RESUMEN
Cytosolic long dsRNA, among the most potent proinflammatory signals, is recognized by melanoma differentiation-associated protein 5 (MDA5). MDA5 binds dsRNA cooperatively forming helical filaments. ATP hydrolysis by MDA5 fulfills a proofreading function by promoting dissociation of shorter endogenous dsRNs from MDA5 while allowing longer viral dsRNAs to remain bound leading to activation of interferon-ß responses. Here, we show that adjacent MDA5 subunits in MDA5-dsRNA filaments hydrolyze ATP cooperatively, inducing cooperative filament disassembly. Consecutive rounds of ATP hydrolysis amplify the filament footprint, displacing tightly bound proteins from dsRNA. Our electron microscopy and biochemical assays show that LGP2 binds to dsRNA at internal binding sites through noncooperative ATP hydrolysis. Unlike MDA5, LGP2 has low nucleic acid selectivity and can hydrolyze GTP and CTP as well as ATP. Binding of LGP2 to dsRNA promotes nucleation of MDA5 filament assembly resulting in shorter filaments. Molecular modeling identifies an internally bound MDA5-LGP2-RNA complex, with the LGP2 C-terminal tail forming the key contacts with MDA5. These contacts are specifically required for NTP-dependent internal RNA binding. We conclude that NTPase-dependent binding of LGP2 to internal dsRNA sites complements NTPase-independent binding to dsRNA ends, via distinct binding modes, to increase the number and signaling output of MDA5-dsRNA complexes.
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ARN Helicasas DEAD-box , Helicasa Inducida por Interferón IFIH1 , ARN Helicasas , ARN Bicatenario , ARN Viral , Adenosina Trifosfato/metabolismo , ARN Helicasas DEAD-box/metabolismo , Hidrólisis , Inmunidad Innata , Helicasa Inducida por Interferón IFIH1/genética , Helicasa Inducida por Interferón IFIH1/metabolismo , Nucleósido-Trifosfatasa/genética , Nucleósido-Trifosfatasa/metabolismo , ARN Helicasas/metabolismo , ARN Bicatenario/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , HumanosRESUMEN
Interactions between pathogens, host microbiota and the immune system influence many physiological and pathological processes. In the 20th century, widespread dermal vaccination with vaccinia virus (VACV) led to the eradication of smallpox but how VACV interacts with the microbiota and whether this influences the efficacy of vaccination are largely unknown. Here we report that intradermal vaccination with VACV induces a large increase in the number of commensal bacteria in infected tissue, which enhance recruitment of inflammatory cells, promote tissue damage and influence the host response. Treatment of vaccinated specific-pathogen-free (SPF) mice with antibiotic, or infection of genetically-matched germ-free (GF) animals caused smaller lesions without alteration in virus titre. Tissue damage correlated with enhanced neutrophil and T cell infiltration and levels of pro-inflammatory tissue cytokines and chemokines. One month after vaccination, GF and both groups of SPF mice had equal numbers of VACV-specific CD8+ T cells and were protected from disease induced by VACV challenge, despite lower levels of VACV-neutralising antibodies observed in GF animals. Thus, skin microbiota may provide an adjuvant-like stimulus during vaccination with VACV and influence the host response to vaccination.
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Viruela , Vaccinia , Animales , Anticuerpos Antivirales , Bacterias , Ratones , Viruela/prevención & control , Vacunación , Virus VacciniaRESUMEN
The interaction between immune cells and virus-infected targets involves multiple plasma membrane (PM) proteins. A systematic study of PM protein modulation by vaccinia virus (VACV), the paradigm of host regulation, has the potential to reveal not only novel viral immune evasion mechanisms, but also novel factors critical in host immunity. Here, >1000 PM proteins were quantified throughout VACV infection, revealing selective downregulation of known T and NK cell ligands including HLA-C, downregulation of cytokine receptors including IFNAR2, IL-6ST and IL-10RB, and rapid inhibition of expression of certain protocadherins and ephrins, candidate activating immune ligands. Downregulation of most PM proteins occurred via a proteasome-independent mechanism. Upregulated proteins included a decoy receptor for TRAIL. Twenty VACV-encoded PM proteins were identified, of which five were not recognised previously as such. Collectively, this dataset constitutes a valuable resource for future studies on antiviral immunity, host-pathogen interaction, poxvirus biology, vector-based vaccine design and oncolytic therapy.
