RESUMO
Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that is causally associated with various malignancies and autoimmune disease. Epstein-Barr Nuclear Antigen 1 (EBNA1) is the viral-encoded DNA binding protein required for viral episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 is known to be a highly stable protein, but the mechanisms regulating protein stability and how this may be linked to EBNA1 function is not fully understood. Proteomic analysis of EBNA1 revealed interaction with Procollagen Lysine-2 Oxoglutarate 5 Dioxygenase (PLOD) family of proteins. Depletion of PLOD1 by shRNA or inhibition with small molecule inhibitors 2,-2' dipyridyl resulted in the loss of EBNA1 protein levels, along with a selective growth inhibition of EBV-positive lymphoid cells. PLOD1 depletion also caused a loss of EBV episomes from latently infected cells and inhibited oriP-dependent DNA replication. Mass spectrometry identified EBNA1 peptides with lysine hydroxylation at K460 or K461. Mutation of K460, but not K461 abrogates EBNA1-driven DNA replication of oriP, but did not significantly affect EBNA1 DNA binding. Mutations in both K460 and K461 perturbed interactions with PLOD1, as well as decreased EBNA1 protein stability. These findings suggest that PLOD1 is a novel interaction partner of EBNA1 that regulates EBNA1 protein stability and function in viral plasmid replication, episome maintenance and host cell survival.
Assuntos
Infecções por Vírus Epstein-Barr , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase , Humanos , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Infecções por Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Lisina/genética , Proteômica , Replicação do DNA , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Replicação Viral , Estabilidade Proteica , Plasmídeos , Origem de ReplicaçãoRESUMO
Aberrant skipping of coding exons in CD19 and CD22 compromises the response to immunotherapy in B-cell malignancies. Here, we showed that the MS4A1 gene encoding human CD20 also produces several messenger RNA (mRNA) isoforms with distinct 5' untranslated regions. Four variants (V1-4) were detected using RNA sequencing (RNA-seq) at distinct stages of normal B-cell differentiation and B-lymphoid malignancies, with V1 and V3 being the most abundant. During B-cell activation and Epstein-Barr virus infection, redirection of splicing from V1 to V3 coincided with increased CD20 positivity. Similarly, in diffuse large B-cell lymphoma, only V3, but not V1, correlated with CD20 protein levels, suggesting that V1 might be translation-deficient. Indeed, the longer V1 isoform contained upstream open reading frames and a stem-loop structure, which cooperatively inhibited polysome recruitment. By modulating CD20 isoforms with splice-switching morpholino oligomers, we enhanced CD20 expression and anti-CD20 antibody rituximab-mediated cytotoxicity in a panel of B-cell lines. Furthermore, reconstitution of CD20-knockout cells with V3 mRNA led to the recovery of CD20 positivity, whereas V1-reconstituted cells had undetectable levels of CD20 protein. Surprisingly, in vitro CD20-directed chimeric antigen receptor T cells were able to kill both V3- and V1-expressing cells, but the bispecific T-cell engager mosunetuzumab was only effective against V3-expressing cells. To determine whether CD20 splicing is involved in immunotherapy resistance, we performed RNA-seq on 4 postmosunetuzumab follicular lymphoma relapses and discovered that in 2 of them, the downregulation of CD20 was accompanied by a V3-to-V1 shift. Thus, splicing-mediated mechanisms of epitope loss extend to CD20-directed immunotherapies.
Assuntos
Infecções por Vírus Epstein-Barr , Neoplasias , Humanos , Processamento Alternativo , RNA Mensageiro/genética , Regiões 5' não Traduzidas , Infecções por Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Antígenos CD20/genética , Isoformas de Proteínas/genética , Imunoterapia , Biossíntese de Proteínas , Neoplasias/genéticaRESUMO
Viruses suppress immune recognition through diverse mechanisms. Epstein-Barr Virus (EBV) establishes latent infection in memory B-lymphocytes and B-cell malignancies where it impacts B-cell immune function. We show here that EBV primary infection of naïve B-cells results in a robust down-regulation of HLA genes. We found that the viral encoded transcriptional regulatory factor EBNA2 bound to multiple regulatory regions in the HLA locus. Conditional expression of EBNA2 correlated with the down regulation of HLA class II transcription. EBNA2 down-regulation of HLA transcription was found to be dependent on CIITA, the major transcriptional activator of HLA class II gene transcription. We identified a major EBNA2 binding site downstream of the CIITA gene and upstream of DEXI, a dexamethasone inducible gene that is oriented head-to-head with CIITA gene transcripts. CRISPR/Cas9 deletion of the EBNA2 site upstream of DEXI attenuated CIITA transcriptional repression. EBNA2 caused an increase in DEXI transcription and a graded change in histone modifications with activation mark H3K27ac near the DEXI locus, and a loss of activation marks at the CIITA locus. A prominent CTCF binding site between CIITA and DEXI enhancers was mutated and further diminished the effects of EBNA2 on CIITA. Analysis of HiC data indicate that DEXI and CIITA enhancers are situated in different chromosome topological associated domains (TADs). These findings suggest that EBNA2 down regulates HLA-II genes through the down regulation of CIITA, and that this down regulation is an indirect consequence of EBNA2 enhancer formation at a neighboring TAD. We propose that enhancer competition between these neighboring chromosome domains represents a novel mechanism for gene regulation demonstrated by EBNA2.
