RESUMO
Viral infections during pregnancy are a considerable cause of adverse outcomes and birth defects, and the underlying mechanisms are poorly understood. Among those, cytomegalovirus (CMV) infection stands out as the most common intrauterine infection in humans, putatively causing early pregnancy loss. We employed murine CMV as a model to study the consequences of viral infection on pregnancy outcome and fertility maintenance. Even though pregnant mice successfully controlled CMV infection, we observed highly selective, strong infection of corpus luteum (CL) cells in their ovaries. High infection densities indicated complete failure of immune control in CL cells, resulting in progesterone insufficiency and pregnancy loss. An abundance of gap junctions, absence of vasculature, strong type I interferon (IFN) responses, and interaction of innate immune cells fully protected the ovarian follicles from viral infection. Our work provides fundamental insights into the effect of CMV infection on pregnancy loss and mechanisms protecting fertility.
Assuntos
Corpo Lúteo/imunologia , Infecções por Citomegalovirus/imunologia , Fertilidade/imunologia , Imunidade Inata/imunologia , Animais , Corpo Lúteo/virologia , Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Feminino , Junções Comunicantes/imunologia , Interferon Tipo I/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Gravidez , Progesterona/imunologiaRESUMO
The retention of intracellular Toll-like receptors (TLRs) in the endoplasmic reticulum prevents their activation under basal conditions. TLR9 is activated by sensing ligands in specific endosomal-lysosomal compartments. Here we identified IRAP+ endosomes as major cellular compartments for the early steps of TLR9 activation in dendritic cells (DCs). Both TLR9 and its ligand, the dinucleotide CpG, were present as cargo in IRAP+ endosomes. In the absence of the aminopeptidase IRAP, the trafficking of CpG and TLR9 to lysosomes and signaling via TLR9 were enhanced in DCs and in mice following bacterial infection. IRAP stabilized CpG-containing endosomes by interacting with the actin-nucleation factor FHOD4, which slowed the trafficking of TLR9 toward lysosomes. Thus, endosomal retention of TLR9 via the interaction of IRAP with the actin cytoskeleton is a mechanism that prevents hyper-activation of TLR9 in DCs.
Assuntos
Cistinil Aminopeptidase/metabolismo , Citoesqueleto/metabolismo , Células Dendríticas/fisiologia , Endossomos/metabolismo , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Receptor Toll-Like 9/metabolismo , Animais , Células Cultivadas , Ilhas de CpG/genética , Cistinil Aminopeptidase/genética , Células Dendríticas/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Oligodesoxirribonucleotídeos/imunologia , Ligação Proteica , Transdução de SinaisRESUMO
Unc-93 homolog B1 (UNC93B1) is a key regulator of nucleic acid (NA)-sensing Toll-like receptors (TLRs). Loss of NA-sensing TLR responses in UNC93B1-deficient patients facilitates Herpes simplex virus type 1 (HSV-1) encephalitis. UNC93B1 is thought to guide NA-sensing TLRs from the endoplasmic reticulum (ER) to their respective endosomal signaling compartments and to guide the flagellin receptor TLR5 to the cell surface, raising the question of how UNC93B1 mediates differential TLR trafficking. Here, we report that UNC93B1 regulates a step upstream of the differential TLR trafficking process. We discovered that UNC93B1 deficiency resulted in near-complete loss of TLR3 and TLR7 proteins in primary splenic mouse dendritic cells and macrophages, showing that UNC93B1 is critical for maintaining TLR expression. Notably, expression of an ER-retained UNC93B1 version was sufficient to stabilize TLRs and largely restore endosomal TLR trafficking and activity. These data are critical for an understanding of how UNC93B1 can regulate the function of a broad subset of TLRs.