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Enfermedades Transmisibles , Poxviridae , Vaccinia , Humanos , Evasión Inmune , Proteínas de la Membrana/metabolismo , Virus VacciniaRESUMEN
There is a paucity of information on dendritic cell (DC) responses to vaccinia virus (VACV), including the traffic of DCs to the draining lymph node (dLN). In this study, using a mouse model of infection, we studied skin DC migration in response to VACV and compared it with the tuberculosis vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG), another live attenuated vaccine administered via the skin. In stark contrast to BCG, skin DCs did not relocate to the dLN in response to VACV. Infection with UV-inactivated VACV or modified VACV Ankara promoted DC movement to the dLN, indicating that interference with skin DC migration requires replication-competent VACV. This suppressive effect of VACV was capable of mitigating responses to a secondary challenge with BCG in the skin, ablating DC migration, reducing BCG transport, and delaying CD4+ T cell priming in the dLN. Expression of inflammatory mediators associated with BCG-triggered DC migration were absent from virus-injected skin, suggesting that other pathways invoke DC movement in response to replication-deficient VACV. Despite adamant suppression of DC migration, VACV was still detected early in the dLN and primed Ag-specific CD4+ T cells. In summary, VACV blocks skin DC mobilization from the site of infection while retaining the ability to access the dLN to prime CD4+ T cells.
Asunto(s)
Movimiento Celular/inmunología , Células Dendríticas/inmunología , Ganglios Linfáticos/inmunología , Piel/inmunología , Virus Vaccinia/inmunología , Vaccinia/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Movimiento Celular/genética , Ratones , Ratones Noqueados , Mycobacterium bovis/inmunología , Vaccinia/genética , Virus Vaccinia/genéticaRESUMEN
Vaccinia virus (VACV) protein N1 is an intracellular immunomodulator that contributes to virus virulence via inhibition of NF-κB. Intradermal infection with a VACV lacking gene N1L (vΔN1) results in smaller skin lesions than infection with wild-type virus (WT VACV), but the impact of N1 deletion on the local microbiota as well as the innate and cellular immune responses in infected ear tissue is mostly uncharacterized. Here, we analysed the bacterial burden and host immune response at the site of infection and report that the presence of protein N1 correlated with enhanced expansion of skin microbiota, even before lesion development. Furthermore, early after infection (days 1-3), prior to lesion development, the levels of inflammatory mediators were higher in vΔN1-infected tissue compared to WT VACV infection. In contrast, infiltration of ear tissue with myeloid and lymphoid cells was greater after WT VACV infection and there was significantly greater secondary bacterial infection that correlated with greater lesion size. We conclude that a more robust innate immune response to vΔN1 infection leads to better control of virus replication, less bacterial growth and hence an overall reduction of tissue damage and lesion size. This analysis shows the potent impact of a single viral immunomodulator on the host immune response and the pathophysiology of VACV infection in the skin.
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Inmunidad Innata , Piel , Virus Vaccinia , Vaccinia , Proteínas Virales , Humanos , Factores Inmunológicos/metabolismo , Vacunación , Virus Vaccinia/genética , Proteínas Virales/genética , Piel/microbiología , MicrobiotaRESUMEN
There are extensive interactions between viruses and the host DNA damage response (DDR) machinery. The outcome of these interactions includes not only direct effects on viral nucleic acids and genome replication, but also the activation of host stress response signalling pathways that can have further, indirect effects on viral life cycles. The non-homologous end-joining (NHEJ) pathway is responsible for the rapid and imprecise repair of DNA double-stranded breaks in the nucleus that would otherwise be highly toxic. Whilst directly repairing DNA, components of the NHEJ machinery, in particular the DNA-dependent protein kinase (DNA-PK), can activate a raft of downstream signalling events that activate antiviral, cell cycle checkpoint and apoptosis pathways. This combination of possible outcomes results in NHEJ being pro- or antiviral depending on the infection. In this review we will describe the broad range of interactions between NHEJ components and viruses and their consequences for both host and pathogen.
