RESUMEN
Background: It remains unclear whether BPIV3 infection leads to stress granules formation and whether G3BP1 plays a role in this process and in viral replication. This study aims to clarify the association between BPIV3 and stress granules, explore the effect of G3BP1 on BPIV3 replication, and provide significant insights into the mechanisms by which BPIV3 evades the host's antiviral immunity to support its own survival. Methods: Here, we use Immunofluorescence staining to observe the effect of BPIV3 infection on the assembly of stress granules. Meanwhile, the expression changes of eIF2α and G3BP1 were determined. Overexpression or siRNA silencing of intracellular G3BP1 levels was examined for its regulatory control of BPIV3 replication. Results: We identify that the BPIV3 infection elicited phosphorylation of the eIF2α protein. However, it did not induce the assembly of stress granules; rather, it inhibited the formation of stress granules and downregulated the expression of G3BP1. G3BP1 overexpression facilitated the formation of stress granules within cells and hindered viral replication, while G3BP1 knockdown enhanced BPIV3 expression. Conclusion: This study suggest that G3BP1 plays a crucial role in BPIV3 suppressing stress granule formation and viral replication.
Asunto(s)
ADN Helicasas , Proteínas de Unión a Poli-ADP-Ribosa , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Gránulos de Estrés , Replicación Viral , Animales , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , ADN Helicasas/metabolismo , ADN Helicasas/genética , ARN Helicasas/metabolismo , ARN Helicasas/genética , Gránulos de Estrés/metabolismo , Bovinos , Factor 2 Eucariótico de Iniciación/metabolismo , Infecciones por Respirovirus/inmunología , Infecciones por Respirovirus/metabolismo , Interacciones Huésped-Patógeno/inmunología , Fosforilación , Línea Celular , Gránulos Citoplasmáticos/metabolismoRESUMEN
Viruses constantly evolve and adapt to the antiviral defenses of their hosts. The biology of viral circumvention of these selective pressures can often be attributed to the acquisition of novel antagonistic gene products or by rapid genome change that prevents host recognition. To study viral evasion of RNA interference (RNAi)-based defenses, we established a robust antiviral system in mammalian cells using recombinant Sendai virus designed to be targeted by endogenous host microRNAs (miRNAs) with perfect complementarity. Using this system, we previously demonstrated the intrinsic ability of positive-strand RNA viruses to escape this selective pressure via homologous recombination, which was not observed in negative-strand RNA viruses. Here, we show that given extensive time, escape of miRNA-targeted Sendai virus was enabled by host adenosine deaminase acting on RNA 1 (ADAR1). Independent of the viral transcript targeted, ADAR1 editing resulted in disruption of the miRNA-silencing motif, suggesting an intolerance for extensive RNA-RNA interactions necessary for antiviral RNAi. This was further supported in Nicotiana benthamiana, where exogenous expression of ADAR1 interfered with endogenous RNAi. Together, these results suggest that ADAR1 diminishes the effectiveness of RNAi and may explain why it is absent in species that utilize this antiviral defense system. IMPORTANCE All life at the cellular level has the capacity to induce an antiviral response. Here, we examine the result of imposing the antiviral response of one branch of life onto another and find evidence for conflict. To determine the consequences of eliciting an RNAi-like defense in mammals, we applied this pressure to a recombinant Sendai virus in cell culture. We find that ADAR1, a host gene involved in regulation of the mammalian response to virus, prevented RNAi-mediated silencing and subsequently allowed for viral replication. In addition, the expression of ADAR1 in Nicotiana benthamiana, which lacks ADARs and has an endogenous RNAi system, suppresses gene silencing. These data indicate that ADAR1 is disruptive to RNAi biology and provide insight into the evolutionary relationship between ADARs and antiviral defenses in eukaryotic life.