Assuntos
Endossomos/imunologia , Proteínas de Membrana Transportadoras/imunologia , Chaperonas Moleculares/imunologia , Receptores Toll-Like/imunologia , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Células HEK293 , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Estabilidade Proteica , Transporte Proteico/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células THP-1 , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
CD8 T cells are the predominant effector cells of adaptive immunity in preventing cytomegalovirus (CMV) multiple-organ disease caused by cytopathogenic tissue infection. The mechanism by which CMV-specific, naïve CD8 T cells become primed and clonally expand is of fundamental importance for our understanding of CMV immune control. For CD8 T-cell priming, two pathways have been identified: direct antigen presentation by infected professional antigen-presenting cells (pAPCs) and antigen cross-presentation by uninfected pAPCs that take up antigenic material derived from infected tissue cells. Studies in mouse models using murine CMV (mCMV) and precluding either pathway genetically or experimentally have shown that, in principle, both pathways can congruently generate the mouse MHC/H-2 class-I-determined epitope-specificity repertoire of the CD8 T-cell response. Recent studies, however, have shown that direct antigen presentation is the canonical pathway when both are accessible. This raised the question of why antigen cross-presentation is ineffective even under conditions of high virus replication thought to provide high amounts of antigenic material for feeding cross-presenting pAPCs. As delivery of antigenic material for cross-presentation is associated with programmed cell death, and as CMVs encode inhibitors of different cell death pathways, we pursued the idea that these inhibitors restrict antigen delivery and thus CD8 T-cell priming by cross-presentation. To test this hypothesis, we compared the CD8 T-cell responses to recombinant mCMVs lacking expression of the apoptosis-inhibiting protein M36 or the necroptosis-inhibiting protein M45 with responses to wild-type mCMV and revertant viruses expressing the respective cell death inhibitors. The data reveal that increased programmed cell death improves CD8 T-cell priming in mice capable of antigen cross-presentation but not in a mutant mouse strain unable to cross-present. These findings strongly support the conclusion that CMV cell death inhibitors restrict the priming of CD8 T cells by antigen cross-presentation.
RESUMO
Merkel cell polyomavirus (MCPyV) is the causative agent of the majority of Merkel cell carcinomas (MCC). The virus has limited coding capacity, with its early viral proteins, large T (LT) and small T (sT), being multifunctional and contributing to infection and transformation. A fundamental difference in early viral gene expression between infection and MCPyV-driven tumorigenesis is the expression of a truncated LT (LTtr) in the tumor. In contrast, sT is expressed in both conditions and contributes significantly to oncogenesis. Here, we identified novel functions of early viral proteins by performing genome-wide transcriptome and chromatin studies in primary human fibroblasts. Due to current limitations in infection and tumorigenesis models, we mimic these conditions by ectopically expressing sT, LT or LTtr, individually or in combination, at different time points. In addition to its known function in cell cycle and inflammation modulation, we reveal a fundamentally new function of sT. We show that sT regulates the type I interferon (IFN) response downstream of the type I interferon receptor (IFNAR) by interfering with the interferon-stimulated gene factor 3 (ISGF3)-induced interferon-stimulated gene (ISG) response. Expression of sT leads to a reduction in the expression of interferon regulatory factor 9 (IRF9) which is a central component of the ISGF3 complex. We further show that this function of sT is conserved in BKPyV. We provide a first mechanistic understanding of which early viral proteins trigger and control the type I IFN response, which may influence MCPyV infection, persistence and, during MCC progression, regulation of the tumor microenvironment.
Assuntos
Carcinoma de Célula de Merkel , Evasão da Resposta Imune , Interferon Tipo I , Poliomavírus das Células de Merkel , Infecções por Polyomavirus , Transdução de Sinais , Infecções Tumorais por Vírus , Humanos , Poliomavírus das Células de Merkel/imunologia , Interferon Tipo I/metabolismo , Interferon Tipo I/imunologia , Carcinoma de Célula de Merkel/virologia , Carcinoma de Célula de Merkel/imunologia , Transdução de Sinais/imunologia , Infecções por Polyomavirus/imunologia , Infecções por Polyomavirus/virologia , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/virologia , Evasão da Resposta Imune/imunologia , Antígenos Virais de Tumores/metabolismo , Antígenos Virais de Tumores/imunologia , Antígenos Virais de Tumores/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/virologia , Neoplasias Cutâneas/metabolismo , Fibroblastos/virologia , Fibroblastos/metabolismo , Fibroblastos/imunologiaRESUMO
Herpesviruses modulate immune control to secure lifelong infection. The mechanisms Human Cytomegalovirus (HCMV) employs in this regard can reveal unanticipated aspects of cellular signaling involved in antiviral immunity. Here, we describe a novel relationship between the TGF-ß family cytokine BMP9 and HCMV infection. We identify a cross-talk between BMP9-induced and IFN receptor-mediated signaling, showing that BMP9 boosts the transcriptional response to and antiviral activity of IFNß, thereby enhancing viral restriction. We also show that BMP9 is secreted by human fibroblasts upon HCMV infection. However, HCMV infection impairs BMP9-induced enhancement of the IFNß response, indicating that this signaling role of BMP9 is actively targeted by HCMV. Indeed, transmembrane proteins US18 and US20, which downregulate type I BMP receptors, are necessary and sufficient to cause inhibition of BMP9-mediated boosting of the antiviral response to IFNß. HCMV lacking US18 and US20 is more sensitive to IFNß. Thus, HCMV has a mutually antagonistic relationship with BMP9, which extends the growing body of evidence that BMP signaling is an underappreciated modulator of innate immunity in response to viral infection.
