RESUMEN
Binding of dsDNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) triggers formation of the metazoan second messenger c[G(2',5')pA(3',5')p], which binds the signaling protein STING with subsequent activation of the interferon (IFN) pathway. We show that human hSTING(H232) adopts a "closed" conformation upon binding c[G(2',5')pA(3',5')p] and its linkage isomer c[G(2',5')pA(2',5')p], as does mouse mSting(R231) on binding c[G(2',5')pA(3',5')p], c[G(3',5')pA(3',5')p] and the antiviral agent DMXAA, leading to similar "closed" conformations. Comparing hSTING to mSting, 2',5'-linkage-containing cGAMP isomers were more specific triggers of the IFN pathway compared to the all-3',5'-linkage isomer. Guided by structural information, we identified a unique point mutation (S162A) placed within the cyclic-dinucleotide-binding site of hSTING that rendered it sensitive to the otherwise mouse-specific drug DMXAA, a conclusion validated by binding studies. Our structural and functional analysis highlights the unexpected versatility of STING in the recognition of natural and synthetic ligands within a small-molecule pocket created by the dimerization of STING.
Asunto(s)
Antivirales/farmacología , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Nucleótidos Cíclicos/metabolismo , Xantonas/farmacología , Animales , Cristalografía por Rayos X , GMP Cíclico/metabolismo , Humanos , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Modelos Moleculares , Mutagénesis , Conformación Proteica , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Modified vaccinia virus Ankara (MVA) is an attenuated poxvirus that has been engineered as a vaccine against infectious agents and cancers. Our goal is to understand how MVA modulates innate immunity in dendritic cells (DCs), which can provide insights to vaccine design. In this study, using murine bone marrow-derived dendritic cells, we assessed type I interferon (IFN) gene induction and protein secretion in response to MVA infection. We report that MVA infection elicits the production of type I IFN in murine conventional dendritic cells (cDCs), but not in plasmacytoid dendritic cells (pDCs). Transcription factors IRF3 (IFN regulatory factor 3) and IRF7, and the positive feedback loop mediated by IFNAR1 (IFN alpha/beta receptor 1), are required for the induction. MVA induction of type I IFN is fully dependent on STING (stimulator of IFN genes) and the newly discovered cytosolic DNA sensor cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase). MVA infection of cDCs triggers phosphorylation of TBK1 (Tank-binding kinase 1) and IRF3, which is abolished in the absence of cGAS and STING. Furthermore, intravenous delivery of MVA induces type I IFN in wild-type mice, but not in mice lacking STING or IRF3. Treatment of cDCs with inhibitors of endosomal and lysosomal acidification or the lysosomal enzyme Cathepsin B attenuated MVA-induced type I IFN production, indicating that lysosomal enzymatic processing of virions is important for MVA sensing. Taken together, our results demonstrate a critical role of the cGAS/STING-mediated cytosolic DNA-sensing pathway for type I IFN induction in cDCs by MVA. We present evidence that vaccinia virulence factors E3 and N1 inhibit the activation of IRF3 and the induction of IFNB gene in MVA-infected cDCs.