Asunto(s)
Adenosina Desaminasa , Interacciones Microbiota-Huesped , MicroARNs , Interferencia de ARN , Infecciones por Respirovirus , Animales , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Antivirales/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Replicación Viral/genética , Virus Sendai/clasificación , Silenciador del Gen , Humanos , Mutación , Sistemas de Lectura Abierta , Evolución Biológica , Interacciones Microbiota-Huesped/genética , Infecciones por Respirovirus/metabolismo , Infecciones por Respirovirus/virologíaRESUMEN
Cells must control genes that are induced by virus infection to mitigate deleterious consequences of inflammation. We investigated the mechanisms whereby Keap1 moderates the transcription of genes that are induced by Sendai virus infection in mouse embryo fibroblasts (MEFs). Keap1-/- deletions increased the transcription of virus induced genes independently of Nrf2. Keap1 moderated early virus induced gene transcription. Virus infection induced Keap1 to bind Ifnb1, Tnf and Il6, and reduced Keap1 binding at Cdkn1a and Ccng1. Virus infection induced G9a-GLP and NFκB p50 recruitment, and H3K9me2 deposition. Keap1-/- deletions eliminated G9a-GLP and NFκB p50 recruitment, and H3K9me2 deposition, but they did not affect NFκB p65, IRF3 or cJun recruitment. G9a-GLP inhibitors (BIX01294, MS012, BRD4770) enhanced virus induced gene transcription in MEFs with intact Keap1, but not in MEFs with Keap1-/- deletions. G9a-GLP inhibitors augmented Keap1 binding to virus induced genes in infected MEFs, and to cell cycle genes in uninfected MEFs. G9a-GLP inhibitors augmented NFκB subunit recruitment in MEFs with intact Keap1. G9a-GLP inhibitors stabilized Keap1 retention in permeabilized MEFs. G9a-GLP lysine methyltransferase activity was required for Keap1 to moderate transcription, and it moderated Keap1 binding to chromatin. The interdependent effects of Keap1 and G9a-GLP on the recruitment of each other and on the moderation of virus induced gene transcription constitute a feedback circuit. Keap1 and the electrophile tBHQ reduced virus induced gene transcription through different mechanisms, and they regulated the recruitment of different NFκB subunits. Characterization of the mechanisms whereby Keap1, G9a-GLP and NFκB p50 moderate virus induced gene transcription can facilitate the development of immunomodulatory agents.
Asunto(s)
N-Metiltransferasa de Histona-Lisina , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2 , Infecciones por Respirovirus/metabolismo , Animales , Cromatina , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Ratones , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , Virus Sendai/fisiologíaRESUMEN
Cystic fibrosis (CF) is an autosomal recessive gene disorder that affects tens of thousands of patients worldwide. Individuals with CF often succumb to progressive lung disease and respiratory failure following recurrent infections with bacteria. Viral infections can also damage the lungs and heighten the CF patient's susceptibility to bacterial infections and long-term sequelae. Vitamin A is a key nutrient important for immune health and epithelial cell integrity, but there is currently no consensus as to whether vitamin A should be monitored in CF patients. Here we evaluate previous literature and present results from a CF mouse model, showing that oral vitamin A supplements significantly reduce lung lesions that would otherwise persist for 5-6 weeks post-virus exposure. Based on these results, we encourage continued research and suggest that programs for the routine monitoring and regulation of vitamin A levels may help reduce virus-induced lung pathology in CF patients.
Asunto(s)
Fibrosis Quística/metabolismo , Pulmón/patología , Infecciones por Respirovirus/metabolismo , Virus Sendai/fisiología , Vitamina A/metabolismo , Animales , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Suplementos Dietéticos , Modelos Animales de Enfermedad , Proteínas de Unión a Ácidos Grasos/genética , Humanos , Pulmón/virología , Ratones , Ratones Endogámicos CFTR , Ratones Transgénicos , Regiones Promotoras Genéticas , Vitamina A/administración & dosificaciónRESUMEN
Human parainfluenza virus type 1 (hPIV1) and 3 (hPIV3) cause seasonal epidemics, but little is known about their interaction with human airway cells. In this study, we determined cytopathology, replication, and progeny virion release from human airway cells during long-term infection in vitro. Both viruses readily established persistent infection without causing significant cytopathic effects. However, assembly and release of hPIV1 rapidly declined in sharp contrast to hPIV3 due to impaired viral ribonucleocapsid (vRNP) trafficking and virus assembly. Transcriptomic analysis revealed that both viruses induced similar levels of type I and III IFNs. However, hPIV1 induced specific ISGs stronger than hPIV3, such as MX2, which bound to hPIV1 vRNPs in infected cells. In addition, hPIV1 but not hPIV3 suppressed genes involved in lipid biogenesis and hPIV1 infection resulted in ubiquitination and degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a rate limiting enzyme in cholesterol biosynthesis. Consequently, formation of cholesterol-rich lipid rafts was impaired in hPIV1 infected cells. These results indicate that hPIV1 is capable of regulating cholesterol biogenesis, which likely together with ISGs contributes to establishment of a quiescent infection.