Assuntos
Fator 2 de Diferenciação de Crescimento , Imunidade Inata , Humanos , Citocinas/metabolismo , Citomegalovirus/metabolismo , Fator 2 de Diferenciação de Crescimento/metabolismo , Transdução de SinaisRESUMO
Induction of type I interferon (IFN) gene expression is among the first lines of cellular defense a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system, showing that M35 interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35's function. Determination of M35's crystal structure combined with reverse genetics revealed that homodimerization is a key feature for M35's immunomodulatory activity. In electrophoretic mobility shift assays (EMSAs), purified M35 protein specifically bound to the regulatory DNA element that governs transcription of the first type I IFN gene induced in nonimmune cells, Ifnb1. DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signaling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the host Ifnb1 promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signaling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labeled transcripts (SLAM-seq) and assessed M35's global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically downregulated basal expression of IRF3-dependent genes. During MCMV infection, M35 impaired expression of IRF3-responsive genes aside of Ifnb1. Our results suggest that M35-DNA binding directly antagonizes gene induction mediated by IRF3 and impairs the antiviral response more broadly than formerly recognized. IMPORTANCE Replication of the ubiquitous human cytomegalovirus (HCMV) in healthy individuals mostly goes unnoticed but can impair fetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry into host cells, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here, we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key cellular factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction.
Assuntos
Infecções por Citomegalovirus , Elementos Facilitadores Genéticos , Fator Regulador 3 de Interferon , Interferon Tipo I , Proteínas da Matriz Viral , Animais , Humanos , Camundongos , Infecções por Citomegalovirus/genética , DNA/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/metabolismo , Interferon beta/genética , Interferon beta/metabolismo , Muromegalovirus/genética , Muromegalovirus/metabolismo , Proteínas da Matriz Viral/metabolismoRESUMO
Cytomegaloviruses (CMVs) are master manipulators of the host immune response. Here, we reveal that the murine CMV (MCMV) protein m152 specifically targets the type I interferon (IFN) response by binding to stimulator of interferon genes (STING), thereby delaying its trafficking to the Golgi compartment from where STING initiates type I IFN signaling. Infection with an MCMV lacking m152 induced elevated type I IFN responses and this leads to reduced viral transcript levels both in vitro and in vivo This effect is ameliorated in the absence of STING Interestingly, while m152 inhibits STING-mediated IRF signaling, it did not affect STING-mediated NF-κB signaling. Analysis of how m152 targets STING translocation reveals that STING activates NF-κB signaling already from the ER prior to its trafficking to the Golgi. Strikingly, this response is important to promote early MCMV replication. Our results show that MCMV has evolved a mechanism to specifically antagonize the STING-mediated antiviral IFN response, while preserving its pro-viral NF-κB response, providing an advantage in the establishment of an infection.