Asunto(s)
Células de la Médula Ósea/metabolismo , Células Dendríticas/metabolismo , Interferón beta/metabolismo , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/metabolismo , Virus Vaccinia/metabolismo , Vaccinia/metabolismo , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/virología , Células Dendríticas/inmunología , Células Dendríticas/virología , Endosomas/genética , Endosomas/inmunología , Endosomas/metabolismo , Femenino , Inmunidad Innata/genética , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/inmunología , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/genética , Interferón beta/inmunología , Lisosomas/genética , Lisosomas/inmunología , Lisosomas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Noqueados , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/inmunología , Fosforilación/genética , Fosforilación/inmunología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/inmunología , Proteínas de Unión al ARN/metabolismo , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/inmunología , Receptor de Interferón alfa y beta/metabolismo , Vaccinia/genética , Vaccinia/inmunología , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismoRESUMEN
Cotton is one of the most economically important crops in the world, and drought is a key abiotic factor that can significantly reduce cotton yield. MADS-box transcription factors play essential roles in various aspects of plant growth and development as well as responses to biotic and abiotic stress. However, the use of MADS-box transcription factors to regulate water stress responses has not been fully explored in cotton. Here, we showed that GhAGL16 acts as a negative regulator of water deficit in cotton, at least in part by regulating ABA signaling. GhAGL16-overexpressing (GhAGL16-OE) transgenic Arabidopsis had lower survival rates and relative water contents (RWCs) under water stress. Isolated leaves of GhAGL16-OE Arabidopsis had increased water loss rates, likely attributable to their increased stomatal density. GhAGL16-OE Arabidopsis also showed reduced primary root lengths in response to mannitol treatment and decreased sensitivity of seed germination to ABA treatment. By contrast, silencing GhAGL16 in cotton enhanced tolerance to water deficit by increasing proline (Pro) content, increasing superoxide dismutase (SOD) and peroxidase (POD) activities, and reducing malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents under water stress. Subcellular localization and transcriptional activation assays confirmed that GhAGL16 is a nuclear protein that lacks transcriptional self-activation activity. The expression of ABA biosynthesis-related genes (GhNCED3/7/14), a catabolism-related gene (GhCYP707A), and a gene related to the ABA signaling pathway (GhABF4) was altered in GhAGL16-silenced plants. Taken together, our data demonstrate that GhAGL16 plays an important role in cotton resistance to water stress.
RESUMEN
The DNA sensor cyclic GMP-AMP synthase (cGAS) is critical in host antiviral immunity. Vaccinia virus (VACV) is a large cytoplasmic DNA virus that belongs to the poxvirus family. How vaccinia virus antagonizes the cGAS-mediated cytosolic DNA-sensing pathway is not well understood. In this study, we screened 80 vaccinia genes to identify potential viral inhibitors of the cGAS/Stimulator of interferon gene (STING) pathway. We discovered that vaccinia E5 is a virulence factor and a major inhibitor of cGAS. E5 is responsible for abolishing cGAMP production during vaccinia virus (Western Reserve strain) infection of dendritic cells. E5 localizes to the cytoplasm and nucleus of infected cells. Cytosolic E5 triggers ubiquitination of cGAS and proteasome-dependent degradation via interacting with cGAS. Deleting the E5R gene from the Modified vaccinia virus Ankara (MVA) genome strongly induces type I IFN production by dendritic cells (DCs) and promotes DC maturation, and thereby improves antigen-specific T cell responses.
Asunto(s)
Células Dendríticas , Nucleotidiltransferasas , Virus Vaccinia , Proteínas Virales , Ratones Endogámicos C57BL , Animales , Ratones , Ratones Noqueados , Femenino , Nucleotidiltransferasas/inmunología , Células Dendríticas/inmunología , Células Dendríticas/virología , Virus Vaccinia/patogenicidad , Factores de Virulencia/inmunología , Ubiquitinación , Proteínas Virales/genética , Proteínas Virales/inmunología , Complejo de la Endopetidasa Proteasomal , Interferón Tipo I/inmunología , Células HEK293 , Humanos , Proteínas de la Membrana/inmunología , Linfocitos T/inmunologíaRESUMEN
Poxviruses are large DNA viruses that replicate in the cytoplasm of infected cells. Myxoma virus is a rabbit poxvirus that belongs to the Leporipoxvirus genus. It causes a lethal disease called myxomatosis in European rabbits but cannot sustain any detectable infection in nonlagomorphs. Vaccinia virus is a prototypal orthopoxvirus that was used as a vaccine to eradicate smallpox. Myxoma virus is nonpathogenic in mice, whereas systemic infection with vaccinia virus can be lethal even in immunocompetent mice. Plasmacytoid dendritic cells (pDCs) are potent type I interferon (IFN)-producing cells that play important roles in antiviral innate immunity. How poxviruses are sensed by pDCs to induce type I IFN production is not well understood. Here we report that infection of primary murine pDCs with myxoma virus, but not with vaccinia virus, induces IFN-α, IFN-ß, tumor necrosis factor (TNF), and interleukin-12p70 (IL-12p70) production. Using pDCs derived from genetic knockout mice, we show that the myxoma virus-induced innate immune response requires the endosomal DNA sensor TLR9 and its adaptor MyD88, transcription factors IRF5 and IRF7, and the type I IFN positive-feedback loop mediated by IFNAR1. It is independent of the cytoplasmic RNA sensing pathway mediated by the mitochondrial adaptor molecule MAVS, the TLR3 adaptor TRIF, or the transcription factor IRF3. Using pharmacological inhibitors, we demonstrate that myxoma virus-induced type I IFN and IL-12p70 production in murine pDCs is also dependent on phosphatidylinositol 3-kinase (PI3K) and Akt. Furthermore, our results reveal that the N-terminal Z-DNA/RNA binding domain of vaccinia virulence factor E3, which is missing in the orthologous M029 protein expressed by myxoma virus, plays an inhibitory role in poxvirus sensing and innate cytokine production by murine pDCs.