Asunto(s)
Colesterol/biosíntesis , Mucosa Respiratoria/virología , Infecciones por Respirovirus/metabolismo , Infecciones por Respirovirus/virología , Células A549 , Humanos , Interferones/inmunología , Virus de la Parainfluenza 1 Humana/inmunología , Virus de la Parainfluenza 1 Humana/metabolismo , Virus de la Parainfluenza 3 Humana/inmunología , Virus de la Parainfluenza 3 Humana/metabolismo , Infecciones por Respirovirus/inmunologíaRESUMEN
Human Parainfluenza Virus Type 3 (HPIV3) is one of the main pathogens that cause acute lower respiratory tract infections in infants and young children. However, there are currently no effective antiviral drugs and vaccines. Herein, we found that a natural compound, curcumin, inhibits HPIV3 infection and has antiviral effects on entry and replication of the virus life cycle. Immunofluorescence and western blotting experiments revealed that curcumin disrupts F-actin and inhibits viral inclusion body (IB) formation, thus inhibiting virus replication. Curcumin can also downregulate cellular PI4KB and interrupt its colocalization in viral IBs. This study verified the antiviral ability of curcumin on HPIV3 infection and preliminarily elucidated its influence on viral replication, providing a theoretical basis for antiviral drug development of HPIV3 and other parainfluenza viruses.
Asunto(s)
Curcumina/farmacología , Cuerpos de Inclusión Viral/metabolismo , Virus de la Parainfluenza 3 Humana/fisiología , Infecciones por Respirovirus/metabolismo , 1-Fosfatidilinositol 4-Quinasa/genética , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Células A549 , Actinas/metabolismo , Animales , Perros , Regulación hacia Abajo , Reducción Gradual de Medicamentos , Células HeLa , Humanos , Cuerpos de Inclusión Viral/efectos de los fármacos , Cuerpos de Inclusión Viral/genética , Células de Riñón Canino Madin Darby , Virus de la Parainfluenza 3 Humana/efectos de los fármacos , Infecciones por Respirovirus/tratamiento farmacológico , Infecciones por Respirovirus/genética , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Accurate control of innate immune responses is required to eliminate invading pathogens and simultaneously avoid autoinflammation and autoimmune diseases. Here, we demonstrate that arginine monomethylation precisely regulates the mitochondrial antiviral-signaling protein (MAVS)-mediated antiviral response. Protein arginine methyltransferase 7 (PRMT7) forms aggregates to catalyze MAVS monomethylation at arginine residue 52 (R52), attenuating its binding to TRIM31 and RIG-I, which leads to the suppression of MAVS aggregation and subsequent activation. Upon virus infection, aggregated PRMT7 is disabled in a timely manner due to automethylation at arginine residue 32 (R32), and SMURF1 is recruited to PRMT7 by MAVS to induce proteasomal degradation of PRMT7, resulting in the relief of PRMT7 suppression of MAVS activation. Therefore, we not only reveal that arginine monomethylation by PRMT7 negatively regulates MAVS-mediated antiviral signaling in vitro and in vivo but also uncover a mechanism by which PRMT7 is tightly controlled to ensure the timely activation of antiviral defense.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Arginina/metabolismo , Interacciones Huésped-Patógeno/fisiología , Inmunidad Innata/fisiología , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Proteína 58 DEAD Box/metabolismo , Fibroblastos/virología , Células HEK293 , Herpes Simple/inmunología , Herpes Simple/metabolismo , Herpes Simple/virología , Humanos , Metilación , Ratones , Ratones Noqueados , Alcamidas Poliinsaturadas , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/inmunología , Receptores Inmunológicos/metabolismo , Infecciones por Respirovirus/inmunología , Infecciones por Respirovirus/metabolismo , Infecciones por Respirovirus/virología , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Viral respiratory infections are a common cause of severe disease, especially in infants, people who are immunocompromised, and in the elderly. Neutrophils, an important innate immune cell, infiltrate the lungs rapidly after an inflammatory insult. The most well-characterized effector mechanisms by which neutrophils contribute to host defense are largely extracellular and the involvement of neutrophils in protection from numerous bacterial and fungal infections is well established. However, the role of neutrophils in responses to viruses, which replicate intracellularly, has been less studied. It remains unclear whether and, by which underlying immunological mechanisms, neutrophils contribute to viral control or confer protection against an intracellular pathogen. Furthermore, neutrophils need to be tightly regulated to avoid bystander damage to host tissues. This is especially relevant in the lung where damage to delicate alveolar structures can compromise gas exchange with life-threatening consequences. It is inherently less clear how neutrophils can contribute to host immunity to viruses without causing immunopathology and/or exacerbating disease severity. In this review, we summarize and discuss the current understanding of how neutrophils in the lung direct immune responses to viruses, control viral replication and spread, and cause pathology during respiratory viral infections.