Assuntos
Infecções por Citomegalovirus/imunologia , Interações Hospedeiro-Patógeno/imunologia , Fatores Reguladores de Interferon/metabolismo , Interferon Tipo I/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , NF-kappa B/metabolismo , Proteínas Virais/metabolismo , Animais , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Fatores Reguladores de Interferon/genética , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Muromegalovirus/genética , Muromegalovirus/isolamento & purificação , Muromegalovirus/patogenicidade , NF-kappa B/genética , Ligação Proteica , Proteínas Virais/genética , Replicação ViralRESUMO
Protein tyrosine phosphatase 1B (PTP1B, also known as PTPN1) is a negative regulator of the leptin and insulin signalling pathways. This phosphatase is of great interest as PTP1B-knockout mice are protected against the development of obesity and diabetes. Here, we provide evidence for a novel function of PTP1B that is independent of its phosphatase activity, but requires its localisation to the membrane of the endoplasmic reticulum. Upon activation of pattern recognition receptors, macrophages and plasmacytoid dendritic cells from PTP1B-knockout mice secrete lower amounts of type I interferon (IFN) than cells from wild-type mice. In contrast, secretion of the proinflammatory cytokines TNFα and IL6 was unaltered. While PTP1B deficiency did not affect Ifnb1 transcription, type I IFN accumulated in macrophages, suggesting a role for PTP1B in mediating secretion of type I IFN. In summary, we have uncovered that PTP1B positively regulates the type I IFN response by promoting secretion of key antiviral cytokines.
Assuntos
Interferon Tipo I , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Viroses , Animais , Insulina , Interferon Tipo I/genética , Camundongos , Camundongos Knockout , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Transdução de Sinais , Viroses/imunologiaRESUMO
Cytomegaloviruses (CMV) infect many different cell types and tissues in their respective hosts. Monocytes and macrophages play an important role in CMV dissemination from the site of infection to target organs. Moreover, macrophages are specialized in pathogen sensing and respond to infection by secreting cytokines and interferons. In murine cytomegalovirus (MCMV), a model for human cytomegalovirus, several genes required for efficient replication in macrophages have been identified, but their specific functions remain poorly understood. Here we show that MCMV m139, a gene of the conserved US22 gene family, encodes a protein that interacts with the DEAD box helicase DDX3, a protein involved in pathogen sensing and interferon (IFN) induction, and the E3 ubiquitin ligase UBR5. DDX3 and UBR5 also participate in the transcription, processing, and translation of a subset of cellular mRNAs. We show that m139 inhibits DDX3-mediated IFN-α and IFN-ß induction and is necessary for efficient viral replication in bone-marrow derived macrophages. In vivo, m139 is crucial for viral dissemination to local lymph nodes and to the salivary glands. An m139-deficient MCMV also replicated to lower titers in SVEC4-10 endothelial cells. This replication defect was not accompanied by increased IFN-ß transcription, but was rescued by knockout of either DDX3 or UBR5. Moreover, m139 co-localized with DDX3 and UBR5 in viral replication compartments in the cell nucleus. These results suggest that m139 inhibits DDX3-mediated IFN production in macrophages and antagonizes DDX3 and UBR5-dependent functions related to RNA metabolism in endothelial cells.