Asunto(s)
Células Dendríticas/inmunología , Factor 7 Regulador del Interferón/inmunología , Factores Reguladores del Interferón/inmunología , Interferón Tipo I/metabolismo , Factor 88 de Diferenciación Mieloide/inmunología , Myxoma virus/inmunología , Receptor Toll-Like 9/inmunología , Animales , Células Cultivadas , Femenino , Factor 7 Regulador del Interferón/metabolismo , Factores Reguladores del Interferón/metabolismo , Interleucina-12/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Interferón alfa y beta/inmunología , Receptor de Interferón alfa y beta/metabolismo , Receptor Toll-Like 9/metabolismo , Virus Vaccinia/inmunologíaRESUMEN
The pulmonary immune system consists of a network of tissue-resident cells as well as immune cells that are recruited to the lungs during infection and/or inflammation. How these immune components function during an acute poxvirus infection is not well understood. Intranasal infection of mice with vaccinia virus causes lethal pneumonia and systemic dissemination. Here we report that vaccinia C7 is a crucial virulence factor that blocks activation of the transcription factor IRF3. We provide evidence that type II alveolar epithelial cells (AECIIs) respond to pulmonary infection of vaccinia virus by inducing IFN-ß and IFN-stimulated genes via the activation of the MDA5 and STING-mediated nucleic acid-sensing pathways and the type I IFN positive feedback loop. This leads to the recruitment and activation of CCR2+ inflammatory monocytes in the infected lungs and subsequent differentiation into Lyve1- interstitial macrophages (Lyve1- IMs), which efficiently engulf viral particles and block viral replication. Our results provide insights into how innate immune sensing of viral infection by lung AECIIs influences the activation and differentiation of CCR2+ inflammatory monocytes to defend against pulmonary poxvirus infection.
Asunto(s)
Infecciones por Poxviridae , Poxviridae , Vaccinia , Células Epiteliales Alveolares/metabolismo , Animales , Pulmón/metabolismo , Ratones , Monocitos/metabolismo , Poxviridae/metabolismo , Receptores CCR2/metabolismo , Virus Vaccinia/genéticaRESUMEN
As a plant-specific Rho-like small G protein, the ROP (Rho-related GTPase of plants) protein regulates the growth and development of plants and various stress responses in the form of molecular switches. Drought is a major abiotic stress that limits cotton yield and fiber quality. In this study, virus-induced gene silencing (VIGS) technology was used to analyze the biological function of GhROP3 in cotton drought stress tolerance. Meanwhile, we used yeast two-hybrid and bimolecular fluorescence complementation assays to examine the interaction between GhROP3 and GhGGB. GhROP3 has a high expression level in cotton true leaves and roots, and responds to drought, high salt, cold, heat stress, and exogenous abscisic acid (ABA) and auxin (IAA) treatments. Silencing GhROP3 improved the drought tolerance of cotton. The water loss rates (WLR) of detached leaves significantly reduced in silenced plants. Also, the relative water content (RWC) and total contents of chlorophyll (Chl) and proline (Pro) of leaves after drought stress and the activities of three antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) significantly increased, whereas the contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA) significantly reduced. In the leaves of silenced plants, the expression of genes related to ABA synthesis and its related pathway was significantly upregulated, and the expression of decomposition-related GhCYP707A gene and genes related to IAA synthesis and its related pathways was significantly downregulated. It indicated that GhROP3 was a negative regulator of cotton response to drought by participating in the negative regulation of the ABA signaling pathway and the positive regulation of the IAA signaling pathway. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that the GhROP3 protein interacted with the GhGGB protein in vivo and in vitro. This study provided a theoretical basis for the in-depth investigation of the drought resistance-related molecular mechanism of the GhROP3 gene and the biological function of the GhGGB gene.