Asunto(s)
Interacciones Huésped-Patógeno , Neutrófilos/inmunología , Neutrófilos/metabolismo , Infecciones por Respirovirus/etiología , Infecciones por Respirovirus/metabolismo , Respirovirus/fisiología , Inmunidad Adaptativa , Animales , Biomarcadores , Comunicación Celular , Coinfección , Citocinas/metabolismo , Resistencia a la Enfermedad/genética , Resistencia a la Enfermedad/inmunología , Susceptibilidad a Enfermedades , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata , Infiltración Neutrófila/genética , Infiltración Neutrófila/inmunología , Infecciones por Respirovirus/patología , Índice de Severidad de la Enfermedad , Replicación ViralRESUMEN
Virus entry into cells is the initial stage of infection and involves multiple steps, and interfering viral entry represents potential antiviral approaches. Ion channels are pore-forming membrane proteins controlling cellular ion homeostasis and regulating many physiological processes, but their roles during viral infection have rarely been explored. Here, the functional Kv1.3 ion channel was found to be expressed in human hepatic cells and tissues. The Kv1.3 was then revealed to restrict HCV entry via inhibiting endosome acidification-mediated viral membrane fusion. The Kv1.3 was also demonstrated to inhibit DENV and ZIKV with an endosome acidification-dependent entry, but have no effect on SeV with a neutral pH penetration. A Kv1.3 antagonist PAP-1 treatment accelerated animal death in ZIKV-infected Ifnar1-/- mice. Moreover, Kv1.3-deletion was found to promote weight loss and reduce survival rate in ZIKV-infected Kv1.3-/- mice. Altogether, the Kv1.3 ion channel behaves as a host factor restricting viral entry. These findings broaden understanding about ion channel biology.
Asunto(s)
Virus del Dengue/fisiología , Dengue/metabolismo , Hepacivirus/fisiología , Hepatitis C/metabolismo , Canal de Potasio Kv1.3/metabolismo , Infecciones por Respirovirus/metabolismo , Virus Sendai/fisiología , Internalización del Virus , Infección por el Virus Zika/metabolismo , Virus Zika/fisiología , Animales , Chlorocebus aethiops , Dengue/virología , Endosomas/metabolismo , Ficusina/farmacología , Células HEK293 , Hepatitis C/virología , Humanos , Concentración de Iones de Hidrógeno , Canal de Potasio Kv1.3/antagonistas & inhibidores , Canal de Potasio Kv1.3/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Respirovirus/virología , Transfección , Células Vero , Internalización del Virus/efectos de los fármacos , Infección por el Virus Zika/virologíaRESUMEN
Respiratory viruses remain a significant cause of morbidity and mortality in the human population, underscoring the importance of ongoing basic research into virus-host interactions. However, many critical aspects of infection are difficult, if not impossible, to probe using standard cell lines, 2D culture formats, or even animal models. In vitro systems such as airway epithelial cultures at air-liquid interface, organoids, or 'on-chip' technologies allow interrogation in human cells and recapitulate emergent properties of the airway epithelium-the primary target for respiratory virus infection. While some of these models have been used for over thirty years, ongoing advancements in both culture techniques and analytical tools continue to provide new opportunities to investigate airway epithelial biology and viral infection phenotypes in both normal and diseased host backgrounds. Here we review these models and their application to studying respiratory viruses. Furthermore, given the ability of these systems to recapitulate the extracellular microenvironment, we evaluate their potential to serve as a platform for studies specifically addressing viral interactions at the mucosal surface and detail techniques that can be employed to expand our understanding.