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Reparación del ADN por Unión de Extremidades , Virus ADN/fisiología , Interacciones Huésped-Patógeno , Virus ARN/fisiología , Virosis/virología , Animales , Roturas del ADN de Doble Cadena , Virus ADN/genética , Interacciones Microbiota-Huesped , Humanos , Inmunidad , Virus ARN/genética , Virosis/genéticaRESUMEN
IFN-stimulated gene 15 (ISG15) deficiency in humans leads to severe IFNopathies and mycobacterial disease, the latter being previously attributed to its extracellular cytokine-like activity. In this study, we demonstrate a novel role for secreted ISG15 as an IL-10 inducer, unique to primary human monocytes. A balanced ISG15-induced monocyte/IL-10 versus lymphoid/IFN-γ expression, correlating with p38 MAPK and PI3K signaling, was found using targeted in vitro and ex vivo systems analysis of human transcriptomic datasets. The specificity and MAPK/PI3K-dependence of ISG15-induced monocyte IL-10 production was confirmed in vitro using CRISPR/Cas9 knockout and pharmacological inhibitors. Moreover, this ISG15/IL-10 axis was amplified in leprosy but disrupted in human active tuberculosis (TB) patients. Importantly, ISG15 strongly correlated with inflammation and disease severity during active TB, suggesting its potential use as a biomarker, awaiting clinical validation. In conclusion, this study identifies a novel anti-inflammatory ISG15/IL-10 myeloid axis that is disrupted in active TB.
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Citocinas/inmunología , Interleucina-10/inmunología , Leucocitos Mononucleares/inmunología , Tuberculosis/inmunología , Ubiquitinas/inmunología , HumanosRESUMEN
Viruses have evolved sophisticated strategies to exploit host cell function for their benefit. Here we show that under physiologically normal oxygen levels (normoxia) vaccinia virus (VACV) infection leads to a rapid stabilization of hypoxia-inducible factor (HIF)-1α, its translocation into the nucleus and the activation of HIF-responsive genes, such as vascular endothelial growth factor (VEGF), glucose transporter-1, and pyruvate dehydrogenase kinase-1. HIF-1α stabilization is mediated by VACV protein C16 that binds the human oxygen sensing enzyme prolyl-hydroxylase domain containing protein (PHD)2 and thereby inhibits PHD2-dependent hydroxylation of HIF-1α. The binding between C16 and PHD2 is direct and specific, and ectopic expression of C16 alone induces transcription of HIF-1α responsive genes. Conversely, a VACV strain lacking the gene for C16, C16L, is unable to induce HIF-1α stabilization. Interestingly, the N-terminal region of C16 is predicted to have a PHD2-like structural fold but lacks the catalytic active site residues of PHDs. The induction of a hypoxic response by VACV is reminiscent of the biochemical consequences of solid tumor formation, and illustrates a poxvirus strategy for manipulation of cellular gene expression and biochemistry.