RESUMEN
Plant virus-mediated sgRNA delivery and expression have great advantages; sgRNA expression can rapidly expand and accumulate along with virus replication and movement, resulting in efficient gene editing efficiency. In this study, a VIGE system based on cotton leaf crumple virus (CLCrV) was established using cotton overexpressing Cas9 (Cas9-OE) as the VIGE receptor. CLCrV-mediated VIGE could not only target and knock out the GhMAPKKK2, GhCLA1 and GhPDS genes subgroup A and D genome sequences but also achieve double mutation of GhCLA1 and GhPDS genes at the same time. These results verified the effectiveness and efficiency of this system. In addition, the off-target effect assay demonstrated that the CLCrV-mediated VIGE system not only has high gene editing efficiency but also high gene editing specificity in cotton. We further explored whether the FT-sgRNA strategy could transport sgRNA to cotton apical meristem (SAM) over long distances to avoid using tissue culture to obtain stable genetic mutants. The results showed that the sgRNA fused with FT mRNA at the 5' end could also efficiently achieve targeted editing of endogenous genes in cotton, but it was difficult to detect heritable mutant progeny. The above results showed that the CLCrV-mediated VIGE system provided an accurate and rapid validation tool for screening effective sgRNAs in cotton.
RESUMEN
BACKGROUND: The virus-induced genome editing (VIGE) system can be used to quickly identify gene functions and generate knock-out libraries as an alternative to the virus-induced gene silencing (VIGS). Although plant virus-mediated VIGE has been shown to have great application prospects, edited genes cannot be transferred to the next generations using this system, as viruses cannot enter into shoot apical meristem (SAM) in plants. RESULTS: We developed a novel cotton leaf crumple virus (CLCrV)-mediated VIGE system designed to target BRI1, GL2, PDS genes, and GUS transgene in A. thaliana by transforming Cas9 overexpression (Cas9-OE) A. thaliana. Given the deficiency of the VIGE system, ProYao::Cas9 and Pro35S::Cas9 A. thaliana were transformed by fusing 102 bp FT mRNAs with sgRNAs so as to explore the function of Flowering Locus T (FT) gene in delivering sgRNAs into SAM, thus avoiding tissue culture and stably acquiring heritable mutant offspring. Our results showed that sgRNAs fused with FT mRNA at the 5' end (FT strategy) effectively enabled gene editing in infected plants and allowed the acquisition of mutations heritable by the next generation, with an efficiency of 4.35-8.79%. In addition, gene-edited offspring by FT-sgRNAs did not contain any components of the CLCrV genome. CONCLUSIONS: FT strategy can be used to acquire heritable mutant offspring avoiding tissue culture and stable transformation based on the CLCrV-mediated VIGE system in A. thaliana.
