RESUMEN
Zika virus (ZIKV) infection in pregnant women causes intrauterine growth restriction, spontaneous abortion, and microcephaly. Here, we describe two mouse models of placental and fetal disease associated with in utero transmission of ZIKV. Female mice lacking type I interferon signaling (Ifnar1(-/-)) crossed to wild-type (WT) males produced heterozygous fetuses resembling the immune status of human fetuses. Maternal inoculation at embryonic day 6.5 (E6.5) or E7.5 resulted in fetal demise that was associated with ZIKV infection of the placenta and fetal brain. We identified ZIKV within trophoblasts of the maternal and fetal placenta, consistent with a trans-placental infection route. Antibody blockade of Ifnar1 signaling in WT pregnant mice enhanced ZIKV trans-placental infection although it did not result in fetal death. These models will facilitate the study of ZIKV pathogenesis, in utero transmission, and testing of therapies and vaccines to prevent congenital malformations.
Asunto(s)
Modelos Animales de Enfermedad , Enfermedades Fetales/virología , Enfermedades Placentarias/virología , Complicaciones Infecciosas del Embarazo/virología , Infección por el Virus Zika/patología , Virus Zika/fisiología , Animales , Apoptosis , Encéfalo/embriología , Encéfalo/patología , Encéfalo/virología , Femenino , Enfermedades Fetales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedades Placentarias/patología , Embarazo , Complicaciones Infecciosas del Embarazo/patología , ARN Viral/aislamiento & purificación , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Infección por el Virus Zika/virologíaRESUMEN
Alternative (M2) activation of macrophages driven via the α-chain of the receptor for interleukin 4 (IL-4Rα) is important for immunity to parasites, wound healing, the prevention of atherosclerosis and metabolic homeostasis. M2 polarization is dependent on fatty acid oxidation (FAO), but the source of the fatty acids that support this metabolic program has not been clear. We found that the uptake of triacylglycerol substrates via the scavenger receptor CD36 and their subsequent lipolysis by lysosomal acid lipase (LAL) was important for the engagement of elevated oxidative phosphorylation, enhanced spare respiratory capacity (SRC), prolonged survival and expression of genes that together define M2 activation. Inhibition of lipolysis suppressed M2 activation during infection with a parasitic helminth and blocked protective responses to this pathogen. Our findings delineate a critical role for cell-intrinsic lysosomal lipolysis in M2 activation.
Asunto(s)
Antígenos CD36/inmunología , Ácidos Grasos/metabolismo , Interleucina-4/inmunología , Lipólisis/inmunología , Lisosomas/inmunología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Fosforilación Oxidativa , Transducción de Señal/inmunología , Esterol Esterasa/inmunología , Animales , Respiración de la Célula , Helmintiasis Animal/inmunología , Humanos , Ratones , Consumo de Oxígeno , Receptores de Interleucina-4/inmunología , TranscriptomaRESUMEN
The ligation of Toll-like receptors (TLRs) leads to rapid activation of dendritic cells (DCs). However, the metabolic requirements that support this process remain poorly defined. We found that DC glycolytic flux increased within minutes of exposure to TLR agonists and that this served an essential role in supporting the de novo synthesis of fatty acids for the expansion of the endoplasmic reticulum and Golgi required for the production and secretion of proteins that are integral to DC activation. Signaling via the kinases TBK1, IKKÉ and Akt was essential for the TLR-induced increase in glycolysis by promoting the association of the glycolytic enzyme HK-II with mitochondria. In summary, we identified the rapid induction of glycolysis as an integral component of TLR signaling that is essential for the anabolic demands of the activation and function of DCs.
Asunto(s)
Células Dendríticas/inmunología , Glucólisis , Quinasa I-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Linfocitos T/inmunología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Ácidos Grasos/biosíntesis , Glucólisis/efectos de los fármacos , Glucólisis/genética , Glucólisis/inmunología , Hexoquinasa/metabolismo , Quinasa I-kappa B/genética , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Receptores Toll-Like/agonistasRESUMEN
Macrophage activation status is intrinsically linked to metabolic remodeling. Macrophages stimulated by interleukin 4 (IL-4) to become alternatively (or, M2) activated increase fatty acid oxidation and oxidative phosphorylation; these metabolic changes are critical for M2 activation. Enhanced glucose utilization is also characteristic of the M2 metabolic signature. Here, we found that increased glucose utilization is essential for M2 activation. Increased glucose metabolism in IL-4-stimulated macrophages required the activation of the mTORC2 pathway, and loss of mTORC2 in macrophages suppressed tumor growth and decreased immunity to a parasitic nematode. Macrophage colony stimulating factor (M-CSF) was implicated as a contributing upstream activator of mTORC2 in a pathway that involved PI3K and AKT. mTORC2 operated in parallel with the IL-4Rα-Stat6 pathway to facilitate increased glycolysis during M2 activation via the induction of the transcription factor IRF4. IRF4 expression required both mTORC2 and Stat6 pathways, providing an underlying mechanism to explain how glucose utilization is increased to support M2 activation.