Asunto(s)
Interacciones Huésped-Patógeno , Mucosa Respiratoria/virología , Infecciones por Respirovirus/metabolismo , Infecciones por Respirovirus/virología , Respirovirus/fisiología , Comunicación Celular , Técnicas de Cultivo de Célula , Células Cultivadas , Espacio Extracelular/metabolismo , Modelos Biológicos , Organoides , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Infecciones por Respirovirus/patología , Ingeniería de Tejidos , ViriónRESUMEN
Human Parainfluenza Virus-3 (HPIV3) causes severe respiratory illness in immunocompromised patients and lacks approved anti-viral therapies. A phase I study of adoptively transferred virus-specific T-cells (VSTs) targeting HPIV3 following bone marrow transplantation is underway (NCT03180216). We sought to identify immunodominant epitopes within HPIV3 Matrix protein and their cross-reactivity against related viral proteins. VSTs were generated from peripheral blood of healthy donors by ex-vivo expansion after stimulation with a 15-mer peptide library encompassing HPIV3 matrix protein. Epitope mapping was performed using IFN-γ ELIspot with combinatorial peptide pools. Flow cytometry was used to characterize products with intracellular cytokine staining. In 10 VST products tested, we discovered 12 novel immunodominant epitopes. All products recognized an epitope at the C-terminus. On IFN-γ ELISpot, individual peptides eliciting activity demonstrated mean IFN-γ spot forming units per well (SFU)/1x105 cells of 115.5 (range 24.5-247.5). VST products were polyfunctional, releasing IFN-γ and TNF-α in response to identified epitopes, which were primarily HLA Class II restricted. Peptides from Human Parainfluenza Virus-1 corresponding to the HPIV3 epitopes showed cross-reactivity for HPIV1 in 11 of 12 tested epitopes (mean cross reactivity index: 1.19). Characterization of HPIV3 epitopes may enable development of third-party VSTs to treat immune suppressed patients with HPIV infection.
Asunto(s)
Traslado Adoptivo , Epítopos Inmunodominantes , Virus de la Parainfluenza 1 Humana/inmunología , Virus de la Parainfluenza 3 Humana/inmunología , Infecciones por Respirovirus/terapia , Linfocitos T/trasplante , Proteínas de la Matriz Viral/inmunología , Células Cultivadas , Ensayos Clínicos Fase I como Asunto , Reacciones Cruzadas , Ensayo de Immunospot Ligado a Enzimas , Mapeo Epitopo , Interacciones Huésped-Patógeno , Humanos , Interferón gamma/metabolismo , Ensayos de Liberación de Interferón gamma , Virus de la Parainfluenza 1 Humana/patogenicidad , Virus de la Parainfluenza 3 Humana/patogenicidad , Infecciones por Respirovirus/inmunología , Infecciones por Respirovirus/metabolismo , Infecciones por Respirovirus/virología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Caprine parainfluenza virus type 3 (CPIV3) is an emerging respiratory pathogen that affects the sheep and goat industry in China and possibly other countries around the world. Accumulating evidence suggests that microRNAs play important roles in regulating virus-host interactions and can suppress or facilitate viral replication. In this study, we showed that CPIV3 infection induced apoptosis in Madin-Darby bovine kidney (MDBK) cells, as determined by morphological changes and flow cytometry. Caspase activity and the expression of pro-apoptotic genes further indicated that CPIV3 induced apoptosis by activating both the intrinsic and extrinsic pathways. We also demonstrated the involvement of bta-microRNA-98 (bta-miR-98) in regulating CPIV3-induced apoptosis. Bta-miR-98 was downregulated in MDBK cells infected with CPIV3. Overexpression of bta-miR-98 significantly decreased the activities of caspase-3, -8, and -9. Conversely, inhibition of bta-miR-98 had completely opposite effects. Furthermore, our data showed that bta-miR-98 markedly affected CPIV3 replication by regulating apoptosis. Importantly, we found that bta-miR-98 modulated CPIV3-induced apoptosis by targeting caspase-3, an effector of apoptosis. Collectively, our results may suggest that CPIV3 infection induced apoptosis and downregulated the levels of bta-miR-98, and this miRNA regulated viral replication through effected apoptosis. This study contributes to our understanding of the molecular mechanisms underlying CPIV3 pathogenesis.
Asunto(s)
Caspasa 3/genética , MicroARNs/genética , Virus de la Parainfluenza 3 Humana/fisiología , Interferencia de ARN , Infecciones por Respirovirus/genética , Infecciones por Respirovirus/virología , Replicación Viral , Animales , Apoptosis/genética , Biomarcadores , Caspasa 3/metabolismo , Línea Celular , Células Cultivadas , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno/inmunología , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Infecciones por Respirovirus/metabolismo , Receptor fas/metabolismoRESUMEN
The phenotypic and functional dichotomy between IRF8+ type 1 and IRF4+ type 2 conventional dendritic cells (cDC1s and cDC2s, respectively) is well accepted; it is unknown how robust this dichotomy is under inflammatory conditions, when additionally monocyte-derived cells (MCs) become competent antigen-presenting cells (APCs). Using single-cell technologies in models of respiratory viral infection, we found that lung cDC2s acquired expression of the Fc receptor CD64 shared with MCs and of IRF8 shared with cDC1s. These inflammatory cDC2s (inf-cDC2s) were superior in inducing CD4+ T helper (Th) cell polarization while simultaneously presenting antigen to CD8+ T cells. When carefully separated from inf-cDC2s, MCs lacked APC function. Inf-cDC2s matured in response to cell-intrinsic Toll-like receptor and type 1 interferon receptor signaling, upregulated an IRF8-dependent maturation module, and acquired antigens via convalescent serum and Fc receptors. Because hybrid inf-cDC2s are easily confused with monocyte-derived cells, their existence could explain why APC functions have been attributed to MCs.