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Hipoxia de la Célula/fisiología , Virus Vaccinia/fisiología , Secuencia de Aminoácidos , Células HEK293 , Humanos , Hidroxilación , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Datos de Secuencia Molecular , Procolágeno-Prolina Dioxigenasa/metabolismo , Homología de Secuencia de Aminoácido , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
Factors influencing T-cell responses are important for vaccine development but are incompletely understood. Here, vaccinia virus (VACV) protein N1 is shown to impair the development of both effector and memory CD8(+) T cells and this correlates with its inhibition of nuclear factor-κB (NF-κB) activation. Infection with VACVs that either have the N1L gene deleted (vΔN1) or contain a I6E mutation (vN1.I6E) that abrogates its inhibition of NF-κB resulted in increased central and memory CD8(+) T-cell populations, increased CD8(+) T-cell cytotoxicity and lower virus titres after challenge. Furthermore, CD8(+) memory T-cell function was increased following infection with vN1.I6E, with more interferon-γ production and greater protection against VACV infection following passive transfer to naive mice, compared with CD8(+) T cells from mice infected with wild-type virus (vN1.WT). This demonstrates the importance of NF-κB activation within infected cells for long-term CD8(+) T-cell memory and vaccine efficacy. Further, it provides a rationale for deleting N1 from VACV vectors to enhance CD8(+) T-cell immunogenicity, while simultaneously reducing virulence to improve vaccine safety.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , FN-kappa B/antagonistas & inhibidores , Virus Vaccinia/inmunología , Vaccinia/inmunología , Proteínas Virales/inmunología , Animales , Linfocitos T CD8-positivos/patología , Proliferación Celular , Femenino , Ratones , Mutación Missense , FN-kappa B/genética , FN-kappa B/inmunología , Vaccinia/genética , Vaccinia/patología , Virus Vaccinia/genética , Proteínas Virales/genética , Vacunas Virales/genética , Vacunas Virales/inmunologíaRESUMEN
The transcription factor NF-κB is essential for immune responses against pathogens and its activation requires the phosphorylation, ubiquitination and proteasomal degradation of IκBα. Here we describe an inhibitor of NF-κB from vaccinia virus that has a closely related counterpart in variola virus, the cause of smallpox, and mechanistic similarity with the HIV protein Vpu. Protein A49 blocks NF-κB activation by molecular mimicry and contains a motif conserved in IκBα which, in IκBα, is phosphorylated by IKKß causing ubiquitination and degradation. Like IκBα, A49 binds the E3 ligase ß-TrCP, thereby preventing ubiquitination and degradation of IκBα. Consequently, A49 stabilised phosphorylated IκBα (p-IκBα) and its interaction with p65, so preventing p65 nuclear translocation. Serine-to-alanine mutagenesis within the IκBα-like motif of A49 abolished ß-TrCP binding, stabilisation of p-IκBα and inhibition of NF-κB activation. Remarkably, despite encoding nine other inhibitors of NF-κB, a VACV lacking A49 showed reduced virulence in vivo.
Asunto(s)
Imitación Molecular , FN-kappa B/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismo , Virus Vaccinia/patogenicidad , Virus de la Viruela/patogenicidad , Proteínas con Repetición de beta-Transducina/metabolismo , Animales , Línea Celular , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Evasión Inmune , Ratones , Ratones Endogámicos BALB C , Mutagénesis Sitio-Dirigida , FN-kappa B/genética , FN-kappa B/metabolismo , Fosforilación , Unión Proteica , Ubiquitina-Proteína Ligasas/genética , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Virus de la Viruela/genética , Virus de la Viruela/inmunología , Virulencia , Proteínas con Repetición de beta-Transducina/genéticaRESUMEN
The innate immune system is critical in the response to infection by pathogens and it is activated by pattern recognition receptors (PRRs) binding to pathogen associated molecular patterns (PAMPs). During viral infection, the direct recognition of the viral nucleic acids, such as the genomes of DNA viruses, is very important for activation of innate immunity. Recently, DNA-dependent protein kinase (DNA-PK), a heterotrimeric complex consisting of the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs was identified as a cytoplasmic PRR for DNA that is important for the innate immune response to intracellular DNA and DNA virus infection. Here we show that vaccinia virus (VACV) has evolved to inhibit this function of DNA-PK by expression of a highly conserved protein called C16, which was known to contribute to virulence but by an unknown mechanism. Data presented show that C16 binds directly to the Ku heterodimer and thereby inhibits the innate immune response to DNA in fibroblasts, characterised by the decreased production of cytokines and chemokines. Mechanistically, C16 acts by blocking DNA-PK binding to DNA, which correlates with reduced DNA-PK-dependent DNA sensing. The C-terminal region of C16 is sufficient for binding Ku and this activity is conserved in the variola virus (VARV) orthologue of C16. In contrast, deletion of 5 amino acids in this domain is enough to knockout this function from the attenuated vaccine strain modified vaccinia virus Ankara (MVA). In vivo a VACV mutant lacking C16 induced higher levels of cytokines and chemokines early after infection compared to control viruses, confirming the role of this virulence factor in attenuating the innate immune response. Overall this study describes the inhibition of DNA-PK-dependent DNA sensing by a poxvirus protein, adding to the evidence that DNA-PK is a critical component of innate immunity to DNA viruses.