Asunto(s)
Factores Reguladores del Interferón/metabolismo , Activación de Macrófagos/fisiología , Macrófagos/fisiología , Complejos Multiproteicos/metabolismo , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Interleucina-4/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción STAT6/metabolismoRESUMEN
Greater understanding of the complex host responses induced by type 1 interferon (IFN) cytokines could allow new therapeutic approaches for diseases in which these cytokines are implicated. We found that in response to the Toll-like receptor-9 agonist CpGA, plasmacytoid dendritic cells (pDC) produced type 1 IFNs, which, through an autocrine type 1 IFN receptor-dependent pathway, induced changes in cellular metabolism characterized by increased fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS). Direct inhibition of FAO and of pathways that support this process, such as fatty acid synthesis, prevented full pDC activation. Type 1 IFNs also induced increased FAO and OXPHOS in non-hematopoietic cells and were found to be responsible for increased FAO and OXPHOS in virus-infected cells. Increased FAO and OXPHOS in response to type 1 IFNs was regulated by PPARα. Our findings reveal FAO, OXPHOS and PPARα as potential targets to therapeutically modulate downstream effects of type 1 IFNs.
Asunto(s)
Células Dendríticas/inmunología , Interferón Tipo I/metabolismo , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , PPAR alfa/metabolismo , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Acetil-CoA C-Aciltransferasa/metabolismo , Animales , Isomerasas de Doble Vínculo Carbono-Carbono/metabolismo , Diferenciación Celular , Células Cultivadas , Islas de CpG/inmunología , Enoil-CoA Hidratasa/metabolismo , Regulación de la Expresión Génica , Inmunidad , Metabolismo de los Lípidos , Ratones , Ratones Endogámicos C57BL , Oligodesoxirribonucleótidos/inmunología , Fosforilación Oxidativa , Racemasas y Epimerasas/metabolismo , Receptores de Interferón/metabolismo , Transducción de Señal , Receptor Toll-Like 9/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.pcbi.1009480.].
RESUMEN
To understand the diversity of immune responses to SARS-CoV-2 and distinguish features that predispose individuals to severe COVID-19, we developed a mechanistic, within-host mathematical model and virtual patient cohort. Our results suggest that virtual patients with low production rates of infected cell derived IFN subsequently experienced highly inflammatory disease phenotypes, compared to those with early and robust IFN responses. In these in silico patients, the maximum concentration of IL-6 was also a major predictor of CD8+ T cell depletion. Our analyses predicted that individuals with severe COVID-19 also have accelerated monocyte-to-macrophage differentiation mediated by increased IL-6 and reduced type I IFN signalling. Together, these findings suggest biomarkers driving the development of severe COVID-19 and support early interventions aimed at reducing inflammation.
Asunto(s)
COVID-19/inmunología , Modelos Inmunológicos , SARS-CoV-2 , Biomarcadores/metabolismo , Linfocitos T CD8-positivos/inmunología , COVID-19/virología , Estudios de Cohortes , Biología Computacional , Simulación por Computador , Susceptibilidad a Enfermedades/inmunología , Interacciones Microbiota-Huesped/inmunología , Humanos , Inmunidad Innata , Terapia de Inmunosupresión , Interferones/metabolismo , Interleucina-6/metabolismo , Macrófagos/inmunología , Pandemias , SARS-CoV-2/inmunología , Índice de Severidad de la Enfermedad , Interfaz Usuario-ComputadorRESUMEN
Nonresolving inflammation expands a heterogeneous population of myeloid suppressor cells capable of inhibiting T cell function. This heterogeneity has confounded the functional dissection of individual myeloid subpopulations and presents an obstacle for antitumor immunity and immunotherapy. Using genetic manipulation of cell death pathways, we found the monocytic suppressor-cell subset, but not the granulocytic subset, requires continuous c-FLIP expression to prevent caspase-8-dependent, RIPK3-independent cell death. Development of the granulocyte subset requires MCL-1-mediated control of the intrinsic mitochondrial death pathway. Monocytic suppressors tolerate the absence of MCL-1 provided cytokines increase expression of the MCL-1-related protein A1. Monocytic suppressors mediate T cell suppression, whereas their granulocytic counterparts lack suppressive function. The loss of the granulocytic subset via conditional MCL-1 deletion did not alter tumor incidence implicating the monocytic compartment as the functionally immunosuppressive subset in vivo. Thus, death pathway modulation defines the development, survival, and function of myeloid suppressor cells.
Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Granulocitos/fisiología , Monocitos/fisiología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Células Mieloides/fisiología , Neoplasias Experimentales/inmunología , Animales , Apoptosis/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Linfocitos T CD8-positivos/inmunología , Carcinogénesis/genética , Caspasa 8/metabolismo , Diferenciación Celular/genética , Línea Celular Tumoral , Linaje de la Célula/genética , Técnicas de Cocultivo , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Tolerancia Inmunológica/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Antígenos de Histocompatibilidad Menor , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Trasplante de Neoplasias , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal/genéticaRESUMEN
Generation of CD8(+) memory T cells requires metabolic reprogramming that is characterized by enhanced mitochondrial fatty-acid oxidation (FAO). However, where the fatty acids (FA) that fuel this process come from remains unclear. While CD8(+) memory T cells engage FAO to a greater extent, we found that they acquired substantially fewer long-chain FA from their external environment than CD8(+) effector T (Teff) cells. Rather than using extracellular FA directly, memory T cells used extracellular glucose to support FAO and oxidative phosphorylation (OXPHOS), suggesting that lipids must be synthesized to generate the substrates needed for FAO. We have demonstrated that memory T cells rely on cell intrinsic expression of the lysosomal hydrolase LAL (lysosomal acid lipase) to mobilize FA for FAO and memory T cell development. Our observations link LAL to metabolic reprogramming in lymphocytes and show that cell intrinsic lipolysis is deterministic for memory T cell fate.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Ácidos Grasos/metabolismo , Memoria Inmunológica/inmunología , Lipólisis/inmunología , Esterol Esterasa/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacología , Traslado Adoptivo , Animales , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ácido Graso Sintasas/antagonistas & inhibidores , Ácido Graso Sintasas/genética , Ácidos Grasos/biosíntesis , Glucosa/metabolismo , Interleucina-15/inmunología , Interleucina-2/inmunología , Lipólisis/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/metabolismo , Oxidación-Reducción , Fosforilación Oxidativa , Oxígeno/metabolismo , Proteínas Quinasas/genética , Interferencia de ARN , ARN Interferente Pequeño , Esterol Esterasa/biosíntesisRESUMEN
Human parainfluenza viruses (HPIVs) are a leading cause of acute respiratory infection hospitalization in children, yet little is known about how dose, strain, tissue tropism, and individual heterogeneity affects the processes driving growth and clearance kinetics. Longitudinal measurements are possible by using reporter Sendai viruses, the murine counterpart of HPIV 1, that express luciferase, where the insertion location yields a wild-type (rSeV-luc(M-F*)) or attenuated (rSeV-luc(P-M)) phenotype. Bioluminescence from individual animals suggests that there is a rapid increase in expression followed by a peak, biphasic clearance, and resolution. However, these kinetics vary between individuals and with dose, strain, and whether the infection was initiated in the upper and/or lower respiratory tract. To quantify the differences, we translated the bioluminescence measurements from the nasopharynx, trachea, and lung into viral loads and used a mathematical model together a nonlinear mixed effects approach to define the mechanisms distinguishing each scenario. The results confirmed a higher rate of virus production with the rSeV-luc(M-F*) virus compared to its attenuated counterpart, and suggested that low doses result in disproportionately fewer infected cells. The analyses indicated faster infectivity and infected cell clearance rates in the lung and that higher viral doses, and concomitantly higher infected cell numbers, resulted in more rapid clearance. This parameter was also highly variable amongst individuals, which was particularly evident during infection in the lung. These critical differences provide important insight into distinct HPIV dynamics, and show how bioluminescence data can be combined with quantitative analyses to dissect host-, virus-, and dose-dependent effects.