Asunto(s)
Plasticidad de la Célula/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Inmunidad , Macrófagos/inmunología , Macrófagos/metabolismo , Infecciones por Respirovirus/etiología , Presentación de Antígeno , Biomarcadores , Susceptibilidad a Enfermedades , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Inmunofenotipificación , Interferón Tipo I/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Especificidad de Órganos/inmunología , Receptores Fc/metabolismo , Infecciones por Respirovirus/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Factores de Transcripción , Virosis/genética , Virosis/inmunología , Virosis/metabolismo , Virosis/virologíaRESUMEN
Mammalian organs consist of diverse, intermixed cell types that signal to each other via ligand-receptor interactions - an interactome - to ensure development, homeostasis and injury-repair. Dissecting such intercellular interactions is facilitated by rapidly growing single-cell RNA sequencing (scRNA-seq) data; however, existing computational methods are often not readily adaptable by bench scientists without advanced programming skills. Here, we describe a quantitative intuitive algorithm, coupled with an optimized experimental protocol, to construct and compare interactomes in control and Sendai virus-infected mouse lungs. A minimum of 90 cells per cell type compensates for the known gene dropout issue in scRNA-seq and achieves comparable sensitivity to bulk RNA sequencing. Cell lineage normalization after cell sorting allows cost-efficient representation of cell types of interest. A numeric representation of ligand-receptor interactions identifies, as outliers, known and potentially new interactions as well as changes upon viral infection. Our experimental and computational approaches can be generalized to other organs and human samples.
Asunto(s)
Perfilación de la Expresión Génica , Pulmón/virología , RNA-Seq , Infecciones por Respirovirus/virología , Virus Sendai/patogenicidad , Análisis de la Célula Individual , Transcriptoma , Animales , Comunicación Celular , Linaje de la Célula , Modelos Animales de Enfermedad , Femenino , Redes Reguladoras de Genes , Interacciones Huésped-Patógeno , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones Endogámicos C57BL , Infecciones por Respirovirus/genética , Infecciones por Respirovirus/metabolismo , Infecciones por Respirovirus/patología , Transducción de SeñalRESUMEN
Quaking (QKI) is an RNA-binding protein (RBP) involved in multiple aspects of RNA metabolism and many biological processes. Despite a known immune function in regulating monocyte differentiation and inflammatory responses, the degree to which QKI regulates the host interferon (IFN) response remains poorly characterized. Here we show that QKI ablation enhances poly(I:C) and viral infection-induced IFNß transcription. Characterization of IFN-related signalling cascades reveals that QKI knockout results in higher levels of IRF3 phosphorylation. Interestingly, complementation with QKI-5 isoform alone is sufficient to rescue this phenotype and reduce IRF3 phosphorylation. Further analysis shows that MAVS, but not RIG-I or MDA5, is robustly upregulated in the absence of QKI, suggesting that QKI downregulates MAVS and thus represses the host IFN response. As expected, MAVS depletion reduces IFNß activation and knockout of MAVS in the QKI knockout cells completely abolishes IFNß induction. Consistently, ectopic expression of RIG-I activates stronger IFNß induction via MAVS-IRF3 pathway in the absence of QKI. Collectively, these findings demonstrate a novel role for QKI in negatively regulating host IFN response by reducing MAVS levels.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Interacciones Huésped-Patógeno/fisiología , Interferón Tipo I/metabolismo , Proteínas de Unión al ARN/metabolismo , Células A549 , Proteínas Adaptadoras Transductoras de Señales/genética , Sistemas CRISPR-Cas , Regulación de la Expresión Génica , Humanos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/genética , Fosforilación , Poli I-C/genética , Poli I-C/metabolismo , Proteínas de Unión al ARN/genética , Infecciones por Respirovirus/metabolismo , Virus Sendai/patogenicidadRESUMEN
