-48. Epub 2015 Mar 16.

From the Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia 30322

Necroptosis is an alternate programmed cell death pathway that is unleashed by caspase-8 compromise and mediated by receptor-interacting protein kinase 3 (RIP3). Murine cytomegalovirus (CMV) and herpes simplex virus (HSV) encode caspase-8 inhibitors that prevent apoptosis together with competitors of RIP homotypic interaction motif (RHIM)-dependent signal transduction to interrupt the necroptosis. Here, we show that pro-necrotic murine CMV M45 mutant virus drives virus-induced necroptosis during nonproductive infection of RIP3-expressing human fibroblasts, whereas WT virus does not. Thus, M45-encoded RHIM competitor, viral inhibitor of RIP activation, sustains viability of human cells like it is known to function in infected mouse cells. Importantly, human CMV is shown to block necroptosis induced by either TNF or M45 mutant murine CMV in RIP3-expressing human cells. Human CMV blocks TNF-induced necroptosis after RIP3 activation and phosphorylation of the mixed lineage kinase domain-like (MLKL) pseudokinase. An early, IE1-regulated viral gene product acts on a necroptosis step that follows MLKL phosphorylation prior to membrane leakage. This suppression strategy is distinct from RHIM signaling competition by murine CMV or HSV and interrupts an execution process that has not yet been fully elaborated.
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http://dx.doi.org/10.1074/jbc.M115.646042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4416866PMC
May 2015

The A, B, Cs of herpesvirus capsids.

Viruses 2015 Feb 26;7(3):899-914. Epub 2015 Feb 26.

Department of Structural Biology, University of Pittsburgh School of Medicine, Biomedical Science Tower 3, 3501 5th Avenue, Pittsburgh, PA 15260, USA.

Assembly of herpesvirus nucleocapsids shares significant similarities with the assembly of tailed dsDNA bacteriophages; however, important differences exist. A unique feature of herpesviruses is the presence of different mature capsid forms in the host cell nucleus during infection. These capsid forms, referred to as A-, B-, and C-capsids, represent empty capsids, scaffold containing capsids and viral DNA containing capsids, respectively. The C-capsids are the closest in form to those encapsidated into mature virions and are considered precursors to infectious virus. The evidence supporting A- and B-capsids as either abortive forms or assembly intermediates has been lacking. Interaction of specific capsid forms with viral tegument proteins has been proposed to be a mechanism for quality control at the point of nuclear egress of mature particles. Here, we will review the available literature on these capsid forms and present data to debate whether A- and B-capsids play an important or an extraneous role in the herpesvirus life cycle.
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http://dx.doi.org/10.3390/v7030899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4379554PMC
February 2015