RESUMEN
BACKGROUND: Viral-based immunotherapy can overcome resistance to immune checkpoint blockade (ICB) and fill the unmet needs of many patients with cancer. Oncolytic viruses (OVs) are defined as engineered or naturally occurring viruses that selectively replicate in and kill cancer cells. OVs also induce antitumor immunity. The purpose of this study was to compare the antitumor effects of live oncolytic vaccinia viruses versus the inactivated versions and elucidate their underlying immunological mechanisms. METHODS: We engineered a replication-competent, oncolytic vaccinia virus (OV-GM) by inserting a murine GM-CSF gene into the thymidine kinase locus of a mutant vaccinia E3L∆83N, which lacks the Z-DNA-binding domain of vaccinia virulence factor E3. We compared the antitumor effects of intratumoral (IT) delivery of live OV-GM versus heat-inactivated OV-GM (heat-iOV-GM) in a murine B16-F10 melanoma bilateral implantation model. We also generated vvDD, a well-studied oncolytic vaccinia virus, and compared the antitumor effects of live vvDD vs heat-inactivated vvDD (heat-ivvDD) in a murine A20 B-cell lymphoma bilateral tumor implantation model. RESULTS: Heat-iOV-GM infection of dendritic cells (DCs) and tumor cells in vitro induced type I interferon and proinflammatory cytokines and chemokines, whereas live OV-GM did not. IT live OV-GM was less effective in generating systemic antitumor immunity compared with heat-iOV-GM. Similar to heat-iOV-GM, the antitumor effects of live OV-GM also require Batf3-dependent CD103+ dendritic cells. When combined with systemic delivery of ICB, IT heat-iOV-GM was more effective in eradicating tumors, compared with live OV-GM. IT heat-ivvDD was also more effective in treating murine A20 B-cell lymphoma, compared with live vvDD. CONCLUSIONS: Tumor lysis induced by the replication of oncolytic vaccinia virus has a limited effect on the generation of systemic antitumor immunity. The activation of Batf3-dependent CD103+ DCs is critical for antitumor effects induced by both live OV-GM and heat-iOV-GM, with the latter being more potent than live OV-GM in inducing innate and adaptive immunity in both locally injected and distant, non-injected tumors. We propose that evaluations of both innate and adaptive immunity, induced by IT oncolytic viral immunotherapy at injected and non-injected tumors, should be included as potential biomarkers for host responses to viral therapy.
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Inmunoterapia/métodos , Viroterapia Oncolítica/métodos , Virus Oncolíticos/metabolismo , Animales , Femenino , Calor , Humanos , Ratones , Microambiente TumoralRESUMEN
Skin keratinocytes provide a first line of defense against invading microorganisms in two ways: (i) by acting as a physical barrier to pathogen entry and (ii) by initiating a vigorous innate immune response upon sensing danger signals. How keratinocytes detect virus infections and generate antiviral immune responses is not well understood. Orthopoxviruses are dermatotropic DNA viruses that cause lethal disease in humans. Virulence in animal models depends on the virus-encoded bifunctional Z-DNA/double-stranded RNA (dsRNA)-binding protein E3. Here, we report that infection of mouse primary keratinocytes with a vaccinia DeltaE3L mutant virus triggers the production of beta interferon (IFN-beta), interleukin-6 (IL-6), CCL4, and CCL5. None of these immune mediators is produced by keratinocytes infected with wild-type vaccinia virus. The dsRNA-binding domain of E3 suffices to prevent activation of the innate immune response. DeltaE3L induction of IFN-beta, IL-6, CCL4, and CCL5 secretion requires mitochondrial antiviral signaling protein (MAVS; an adaptor for the cytoplasmic viral RNA sensors RIG-I and MDA5) and the transcription factor IRF3. IRF3 phosphorylation is induced in keratinocytes infected with DeltaE3L, an event that depends on MAVS. The response of keratinocytes to DeltaE3L is unaffected by genetic ablation of Toll-like receptor 3 (TLR3), TRIF, TLR9, and MyD88.