Asunto(s)
Pulmón/virología , Infecciones por Paramyxoviridae/fisiopatología , Infecciones del Sistema Respiratorio/virología , Animales , Interacciones Huésped-Patógeno , Humanos , Luciferasas/genética , Luminiscencia , Ratones , Infecciones del Sistema Respiratorio/fisiopatología , Virus Sendai/genéticaRESUMEN
The endpoint dilution assay's output, the 50% infectious dose (ID50), is calculated using the Reed-Muench or Spearman-Kärber mathematical approximations, which are biased and often miscalculated. We introduce a replacement for the ID50 that we call Specific INfection (SIN) along with a free and open-source web-application, midSIN (https://midsin.physics.ryerson.ca) to calculate it. midSIN computes a virus sample's SIN concentration using Bayesian inference based on the results of a standard endpoint dilution assay, and requires no changes to current experimental protocols. We analyzed influenza and respiratory syncytial virus samples using midSIN and demonstrated that the SIN/mL reliably corresponds to the number of infections a sample will cause per mL. It can therefore be used directly to achieve a desired multiplicity of infection, similarly to how plaque or focus forming units (PFU, FFU) are used. midSIN's estimates are shown to be more accurate and robust than the Reed-Muench and Spearman-Kärber approximations. The impact of endpoint dilution plate design choices (dilution factor, replicates per dilution) on measurement accuracy is also explored. The simplicity of SIN as a measure and the greater accuracy provided by midSIN make them an easy and superior replacement for the TCID50 and other in vitro culture ID50 measures. We hope to see their universal adoption to measure the infectivity of virus samples.
Asunto(s)
Bioensayo/métodos , Biología Computacional/métodos , Ensayo de Placa Viral/métodos , Virosis/virología , Teorema de BayesRESUMEN
Influenza virus infections are a leading cause of morbidity and mortality worldwide. This is due in part to the continual emergence of new viral variants and to synergistic interactions with other viruses and bacteria. There is a lack of understanding about how host responses work to control the infection and how other pathogens capitalize on the altered immune state. The complexity of multi-pathogen infections makes dissecting contributing mechanisms, which may be non-linear and occur on different time scales, challenging. Fortunately, mathematical models have been able to uncover infection control mechanisms, establish regulatory feedbacks, connect mechanisms across time scales, and determine the processes that dictate different disease outcomes. These models have tested existing hypotheses and generated new hypotheses, some of which have been subsequently tested and validated in the laboratory. They have been particularly a key in studying influenza-bacteria coinfections and will be undoubtedly be useful in examining the interplay between influenza virus and other viruses. Here, I review recent advances in modeling influenza-related infections, the novel biological insight that has been gained through modeling, the importance of model-driven experimental design, and future directions of the field.
Asunto(s)
Infecciones Bacterianas/inmunología , Gripe Humana/inmunología , Modelos Inmunológicos , Infecciones por Orthomyxoviridae/inmunología , Orthomyxoviridae/fisiología , Animales , Coinfección , Interacciones Huésped-Patógeno , Humanos , Modelos TeóricosRESUMEN
BACKGROUND: Secondary bacterial coinfections are ranked as a leading cause of hospitalization and morbid conditions associated with influenza. Because vitamin A deficiency (VAD) and insufficiency are frequent in both developed and developing countries, we asked how VAD influences coinfection severity. METHODS: VAD and control mice were infected with influenza virus for evaluation of inflammatory cytokines, cellular immune responses, and viral clearance. Influenza-infected mice were coinfected with Streptococcus pneumoniae to study weight loss and survival. RESULTS: Naive VAD mouse lungs exhibited dysregulated immune function. Neutrophils were enhanced in frequency and there was a significant reduction in RANTES (regulated on activation of normal T cells expressed and secreted), a chemokine instrumental in T-cell homing and recruitment. After influenza virus infection, VAD mice experienced failures in CD4+ T-cell recruitment and B-cell organization into lymphoid structures in the lung. VAD mice exhibited higher viral titers than controls and slow viral clearance. There were elevated levels of inflammatory cytokines and innate cell subsets in the lungs. However, arginase, a marker of alternatively activated M2 macrophages, was rare. When influenza-infected VAD animals were exposed to bacteria, they experienced a 100% mortality rate. CONCLUSION: Data showed that VAD dysregulated the immune response. Consequently, secondary bacterial infections were 100% lethal in influenza-infected VAD mice.