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Proteína Quinasa Activada por ADN/inmunología , Proteínas de Unión al ADN/inmunología , Regulación Enzimológica de la Expresión Génica/inmunología , Inmunidad Innata , Proteínas Nucleares/inmunología , Virus Vaccinia/inmunología , Vaccinia/inmunología , Proteínas Virales/inmunología , Animales , Antígenos Nucleares/genética , Antígenos Nucleares/inmunología , Antígenos Nucleares/metabolismo , Línea Celular , Proteína Quinasa Activada por ADN/biosíntesis , Proteína Quinasa Activada por ADN/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Autoantígeno Ku , Ratones Endogámicos BALB C , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Unión Proteica , Vaccinia/genética , Vaccinia/metabolismo , Virus Vaccinia/genética , Virus Vaccinia/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Preeclampsia is a syndrome occurring only in pregnancy characterized by systemic maternal inflammation and associated with the presence of the placenta. How these 2 aspects of the disease are linked has been the subject of numerous theories and ideas. Recently, there has been increasing interest in DNA shed from the placenta into the maternal circulation as a potential agent initiating the inflammatory response. This review will discuss the current evidence and future directions for placental DNA as the linking factor in preeclampsia in the context of other hypotheses.
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ADN/metabolismo , Placenta/metabolismo , Preeclampsia/metabolismo , Síndrome de Respuesta Inflamatoria Sistémica/metabolismo , Micropartículas Derivadas de Células/inmunología , Micropartículas Derivadas de Células/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , ADN/inmunología , Femenino , Humanos , Hipoxia/inmunología , Hipoxia/metabolismo , Leptina/inmunología , Leptina/metabolismo , Placenta/irrigación sanguínea , Placenta/inmunología , Preeclampsia/inmunología , Embarazo , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , Trofoblastos/citología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
To mount an efficient interferon response to virus infection, intracellular pattern recognition receptors (PRRs) sense viral nucleic acids and activate anti-viral gene transcription. The mechanisms by which intracellular DNA and DNA viruses are sensed are relevant not only to anti-viral innate immunity, but also to autoinflammation and anti-tumour immunity through the initiation of sterile inflammation by self-DNA recognition. The PRRs that directly sense and respond to viral or damaged self-DNA function by signaling to activate interferon regulatory factor (IRF)-dependent type one interferon (IFN-I) transcription. We and others have previously defined DNA-dependent protein kinase (DNA-PK) as an essential component of the DNA-dependent anti-viral innate immune system. Here, we show that DNA-PK is essential for cyclic GMP-AMP synthase (cGAS)- and stimulator of interferon genes (STING)-dependent IFN-I responses in human cells during stimulation with exogenous DNA and infection with DNA viruses.
RESUMEN
The innate immune response relies on the ability of host cells to rapidly detect and respond to microbial nucleic acids. Toll-like receptors (TLRs), a class of pattern recognition receptors (PRRs), play a fundamental role in distinguishing self from non-self at the molecular level. In this study, we focused on TLR21, an avian TLR that recognizes DNA motifs commonly found in bacterial genomic DNA, specifically unmethylated CpG motifs. TLR21 is believed to act as a functional homologue to mammalian TLR9. By analysing TLR21 signalling in chickens, we sought to elucidate avian TLR21 activation outputs in parallel to that of other nucleic acid species. Our analyses revealed that chicken TLR21 (chTLR21) triggers the activation of NF-κB and induces a potent type-I interferon response in chicken macrophages, similar to the signalling cascades observed in mammalian TLR9 activation. Notably, the transcription of interferon beta (IFNB) by chTLR21 was found to be dependent on both NF-κB and IRF7 signalling, but independent of the TBK1 kinase, a distinctive feature of mammalian TLR9 signalling. These findings highlight the conservation of critical signalling components and downstream responses between avian TLR21 and mammalian TLR9, despite their divergent evolutionary origins. These insights into the evolutionarily conserved mechanisms of nucleic acid sensing contribute to the broader understanding of host-pathogen interactions across species.