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Queratinocitos/virología , Proteínas de Unión al ARN/inmunología , Proteínas de Unión al ARN/fisiología , Virus Vaccinia/inmunología , Virus Vaccinia/fisiología , Proteínas Virales/inmunología , Proteínas Virales/fisiología , Animales , Sitios de Unión , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Citocinas/metabolismo , Femenino , Eliminación de Gen , Humanos , Ratones , Ratones Endogámicos BALB C , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/genética , Conejos , Proteínas Virales/genéticaRESUMEN
The bioterror threat of a smallpox outbreak in an unvaccinated population has mobilized efforts to develop new antipoxviral agents. By screening a library of known drugs, we identified 13 compounds that inhibited vaccinia virus replication at noncytotoxic doses. The anticancer drug mitoxantrone is unique among the inhibitors identified in that it has no apparent impact on viral gene expression. Rather, it blocks processing of viral structural proteins and assembly of mature progeny virions. The isolation of mitoxantrone-resistant vaccinia strains underscores that a viral protein is the likely target of the drug. Whole-genome sequencing of mitoxantrone-resistant viruses pinpointed missense mutations in the N-terminal domain of vaccinia DNA ligase. Despite its favorable activity in cell culture, mitoxantrone administered intraperitoneally at the maximum tolerated dose failed to protect mice against a lethal intranasal infection with vaccinia virus.
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Antineoplásicos , Antivirales , Mitoxantrona , Virus Vaccinia/efectos de los fármacos , Virión/metabolismo , Ensamble de Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Antivirales/administración & dosificación , Antivirales/farmacología , Antivirales/uso terapéutico , Línea Celular , Evaluación Preclínica de Medicamentos , Farmacorresistencia Viral , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Mitoxantrona/administración & dosificación , Mitoxantrona/farmacología , Mitoxantrona/uso terapéutico , Organismos Libres de Patógenos Específicos , Vaccinia/prevención & control , Vaccinia/virología , Virus Vaccinia/genéticaRESUMEN
Advanced cancers remain a therapeutic challenge despite recent progress in targeted therapy and immunotherapy. Novel approaches are needed to alter the tumor immunosuppressive microenvironment and to facilitate the recognition of tumor antigens that leads to antitumor immunity. Poxviruses, such as modified vaccinia virus Ankara (MVA), have potential as immunotherapeutic agents. We show that infection of conventional dendritic cells (DCs) with heat- or ultraviolet-inactivated MVA leads to higher levels of interferon induction than MVA alone through the cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase)-STING cytosolic DNA-sensing pathway. Intratumoral injection of inactivated MVA (iMVA) was effective and generated adaptive antitumor immunity in murine melanoma and colon cancer models. iMVA-induced antitumor therapy was less effective in STING- or Batf3-deficient mice than in wild-type mice, indicating that both cytosolic DNA sensing and Batf3-dependent CD103+/CD8α+ DCs are essential for iMVA immunotherapy. The combination of intratumoral delivery of iMVA and systemic delivery of immune checkpoint blockade generated synergistic antitumor effects in bilateral tumor implantation models as well as in a unilateral large established tumor model. Our results suggest that inactivated vaccinia virus could be used as an immunotherapeutic agent for human cancers.
RESUMEN
The drug DMXAA (5,6-dimethylxanthenone-4-acetic acid) showed therapeutic promise against solid tumors in mouse models but subsequently failed in human clinical trials. DMXAA was later discovered to activate mouse, but not human, STING, an adaptor protein in the cyclic dinucleotide cGAMP-mediated signaling pathway, inducing type I interferon expression. To facilitate the development of compounds that target human STING, we combined structural, biophysical, and cellular assays to study mouse and human chimeric proteins and their interaction with DMXAA. We identified a single substitution (G230I) that enables a DMXAA-induced conformational transition of hSTING from an inactive "open" to an active "closed" state. We also identified a substitution within the binding pocket (Q266I) that cooperates with G230I and the previously identified S162A binding-pocket point substitution, rendering hSTING highly sensitive to DMXAA. These findings should facilitate the reciprocal engineering of DMXAA analogs that bind and stimulate wild-type hSTING and their exploitation for vaccine-adjuvant and anticancer drug development.