Asunto(s)
Coinfección , Infecciones por Orthomyxoviridae , Infecciones Neumocócicas/complicaciones , Deficiencia de Vitamina A , Animales , Citocinas , Inmunidad , Pulmón , Ratones , Ratones Endogámicos C57BL , Orthomyxoviridae , Infecciones por Orthomyxoviridae/complicaciones , Infecciones Neumocócicas/mortalidad , Streptococcus pneumoniae , Deficiencia de Vitamina A/complicacionesRESUMEN
Streptococcus pneumoniae (pneumococcus) is one of the primary bacterial pathogens that complicates influenza virus infections. These bacterial coinfections increase influenza-associated morbidity and mortality through a number of immunological and viral-mediated mechanisms, but the specific bacterial genes that contribute to postinfluenza pathogenicity are not known. Here, we used genome-wide transposon mutagenesis (Tn-Seq) to reveal bacterial genes that confer improved fitness in influenza virus-infected hosts. The majority of the 32 genes identified are involved in bacterial metabolism, including nucleotide biosynthesis, amino acid biosynthesis, protein translation, and membrane transport. We generated mutants with single-gene deletions (SGD) of five of the genes identified, SPD1414, SPD2047 (cbiO1), SPD0058 (purD), SPD1098, and SPD0822 (proB), to investigate their effects on in vivo fitness, disease severity, and host immune responses. The growth of the SGD mutants was slightly attenuated in vitro and in vivo, but each still grew to high titers in the lungs of mock- and influenza virus-infected hosts. Despite high bacterial loads, mortality was significantly reduced or delayed with all SGD mutants. Time-dependent reductions in pulmonary neutrophils, inflammatory macrophages, and select proinflammatory cytokines and chemokines were also observed. Immunohistochemical staining further revealed altered neutrophil distribution with reduced degeneration in the lungs of influenza virus-SGD mutant-coinfected animals. These studies demonstrate a critical role for specific bacterial genes and for bacterial metabolism in driving virulence and modulating immune function during influenza-associated bacterial pneumonia.
Asunto(s)
Coinfección , Aptitud Genética , Interacciones Huésped-Patógeno , Virus de la Influenza A , Gripe Humana/virología , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/fisiología , Proteínas Bacterianas/genética , Citocinas/metabolismo , Interacciones Huésped-Patógeno/inmunología , Humanos , Mediadores de Inflamación , Virus de la Influenza A/inmunología , Leucocitos/inmunología , Leucocitos/metabolismo , Mutación , Infecciones Neumocócicas/inmunología , Infecciones Neumocócicas/patologíaRESUMEN
We previously generated STING N153S knock-in mice that have a human disease-associated gain-of-function mutation in STING. Patients with this mutation (STING N154S in humans) develop STING-associated vasculopathy with onset in infancy (SAVI), a severe pediatric autoinflammatory disease characterized by pulmonary fibrosis. Since this mutation promotes the upregulation of antiviral type I interferon-stimulated genes (ISGs), we hypothesized that STING N153S knock-in mice may develop more severe autoinflammatory disease in response to a virus challenge. To test this hypothesis, we infected heterozygous STING N153S mice with murine gammaherpesvirus 68 (γHV68). STING N153S mice were highly vulnerable to infection and developed pulmonary fibrosis after infection. In addition to impairing CD8+ T cell responses and humoral immunity, STING N153S also promoted the replication of γHV68 in cultured macrophages. In further support of a combined innate and adaptive immunodeficiency, γHV68 infection was more severe in Rag1-/- STING N153S mice than in Rag1-/- littermate mice, which completely lack adaptive immunity. Thus, a gain-of-function STING mutation creates a combined innate and adaptive immunodeficiency that leads to virus-induced pulmonary fibrosis.IMPORTANCE A variety of human rheumatologic disease-causing mutations have recently been identified. Some of these mutations are found in viral nucleic acid-sensing proteins, but whether viruses can influence the onset or progression of these human diseases is less well understood. One such autoinflammatory disease, called STING-associated vasculopathy with onset in infancy (SAVI), affects children and leads to severe lung disease. We generated mice with a SAVI-associated STING mutation and infected them with γHV68, a common DNA virus that is related to human Epstein-Barr virus. Mice with the human disease-causing STING mutation were more vulnerable to infection than wild-type littermate control animals. Furthermore, the STING mutant mice developed lung fibrosis similar to that of patients with SAVI. These findings reveal that a human STING mutation creates severe immunodeficiency, leading to virus-induced lung disease in mice.