Asunto(s)
Interferón Tipo I , Ácidos Nucleicos , Animales , Pollos , Receptor Toll-Like 9 , FN-kappa B , Oligodesoxirribonucleótidos , MamíferosRESUMEN
The ability of cells to mount an interferon response to virus infections depends on intracellular nucleic acid sensing pattern recognition receptors (PRRs). RIG-I is an intracellular PRR that binds short double-stranded viral RNAs to trigger MAVS-dependent signalling. The RIG-I/MAVS signalling complex requires the coordinated activity of multiple kinases and E3 ubiquitin ligases to activate the transcription factors that drive type I and type III interferon production from infected cells. The linear ubiquitin chain assembly complex (LUBAC) regulates the activity of multiple receptor signalling pathways in both ligase-dependent and -independent ways. Here, we show that the three proteins that constitute LUBAC have separate functions in regulating RIG-I signalling. Both HOIP, the E3 ligase capable of generating M1-ubiquitin chains, and LUBAC accessory protein HOIL-1 are required for viral RNA sensing by RIG-I. The third LUBAC component, SHARPIN, is not required for RIG-I signalling. These data cement the role of LUBAC as a positive regulator of RIG-I signalling and as an important component of antiviral innate immune responses.
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Virus ARN , Ubiquitina-Proteína Ligasas , Ubiquitinación , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Transducción de Señal , Proteína 58 DEAD Box/genética , Virus ARN/metabolismoRESUMEN
Virus infection of mammalian cells is sensed by pattern recognition receptors and leads to an innate immune response that restricts virus replication and induces adaptive immunity. In response, viruses have evolved many countermeasures that enable them to replicate and be transmitted to new hosts, despite the host innate immune response. Poxviruses, such as vaccinia virus (VACV), have large DNA genomes and encode many proteins that are dedicated to host immune evasion. Some of these proteins are secreted from the infected cell, where they bind and neutralize complement factors, interferons, cytokines and chemokines. Other VACV proteins function inside cells to inhibit apoptosis or signalling pathways that lead to the production of interferons and pro-inflammatory cytokines and chemokines. In this review, these VACV immunomodulatory proteins are described and the potential to create more immunogenic VACV strains by manipulation of the gene encoding these proteins is discussed.
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Evasión Inmune/inmunología , Virus Vaccinia/inmunología , Virus Vaccinia/patogenicidad , Proteínas Virales/metabolismo , Animales , Humanos , Inmunomodulación , Vaccinia/inmunología , Vaccinia/virología , Virus Vaccinia/metabolismo , Proteínas Virales/genética , VirulenciaRESUMEN
Vaccinia virus (VACV) expresses many proteins that are non-essential for virus replication but promote virulence by inhibiting components of the host immune response to infection. These immunomodulators include a family of proteins that have, or are predicted to have, a structure related to the B-cell lymphoma (Bcl)-2 protein. Five members of the VACV Bcl-2 family (N1, B14, A52, F1 and K7) have had their crystal structure solved, others have been characterized and a function assigned (C6, A46), and others are predicted to be Bcl-2 proteins but are uncharacterized hitherto (N2, B22, C1). Data presented here show that N2 is a nuclear protein that is expressed early during infection and inhibits the activation of interferon regulatory factor (IRF)3. Consistent with its nuclear localization, N2 inhibits IRF3 downstream of the TANK-binding kinase (TBK)-1 and after IRF3 translocation into the nucleus. A mutant VACV strain Western Reserve lacking the N2L gene (vΔN2) showed normal replication and spread in cultured cells compared to wild-type parental (vN2) and revertant (vN2-rev) viruses, but was attenuated in two murine models of infection. After intranasal infection, the vΔN2 mutant induced lower weight loss and signs of illness, and virus was cleared more rapidly from the infected tissue. In the intradermal model of infection, vΔN2 induced smaller lesions that were resolved more rapidly. In summary, the N2 protein is an intracellular virulence factor that inhibits IRF3 activity in the nucleus.