Assuntos
RNA Helicases DEAD-box/metabolismo , Células Endoteliais/virologia , Infecções por Herpesviridae/microbiologia , Interferon beta/metabolismo , Macrófagos/virologia , Muromegalovirus/fisiologia , Replicação Viral , Animais , Células Cultivadas , RNA Helicases DEAD-box/genética , Feminino , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/patologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Toll-like receptor-9 (TLR9) is largely responsible for discriminating self from pathogenic DNA. However, association of host DNA with autoantibodies activates TLR9, inducing the pathogenic secretion of type I interferons (IFNs) from plasmacytoid dendritic cells (pDCs). Here, we found that in response to DNA-containing immune complexes (DNA-IC), but not to soluble ligands, IFN-α production depended upon the convergence of the phagocytic and autophagic pathways, a process called microtubule-associated protein 1A/1B-light chain 3 (LC3)-associated phagocytosis (LAP). LAP was required for TLR9 trafficking into a specialized interferon signaling compartment by a mechanism that involved autophagy-related proteins, but not the conventional autophagic preinitiation complex, or adaptor protein-3 (AP-3). Our findings unveil a new role for nonconventional autophagy in inflammation and provide one mechanism by which anti-DNA autoantibodies, such as those found in several autoimmune disorders, bypass the controls that normally restrict the apportionment of pathogenic DNA and TLR9 to the interferon signaling compartment.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Autofagia/imunologia , DNA/imunologia , Interferon Tipo I/biossíntese , Animais , Humanos , Imunoglobulina G/imunologia , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Fagocitose/imunologia , Fagossomos/metabolismo , Transporte Proteico , Receptor Toll-Like 9/imunologia , Receptor Toll-Like 9/metabolismoRESUMO
Murine gammaherpesvirus 68 (MHV68) is a small-animal model suitable for study of the human pathogens Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. Here, we have characterized the roles of the endosomal Toll-like receptor (TLR) escort protein UNC93B, endosomal TLR7, -9, and -13, and cell surface TLR2 in MHV68 detection. We found that the alpha interferon (IFN-α) response of plasmacytoid dendritic cells (pDC) to MHV68 was reduced in Tlr9-/- cells compared to levels in wild type (WT) cells but not completely lost. Tlr7-/- pDC responded similarly to WT. However, we found that in Unc93b-/- pDC, as well as in Tlr7-/-Tlr9-/- double-knockout pDC, the IFN-α response to MHV68 was completely abolished. Thus, the only pattern recognition receptors contributing to the IFN-α response to MHV68 in pDC are TLR7 and TLR9, but the contribution of TLR7 is masked by the presence of TLR9. To address the role of UNC93B and TLR for MHV68 infection in vivo, we infected mice with MHV68. Lytic replication of MHV68 after intravenous infection was enhanced in the lungs, spleen, and liver of UNC93B-deficient mice, in the spleen of TLR9-deficient mice, and in the liver and spleen of Tlr7-/-Tlr9-/- mice. The absence of TLR2 or TLR13 did not affect lytic viral titers. We then compared reactivation of MHV68 from latently infected WT, Unc93b-/-, Tlr7-/-Tlr9-/-, Tlr7-/-, and Tlr9-/- splenocytes. We observed enhanced reactivation and latent viral loads, particularly from Tlr7-/-Tlr9-/- splenocytes compared to levels in the WT. Our data show that UNC93B-dependent TLR7 and TLR9 cooperate in and contribute to detection and control of MHV68 infection.IMPORTANCE The two human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), can cause aggressive forms of cancer. These herpesviruses are strictly host specific, and therefore the homolog murine gammaherpesvirus 68 (MHV68) is a widely used model to obtain in vivo insights into the interaction between these two gammaherpesviruses and their host. Like EBV and KSHV, MHV68 establishes lifelong latency in B cells. The innate immune system serves as one of the first lines of host defense, with pattern recognition receptors such as the Toll-like receptors playing a crucial role in mounting a potent antiviral immune response to various pathogens. Here, we shed light on a yet unanticipated role of Toll-like receptor 7 in the recognition of MHV68 in a subset of immune cells called plasmacytoid dendritic cells, as well as on the control of this virus in its host.
Assuntos
Células Dendríticas/imunologia , Endossomos/imunologia , Gammaherpesvirinae/patogenicidade , Infecções por Herpesviridae/diagnóstico , Glicoproteínas de Membrana/fisiologia , Células-Tronco Mesenquimais/imunologia , Receptor 7 Toll-Like/fisiologia , Receptor Toll-Like 9/fisiologia , Animais , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Endossomos/metabolismo , Endossomos/virologia , Feminino , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/virologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Ativação Viral , Latência Viral , Replicação ViralRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the few oncogenic human viruses known to date. Its large genome encodes more than 85 proteins and includes both unique viral proteins as well as proteins conserved amongst herpesviruses. KSHV ORF20 is a member of the herpesviral core UL24 family, but the function of ORF20 and its role in the viral life cycle is not well understood. ORF20 encodes three largely uncharacterized isoforms, which we found were localized predominantly in the nuclei and nucleoli. Quantitative affinity purification coupled to mass spectrometry (q-AP-MS) identified numerous specific interacting partners of ORF20, including ribosomal proteins and the interferon-stimulated gene product (ISG) oligoadenylate synthetase-like protein (OASL). Both endogenous and transiently transfected OASL co-immunoprecipitated with ORF20, and this interaction was conserved among all ORF20 isoforms and multiple ORF20 homologs of the UL24 family in other herpesviruses. Characterization of OASL interacting partners by q-AP-MS identified a very similar interactome to that of ORF20. Both ORF20 and OASL copurified with 40S and 60S ribosomal subunits, and when they were co-expressed, they associated with polysomes. Although ORF20 did not have a global effect on translation, ORF20 enhanced RIG-I induced expression of endogenous OASL in an IRF3-dependent but IFNAR-independent manner. OASL has been characterized as an ISG with antiviral activity against some viruses, but its role for gammaherpesviruses was unknown. We show that OASL and ORF20 mRNA expression were induced early after reactivation of latently infected HuARLT-rKSHV.219 cells. Intriguingly, we found that OASL enhanced infection of KSHV. During infection with a KSHV ORF20stop mutant, however, OASL-dependent enhancement of infectivity was lost. Our data have characterized the interaction of ORF20 with OASL and suggest ORF20 usurps the function of OASL to benefit KSHV infection.
Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/patogenicidade , Fases de Leitura Aberta/genética , Proteínas Virais/metabolismo , Replicação Viral , 2',5'-Oligoadenilato Sintetase/genética , Sequência de Aminoácidos , Células Cultivadas , Regulação Viral da Expressão Gênica , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/metabolismo , Humanos , Interferons/farmacologia , Proteínas Ribossômicas , Proteínas Virais/genéticaRESUMO
Toll-like receptors (TLRs) activate the innate immune system in response to pathogens. Here we show that TLR9 proteolytic cleavage is a prerequisite for TLR9 signaling. Inhibition of lysosomal proteolysis rendered TLR9 inactive. The carboxy-terminal fragment of TLR9 thus generated included a portion of the TLR9 ectodomain, as well as the transmembrane and cytoplasmic domains. This cleavage fragment bound to the TLR9 ligand CpG DNA and, when expressed in Tlr9(-/-) dendritic cells, restored CpG DNA-induced cytokine production. Although cathepsin L generated the requisite TLR9 cleavage products in a cell-free in vitro system, several proteases influenced TLR9 cleavage in intact cells. Lysosomal proteolysis thus contributes to innate immunity by facilitating specific cleavage of TLR9.
Assuntos
Ativação Enzimática/imunologia , Imunidade Inata/fisiologia , Lisossomos/metabolismo , Transdução de Sinais/imunologia , Receptor Toll-Like 9/metabolismo , Animais , Western Blotting , Catepsinas/imunologia , Catepsinas/metabolismo , Linhagem Celular , Ilhas de CpG , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunoprecipitação , Lisossomos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico/imunologia , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologiaRESUMO
The tetraspanin CD82 is a potent suppressor of tumor metastasis and regulates several processes including signal transduction, cell adhesion, motility, and aggregation. However, the mechanisms by which CD82 participates in innate immunity are unknown. We report that CD82 is a key regulator of TLR9 trafficking and signaling. TLR9 recognizes unmethylated cytosine-phosphate-guanine (CpG) motifs present in viral, bacterial, and fungal DNA. We demonstrate that TLR9 and CD82 associate in macrophages, which occurs in the endoplasmic reticulum (ER) and post-ER. Moreover, CD82 is essential for TLR9-dependent myddosome formation in response to CpG stimulation. Finally, CD82 modulates TLR9-dependent NF-κB nuclear translocation, which is critical for inflammatory cytokine production. To our knowledge, this is the first time a tetraspanin has been implicated as a key regulator of TLR signaling. Collectively, our study demonstrates that CD82 is a specific regulator of TLR9 signaling, which may be critical in cancer immunotherapy approaches and coordinating the innate immune response to pathogens.-Khan, N. S., Lukason, D. P., Feliu, M., Ward, R. A., Lord, A. K., Reedy, J. L., Ramirez-Ortiz, Z. G., Tam, J. M., Kasperkovitz, P. V., Negoro, P. E., Vyas, T. D., Xu, S., Brinkmann, M. M., Acharaya, M., Artavanis-Tsakonas, K., Frickel, E.-M., Becker, C. E., Dagher, Z., Kim, Y.-M., Latz, E., Ploegh, H. L., Mansour, M. K., Miranti, C. K., Levitz, S. M., Vyas, J. M. CD82 controls CpG-dependent TLR9 signaling.