MicroRNAs (miRNAs) are small noncoding RNAs that suppress the expression of multiple genes and are involved in numerous biologic functions and disorders, including human diseases. Here, we report that two miRNAs, miR-302b and miR-372, target mitochondrial-mediated antiviral innate immunity by regulating mitochondrial dynamics and metabolic demand. Using human cell lines transfected with the synthetic analog of viral dsRNA, poly(I-C), or challenged with Sendai virus, we found that both miRNAs are up-regulated in the cells late after viral infection and ultimately terminate the production of type I interferons and inflammatory cytokines. We found that miR-302b and miR-372 are involved in dynamin-related protein 1 (DRP1)-dependent mitochondrial fragmentation and disrupt mitochondrial metabolism by attenuating solute carrier family 25 member 12 (SLC25A12), a member of the SLC25 family. Neutralizing the effects of the two miRNAs through specific inhibitors re-established the mitochondrial dynamics and the antiviral responses. We found that SLC25A12 contributes to regulating the antiviral response by inducing mitochondrial-related metabolite changes in the organelle. Structure-function analysis indicated that SLC25A12, as part of a prohibitin complex, associates with the mitochondrial antiviral-signaling protein in mitochondria, providing structural insight into the regulation of the mitochondrial-mediated antiviral response. Our results contribute to the understanding of how miRNAs modulate the innate immune response by altering mitochondrial dynamics and metabolic demand. Manipulating the activities of miR-302b and miR-372 may be a potential therapeutic approach to target RNA viruses.
Asunto(s)
MicroARNs/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Infecciones por Respirovirus/metabolismo , Virus Sendai/fisiología , Línea Celular , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , MicroARNs/inmunología , Mitocondrias/inmunología , Mitocondrias/virología , Proteínas de Transporte de Membrana Mitocondrial/inmunología , Membranas Mitocondriales/inmunología , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/virología , Infecciones por Respirovirus/inmunología , Infecciones por Respirovirus/virología , Virus Sendai/inmunologíaRESUMEN
Recent studies have begun to elucidate a role for E3 ubiquitin ligases as important mediators of the innate immune response. Our previous work defined a role for the ubiquitin ligase natural killer lytic-associated molecule (NKLAM/RNF19b) in mouse and human innate immunity. Here, we present novel data describing a role for NKLAM in regulating the immune response to Sendai virus (SeV), a murine model of paramyxoviral pneumonia. NKLAM expression was significantly upregulated by SeV infection. SeV-infected mice that are deficient in NKLAM demonstrated significantly less weight loss than wild type mice. In vivo, Sendai virus replication was attenuated in NKLAM-/- mice. Autophagic flux and the expression of autophagy markers LC3 and p62/SQSTM1 were also less in NKLAM-/- mice. Using flow cytometry, we observed less neutrophils and macrophages in the lungs of NKLAM-/- mice during SeV infection. Additionally, phosphorylation of STAT1 and NFκB p65 was lower in NKLAM-/- than wild type mice. The dysregulated phosphorylation profile of STAT1 and NFκB in NKLAM-/- mice correlated with decreased expression of numerous proinflammatory cytokines that are regulated by STAT1 and/or NFκB. The lack of NKLAM and the resulting attenuated immune response is favorable to NKLAM-/- mice receiving a low dose of SeV; however, at a high dose of virus, NKLAM-/- mice succumbed to the infection faster than wild type mice. In conclusion, our novel results indicate that NKLAM plays a role in regulating the production of pro-inflammatory cytokines during viral infection.