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Interacciones Huésped-Patógeno , Factor 3 Regulador del Interferón/antagonistas & inhibidores , Virus Vaccinia/patogenicidad , Proteínas Virales/metabolismo , Factores de Virulencia/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Ratones , Ratones Endogámicos BALB C , Vaccinia/patología , Vaccinia/virología , Virus Vaccinia/genética , Virus Vaccinia/fisiología , Virulencia , Replicación ViralRESUMEN
Natural killer (NK) cells have an established role in controlling poxvirus infection and there is a growing interest to exploit their capabilities in the context of poxvirus-based oncolytic therapy and vaccination. How NK cells respond to poxvirus-infected cells to become activated is not well established. To address this knowledge gap, we studied the NK cell response to vaccinia virus (VACV) in vivo, using a systemic infection murine model. We found broad alterations in NK cells transcriptional activity in VACV-infected mice, consistent with both direct target cell recognition and cytokine exposure. There were also alterations in the expression levels of specific NK surface receptors (NKRs), including the Ly49 family and SLAM receptors, as well as upregulation of memory-associated NK markers. Despite the latter observation, adoptive transfer of VACV-expercienced NK populations did not confer protection from infection. Comparison with the NK cell response to murine cytomegalovirus (MCMV) infection highlighted common features, but also distinct NK transcriptional programmes initiated by VACV. Finally, there was a clear overlap between the NK transcriptional response in humans vaccinated with an attenuated VACV, modified vaccinia Ankara (MVA), demonstrating conservation between the NK response in these different host species. Overall, this study provides new data about NK cell activation, function, and homeostasis during VACV infection, and may have implication for the design of VACV-based therapeutics.
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Poxviridae , Vaccinia , Ratones , Humanos , Animales , Virus Vaccinia/fisiología , Células Asesinas Naturales/metabolismo , Citocinas/metabolismoRESUMEN
IMPORTANCE: One of the fundamental features that make viruses intracellular parasites is the necessity to use cellular translational machinery. Hence, this is a crucial checkpoint for controlling infections. Here, we show that dengue and Zika viruses, responsible for nearly 400 million infections every year worldwide, explore such control for optimal replication. Using immunocompetent cells, we demonstrate that arrest of protein translations happens after sensing of dsRNA and that the information required to avoid this blocking is contained in viral 5'-UTR. Our work, therefore, suggests that the non-canonical translation described for these viruses is engaged when the intracellular stress response is activated.
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Virus del Dengue , Estrés Fisiológico , Replicación Viral , Virus Zika , eIF-2 Quinasa , Animales , Humanos , Células A549 , Chlorocebus aethiops , Dengue/inmunología , Dengue/virología , Virus del Dengue/fisiología , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Eliminación de Gen , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/inmunología , Estrés Fisiológico/genética , Estrés Fisiológico/inmunología , Células Vero , Replicación Viral/genética , Replicación Viral/inmunología , Virus Zika/fisiología , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/virología , ARN Bicatenario/metabolismoRESUMEN
The IκB kinase (IKK) complex regulates activation of NF-κB, a critical transcription factor in mediating inflammatory and immune responses. Not surprisingly, therefore, many viruses seek to inhibit NF-κB activation. The vaccinia virus B14 protein contributes to virus virulence by binding to the IKKß subunit of the IKK complex and preventing NF-κB activation in response to pro-inflammatory stimuli. Previous crystallographic studies showed that the B14 protein has a Bcl-2-like fold and forms homodimers in the crystal. However, multi-angle light scattering indicated that B14 is in monomer-dimer equilibrium in solution. This transient self-association suggested that the hydrophobic dimerization interface of B14 might also mediate its interaction with IKKß, and this was investigated by introducing amino acid substitutions on the dimer interface. One mutant (Y35E) was entirely monomeric but still co-immunoprecipitated with IKKß and blocked both NF-κB nuclear translocation and NF-κB-dependent gene expression. Therefore, B14 homodimerization is nonessential for binding and inhibition of IKKß. In contrast, a second monomeric mutant (F130K) neither bound IKKß nor inhibited NF-κB-dependent gene expression, demonstrating that this residue is required for the B14-IKKß interaction. Thus, the dimerization and IKKß-binding interfaces overlap and lie on a surface used for protein-protein interactions in many viral and cellular Bcl-2-like proteins.