Assuntos
Núcleo Celular/imunologia , Proteína Kangai-1/imunologia , Macrófagos/imunologia , Oligodesoxirribonucleotídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptor Toll-Like 9/imunologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/imunologia , Animais , Núcleo Celular/genética , Citocinas/genética , Citocinas/imunologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/patologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Proteína Kangai-1/genética , Macrófagos/patologia , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/imunologia , Células RAW 264.7 , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor Toll-Like 9/genéticaRESUMO
Toll-like receptor (TLR) signaling plays a critical role in innate and adaptive immune responses and must be tightly controlled. TLR4 uses LPS binding protein, MD-2, and CD14 as accessories to respond to LPS. We therefore investigated the presence of an analagous soluble cofactor that might assist in the recruitment of CpG oligonucleotides (CpG-ODNs) to TLR9. We report the identification of granulin as an essential secreted cofactor that potentiates TLR9-driven responses to CpG-ODNs. Granulin, an unusual cysteine-rich protein, bound to CpG-ODNs and interacted with TLR9. Macrophages from granulin-deficient mice showed not only impaired delivery of CpG-ODNs to endolysosomal compartments, but also decreased interaction of TLR9 with CpG-ODNs. As a consequence, granulin-deficient macrophages showed reduced responses to stimulation with CpG-ODNs, a trait corrected by provision of exogenous granulin. Thus, we propose that granulin contributes to innate immunity as a critical soluble cofactor for TLR9 signaling.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Transdução de Sinais , Receptor Toll-Like 9/imunologia , Animais , Células Cultivadas , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligodesoxirribonucleotídeos/imunologia , Progranulinas , Ligação Proteica , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismoRESUMO
The type I interferon (IFN) response is imperative for the establishment of the early antiviral immune response. Here we report the identification of the first type I IFN antagonist encoded by murine cytomegalovirus (MCMV) that shuts down signaling following pattern recognition receptor (PRR) sensing. Screening of an MCMV open reading frame (ORF) library identified M35 as a novel and strong negative modulator of IFNß promoter induction following activation of both RNA and DNA cytoplasmic PRR. Additionally, M35 inhibits the proinflammatory cytokine response downstream of Toll-like receptors (TLR). Using a series of luciferase-based reporters with specific transcription factor binding sites, we determined that M35 targets NF-κB-, but not IRF-mediated, transcription. Expression of M35 upon retroviral transduction of immortalized bone marrow-derived macrophages (iBMDM) led to reduced IFNß transcription and secretion upon activation of stimulator of IFN genes (STING)-dependent signaling. On the other hand, M35 does not antagonize interferon-stimulated gene (ISG) 56 promoter induction or ISG transcription upon exogenous stimulation of the type I IFN receptor (IFNAR). M35 is present in the viral particle and, upon MCMV infection of fibroblasts, is immediately shuttled to the nucleus where it exerts its immunomodulatory effects. Deletion of M35 from the MCMV genome and hence from the viral particle resulted in elevated type I IFN transcription and secretion in vitro and in vivo. In the absence of M35, lower viral titers are observed during acute infection of the host, and productive infection in the salivary glands was not detected. In conclusion, the M35 protein is released by MCMV immediately upon infection in order to deftly inhibit the antiviral type I IFN response by targeting NF-κB-mediated transcription. The identification of this novel viral protein reinforces the importance of timely countermeasures in the complex relationship between virus and host.