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Proteínas de la Membrana/metabolismo , Xantonas/metabolismo , Secuencia de Aminoácidos , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/uso terapéutico , Sitios de Unión , Quimiocinas/metabolismo , Cristalografía por Rayos X , Células HEK293 , Humanos , Interferón Tipo I/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Neoplasias/tratamiento farmacológico , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Alineación de Secuencia , Xantonas/química , Xantonas/uso terapéuticoRESUMEN
Plasmacytoid dendritic cells (pDCs) play important roles in antiviral innate immunity by producing type I interferon (IFN). In this study, we assess the immune responses of primary human pDCs to two poxviruses, vaccinia and myxoma virus. Vaccinia, an orthopoxvirus, was used for immunization against smallpox, a contagious human disease with high mortality. Myxoma virus, a Leporipoxvirus, causes lethal disease in rabbits, but is non-pathogenic in humans. We report that myxoma virus infection of human pDCs induces IFN-α and TNF production, whereas vaccinia infection does not. Co-infection of pDCs with myxoma virus plus vaccinia blocks myxoma induction effects. We find that heat-inactivated vaccinia (Heat-VAC; by incubating the virus at 55°C for 1â h) gains the ability to induce IFN-α and TNF in primary human pDCs. Induction of IFN-α in pDCs by myxoma virus or Heat-VAC is blocked by chloroquine, which inhibits endosomal acidification required for TLR7/9 signaling, and by inhibitors of cellular kinases PI3K and Akt. Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1. These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway. Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists. The myxoma virus ortholog of vaccinia E3 (M029) lacks the N-terminal Z-DNA/RNA binding domain, which might contribute to the immunostimulating properties of myxoma virus.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/virología , Inmunidad Innata , Proteínas de Unión al ARN/inmunología , Virus Vaccinia/inmunología , Proteínas Virales/inmunología , Animales , Cloroquina/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Regulación hacia Abajo , Humanos , Interferón-alfa/biosíntesis , Glicoproteínas de Membrana/metabolismo , Ratones , Factor 88 de Diferenciación Mieloide/metabolismo , Myxoma virus/genética , Myxoma virus/inmunología , Myxoma virus/patogenicidad , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Conejos , Receptor Toll-Like 7/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Virus Vaccinia/genética , Virus Vaccinia/patogenicidad , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Langerhans cells (LCs) are antigen-presenting cells in the skin that play sentinel roles in host immune defense by secreting proinflammatory molecules and activating T cells. Here we studied the interaction of vaccinia virus with XS52 cells, a murine epidermis-derived dendritic cell line that serves as a surrogate model for LCs. We found that vaccinia virus productively infects XS52 cells, yet this infection displays an atypical response to anti-poxvirus agents. Whereas adenosine N1-oxide blocked virus production and viral protein synthesis during a synchronous infection, cytosine arabinoside had no effect at concentrations sufficient to prevent virus replication in BSC40 monkey kidney cells. Vaccinia virus infection of XS52 cells not only failed to elicit the production of various cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, IL-10, IL-12 p40, alpha interferon (IFN-alpha), and IFN-gamma, it actively inhibited the production of proinflammatory cytokines TNF-alpha and IL-6 by XS52 cells in response to exogenous lipopolysaccharide (LPS) or poly(I:C). Infection with a vaccinia virus mutant lacking the E3L gene resulted in TNF-alpha secretion in the absence of applied stimuli. Infection of XS52 cells or BSC40 cells with the DeltaE3L virus, but not wild-type vaccinia virus, triggered proteolytic decay of IkappaBalpha. These results suggest a novel role for the E3L protein as an antagonist of the NF-kappaB signaling pathway. DeltaE3L-infected XS52 cells secreted higher levels of TNF-alpha and IL-6 in response to LPS and poly(I:C) than did cells infected with the wild-type virus. XS52 cells were productively infected by a vaccinia virus mutant lacking the K1L gene. DeltaK1L-infected cells secreted higher levels of TNF-alpha and IL-6 in response to LPS than wild-type virus-infected cells. Vaccinia virus infection of primary LCs harvested from mouse epidermis was nonpermissive, although a viral reporter protein was expressed in the infected LCs. Vaccinia virus infection of primary LCs strongly inhibited their capacity for antigen-specific activation of T cells. Our results highlight suppression of the skin immune response as a feature of orthopoxvirus infection.