Asunto(s)
Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Fibrosis Pulmonar/genética , Inmunidad Adaptativa/genética , Animales , Mutación con Ganancia de Función/genética , Gammaherpesvirinae/metabolismo , Gammaherpesvirinae/fisiología , Síndromes de Inmunodeficiencia , Inflamación/genética , Pulmón/virología , Macrófagos/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación , Fibrosis Pulmonar/metabolismo , Transducción de Señal , Linfocitos T/metabolismoRESUMEN
The lentiviral protein Viral Infectivity Factor (Vif) counteracts the antiviral effects of host APOBEC3 (A3) proteins and contributes to persistent HIV infection. Vif targets A3 restriction factors for ubiquitination and proteasomal degradation by recruiting them to a multi-protein ubiquitin E3 ligase complex. Here, we describe a degradation-independent mechanism of Vif-mediated antagonism that was revealed through detailed structure-function studies of antibody antigen-binding fragments (Fabs) to the Vif complex. Two Fabs were found to inhibit Vif-mediated A3 neutralization through distinct mechanisms: shielding A3 from ubiquitin transfer and blocking Vif E3 assembly. Combined biochemical, cell biological and structural studies reveal that disruption of Vif E3 assembly inhibited A3 ubiquitination but was not sufficient to restore its packaging into viral particles and antiviral activity. These observations establish that Vif can neutralize A3 family members in a degradation-independent manner. Additionally, this work highlights the potential of Fabs as functional probes, and illuminates how Vif uses a multi-pronged approach involving both degradation dependent and independent mechanisms to suppress A3 innate immunity.
Asunto(s)
Antivirales/farmacología , Citosina Desaminasa/metabolismo , Fragmentos Fab de Inmunoglobulinas/química , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/inmunología , Desaminasas APOBEC , Antivirales/química , Proteínas Cullin/química , Proteínas Cullin/metabolismo , Citidina Desaminasa , Células HEK293 , Infecciones por VIH/inmunología , Infecciones por VIH/terapia , Infecciones por VIH/virología , VIH-1/inmunología , VIH-1/metabolismo , Humanos , Ubiquitina/metabolismo , Ubiquitinación , Ensamble de Virus , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
Toll-like receptor (TLR) signaling in macrophages is required for antipathogen responses, including the biosynthesis of nitric oxide from arginine, and is essential for immunity to Mycobacterium tuberculosis, Toxoplasma gondii and other intracellular pathogens. Here we report a 'loophole' in the TLR pathway that is advantageous to these pathogens. Intracellular pathogens induced expression of the arginine hydrolytic enzyme arginase 1 (Arg1) in mouse macrophages through the TLR pathway. In contrast to diseases dominated by T helper type 2 responses in which Arg1 expression is greatly increased by interleukin 4 and 13 signaling through the transcription factor STAT6, TLR-mediated Arg1 induction was independent of the STAT6 pathway. Specific elimination of Arg1 in macrophages favored host survival during T. gondii infection and decreased lung bacterial load during tuberculosis infection.
Asunto(s)
Arginasa/inmunología , Infecciones Bacterianas/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Receptores Toll-Like/inmunología , Animales , Arginasa/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/inmunología , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Immunoblotting , Inmunohistoquímica , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Factor de Transcripción STAT6/inmunología , Factor de Transcripción STAT6/metabolismo , Receptores Toll-Like/metabolismoRESUMEN
Viruses are obligate parasites that rely heavily on host cellular processes for replication. The small number of proteins typically encoded by a virus is faced with selection pressures that lead to the evolution of distinctive structural properties, allowing each protein to maintain its function under constraints such as small genome size, high mutation rate, and rapidly changing fitness conditions. One common strategy for this evolution is to utilize small building blocks to generate protein oligomers that assemble in multiple ways, thereby diversifying protein function and regulation. In this review, we discuss specific cases that illustrate how oligomerization is used to generate a single defined functional state, to modulate activity via different oligomeric states, or to generate multiple functional forms via different oligomeric states.