Asunto(s)
Citocinas/metabolismo , Modelos Animales de Enfermedad , Mediadores de Inflamación/metabolismo , Proteínas de la Membrana/deficiencia , Neumonía/metabolismo , Infecciones por Respirovirus/metabolismo , Animales , Citocinas/genética , Humanos , Inmunidad Innata/genética , Macrófagos/metabolismo , Macrófagos/virología , Proteínas de la Membrana/genética , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Neumonía/genética , Neumonía/virología , Infecciones por Respirovirus/genética , Infecciones por Respirovirus/virología , Virus Sendai/fisiología , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismoRESUMEN
BACKGROUND: Caprine parainfluenza virus type 3 (CPIV3) is major pathogen of goat herds causing serious respiratory tract disease and economic losses to the goat industry in China. We analyzed the differential proteomics of CPIV3-infected Madin-Darby bovine kidney (MDBK) cells using quantitative iTRAQ coupled LC-MS/MS. In addition, four DEPs were validated by qRT-PCR and western blot analysis. RESULTS: Quantitative proteomics analysis revealed 163 differentially expressed proteins (DEPs) between CPIV3-infected and mock-infected groups (p-value < 0.05 and fold change > 1.2), among which 91 were down-regulated and 72 were up-regulated. Gene ontology (GO) analysis showed that these DEPs were involved in molecular functions, cellular components and biological processes. Biological functions in which the DEPs were involved in included diseases, genetic information processing, metabolism, environmental information processing, cellular processes, and organismal systems. STRING analysis revealed that four heat shock proteins (HSPs) included HSPA5, HSPA1B, HSP90B1 and HSPA6 may be associated with proliferation of CPIV3 in MDBK cells. qRT-PCR and western blot analysis showed that the selected HSPs were identical to the quantitative proteomics data. CONCLUSION: To our knowledge, this is the first report of the proteomic changes in MDBK cells after CPIV3 infection.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Proteómica , Infecciones por Respirovirus/veterinaria , Respirovirus/fisiología , Animales , Western Blotting , Bovinos , Línea Celular , Cromatografía Liquida , Perfilación de la Expresión Génica , Proteínas de Choque Térmico/genética , Riñón/virología , Reacción en Cadena en Tiempo Real de la Polimerasa , Respirovirus/genética , Infecciones por Respirovirus/genética , Infecciones por Respirovirus/metabolismo , Espectrometría de Masas en Tándem , Replicación Viral/fisiologíaRESUMEN
The matrix (M) proteins of paramyxoviruses bind to the nucleocapsids and cytoplasmic tails of glycoproteins, thus mediating the assembly and budding of virions. We first determined the budding characterization of the HPIV3 Fusion (F) protein to investigate the assembly mechanism of human parainfluenza virus type 3 (HPIV3). Our results show that expression of the HPIV3 F protein alone is sufficient to initiate the release of virus-like particles (VLPs), and the F protein can regulate the VLP-forming ability of the M protein. Furthermore, HPIV3F-Flag, which is a recombinant HPIV3 with a Flag tag at the C-terminus of the F protein, was constructed and recovered. We found that the M, F, and hemagglutinin-neuraminidase (HN) proteins and the viral genome can accumulate in lipid rafts in HPIV3F-Flag-infected cells, and the F protein mainly exists in the form of F1 in VLPs, lipid rafts, and purified virions. Furthermore, the function of cholesterol in the viral envelope and cell membrane was assessed via the elimination of cholesterol by methyl-ß-cyclodextrin (MßCD). Our results suggest that the infectivity of HPIV3 was markedly reduced, due to defective internalization ability in the absence of cholesterol. These results reveal that HPIV3 might assemble in the lipid rafts to acquire cholesterol for the envelope of HPIV3, which suggests the that disruption of the cholesterol composition of HPIV3 virions might be a useful method for the design of anti-HPIV3 therapy.
Asunto(s)
Colesterol/metabolismo , Virus de la Parainfluenza 3 Humana/fisiología , Infecciones por Respirovirus/metabolismo , Virión/metabolismo , Línea Celular , Proteína HN/genética , Proteína HN/metabolismo , Humanos , Infecciones por Respirovirus/virología , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo , Proteínas de la Matriz Viral/metabolismo , Ensamble de VirusRESUMEN
Sendai virus (SeV) has been used as a model strain to reveal molecular features of paramyxovirus biology. In this study, we comprehensively analyzed the gene profiling of murine macrophages and airway epithelial cells in response to SeV using gene expression data. The significantly differentially expressed genes (DEGs) were screened by GEO2R. Gene ontology (GO) and pathway enrichment analyses were performed by DAVID. The protein-protein interaction (PPI) map of DEGs was constructed by STRING. The modules of PPI network are produced by molecular complex detection (MCODE) plug-in of Cytoscape. In total, 241 up- and 83 downregulated DEGs were identified in airway epithelial cells while 130 up- and 148 downregulated in macrophage. Particularly, Tmem119 and Colla2 are significantly downregulated in airway epithelial cells and macrophages, respectively. Functional enrichment analysis showed that upregulated DEGs are clustered in innate immunity and inflammatory response in both cell types, whereas downregulated DEGs are involved in host metabolic pathway in airway epithelial cells. PI3K-AKT signaling pathway is downregulated in macrophages. PPI network analysis indicated that some high degree of nodes exist in both cell types, such as Stat1, Tnf, and Cxcl10. In conclusion, SeV infection can induce different host cell responses in airway epithelial cells and macrophages.