Assuntos
Infecções por Citomegalovirus/imunologia , Interferon Tipo I/antagonistas & inibidores , Muromegalovirus/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais , Proteínas Virais/metabolismo , Animais , Infecções por Citomegalovirus/virologia , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Interferon beta/genética , Interferon beta/metabolismo , Macrófagos/imunologia , Macrófagos/virologia , Camundongos , Muromegalovirus/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Ligação Proteica , Receptores de Reconhecimento de Padrão/genética , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Proteínas Virais/genéticaRESUMO
Early detection of viral invasion by pattern recognition receptors (PRR) is crucial for the induction of a rapid and efficient immune response. Cytosolic DNA sensors are the most recently described class of PRR, and induce transcription of type I interferons (IFN) and proinflammatory cytokines via the key adaptor protein stimulator of interferon genes (STING). Herpesviruses are a family of large DNA viruses widely known for their immense arsenal of proteins dedicated to manipulating and evading host immune responses. Tantamount to the significant role played by DNA sensors and STING in innate immune responses, herpesviruses have in turn evolved a range of mechanisms targeting virtually every step of this key signaling pathway. Strikingly, some herpesviruses also take advantage of this pathway to promote their own replication. In this review, we will summarize the current understanding of DNA sensing and subsequent induction of signaling and transcription, and showcase the close adaptation of herpesviruses to their host reflected by the myriad of viral proteins dedicated to modulating this critical innate immune pathway.
Assuntos
Evolução Biológica , DNA Viral/imunologia , Herpesviridae/crescimento & desenvolvimento , Herpesviridae/imunologia , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Imunidade Inata , Animais , Humanos , Receptores Imunológicos/metabolismoRESUMO
The latency-associated nuclear antigen (LANA) of Kaposi sarcoma herpesvirus (KSHV) is mainly localized and functions in the nucleus of latently infected cells, playing a pivotal role in the replication and maintenance of latent viral episomal DNA. In addition, N-terminally truncated cytoplasmic isoforms of LANA, resulting from internal translation initiation, have been reported, but their function is unknown. Using coimmunoprecipitation and MS, we found the cGMP-AMP synthase (cGAS), an innate immune DNA sensor, to be a cellular interaction partner of cytoplasmic LANA isoforms. By directly binding to cGAS, LANA, and particularly, a cytoplasmic isoform, inhibit the cGAS-STING-dependent phosphorylation of TBK1 and IRF3 and thereby antagonize the cGAS-mediated restriction of KSHV lytic replication. We hypothesize that cytoplasmic forms of LANA, whose expression increases during lytic replication, inhibit cGAS to promote the reactivation of the KSHV from latency. This observation points to a novel function of the cytoplasmic isoforms of LANA during lytic replication and extends the function of LANA from its role during latency to the lytic replication cycle.
Assuntos
Antígenos Virais/metabolismo , Citoplasma/metabolismo , Herpesvirus Humano 8/fisiologia , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/metabolismo , Replicação Viral/fisiologia , Animais , Antígenos Virais/genética , Chlorocebus aethiops , Citoplasma/genética , Citoplasma/virologia , Células HeLa , Humanos , Proteínas Nucleares/genética , Nucleotidiltransferases/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Células VeroRESUMO
The receptor-like protein tyrosine phosphatase CD45 is expressed on the surface of cells of hematopoietic origin and has a pivotal role for the function of these cells in the immune response. Here we report that following infection of macrophages with mouse cytomegalovirus (MCMV) the cell surface expression of CD45 is drastically diminished. Screening of a set of MCMV deletion mutants allowed us to identify the viral gene m42 of being responsible for CD45 down-modulation. Moreover, expression of m42 independent of viral infection upon retroviral transduction of the RAW264.7 macrophage cell line led to comparable regulation of CD45 expression. In immunocompetent mice infected with an m42 deletion mutant lower viral titers were observed in all tissues examined when compared to wildtype MCMV, indicating an important role of m42 for viral replication in vivo. The m42 gene product was identified as an 18 kDa protein expressed with early kinetics and is predicted to be a tail-anchored membrane protein. Tracking of surface-resident CD45 molecules revealed that m42 induces internalization and degradation of CD45. The observation that the amounts of the E3 ubiquitin ligases Itch and Nedd4 were diminished in cells expressing m42 and that disruption of a PY motif in the N-terminal part of m42 resulted in loss of function, suggest that m42 acts as an activator or adaptor for these Nedd4-like ubiquitin ligases, which mark CD45 for lysosomal degradation. In conclusion, the down-modulation of CD45 expression in MCMV-infected myeloid cells represents a novel pathway of virus-host interaction.