Assuntos
Linfócitos B/virologia , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Genes MHC da Classe II/fisiologia , Proteínas de Membrana/genética , Proteínas Nucleares/genética , Transativadores/genética , Proteínas Virais/metabolismo , Linfócitos B/metabolismo , Proteínas de Ligação a DNA/metabolismo , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/genética , Regulação da Expressão Gênica , Herpesvirus Humano 4/fisiologia , Humanos , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Transativadores/metabolismo , Proteínas Virais/genéticaRESUMO
Epstein-Barr virus (EBV) immortalizes resting B-lymphocytes through a highly orchestrated reprogramming of host chromatin structure, transcription and metabolism. Here, we use a multi-omics-based approach to investigate these underlying mechanisms. ATAC-seq analysis of cellular chromatin showed that EBV alters over a third of accessible chromatin during the infection time course, with many of these sites overlapping transcription factors such as PU.1, Interferon Regulatory Factors (IRFs), and CTCF. Integration of RNA-seq analysis identified a complex transcriptional response and associations with EBV nuclear antigens (EBNAs). Focusing on EBNA1 revealed enhancer-binding activity at gene targets involved in nucleotide metabolism, supported by metabolomic analysis which indicated that adenosine and purine metabolism are significantly altered by EBV immortalization. We further validated that adenosine deaminase (ADA) is a direct and critical target of the EBV-directed immortalization process. These findings reveal that purine metabolism and ADA may be useful therapeutic targets for EBV-driven lymphoid cancers.
Assuntos
Linfócitos B/patologia , Transformação Celular Viral , Cromatina/genética , Infecções por Vírus Epstein-Barr/patologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Nucleotídeos/metabolismo , Proteínas Virais/metabolismo , Linfócitos B/metabolismo , Linfócitos B/virologia , Cromatina/metabolismo , Epigênese Genética , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Metaboloma , Transcriptoma , Proteínas Virais/genéticaRESUMO
Subpopulations of B-lymphocytes traffic to different sites and organs to provide diverse and tissue-specific functions. Here, we provide evidence that epigenetic differences confer a neuroinvasive phenotype. An EBV+ B cell lymphoma cell line (M14) with low frequency trafficking to the CNS was neuroadapted to generate a highly neuroinvasive B-cell population (MUN14). MUN14 B cells efficiently infiltrated the CNS within one week and produced neurological pathologies. We compared the gene expression profiles of viral and cellular genes using RNA-Seq and identified one viral (EBNA1) and several cellular gene candidates, including secreted phosphoprotein 1/osteopontin (SPP1/OPN), neuron navigator 3 (NAV3), CXCR4, and germinal center-associated signaling and motility protein (GCSAM) that were selectively upregulated in MUN14. ATAC-Seq and ChIP-qPCR revealed that these gene expression changes correlated with epigenetic changes at gene regulatory elements. The neuroinvasive phenotype could be attenuated with a neutralizing antibody to OPN, confirming the functional role of this protein in trafficking EBV+ B cells to the CNS. These studies indicate that B-cell trafficking to the CNS can be acquired by epigenetic adaptations and provide a new model to study B-cell neuroinvasion associated CNS lymphoma and autoimmune disease of the CNS, including multiple sclerosis (MS).
Assuntos
Linfócitos B/patologia , Linfócitos B/virologia , Neoplasias do Sistema Nervoso Central/virologia , Epigênese Genética , Infecções por Vírus Epstein-Barr/patologia , Animais , Linfócitos B/metabolismo , Transformação Celular Viral/fisiologia , Neoplasias do Sistema Nervoso Central/metabolismo , Neoplasias do Sistema Nervoso Central/patologia , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4 , Linfoma/metabolismo , Linfoma/patologia , Linfoma/virologia , Camundongos , Osteopontina/metabolismoRESUMO
Autophagy has been implicated as a component of host defense, but the significance of antimicrobial autophagy in vivo and the mechanism by which it is regulated during infection are poorly defined. Here we found that antiviral autophagy was conserved in flies and mammals during infection with Rift Valley fever virus (RVFV), a mosquito-borne virus that causes disease in humans and livestock. In Drosophila, Toll-7 limited RVFV replication and mortality through activation of autophagy. RVFV infection also elicited autophagy in mouse and human cells, and viral replication was increased in the absence of autophagy genes. The mammalian Toll-like receptor adaptor, MyD88, was required for anti-RVFV autophagy, revealing an evolutionarily conserved requirement for pattern-recognition receptors in antiviral autophagy. Pharmacologic activation of autophagy inhibited RVFV infection in mammalian cells, including primary hepatocytes and neurons. Thus, autophagy modulation might be an effective strategy for treating RVFV infection, which lacks approved vaccines and therapeutics.
Assuntos
Autofagia/imunologia , Febre do Vale de Rift/imunologia , Vírus da Febre do Vale do Rift/imunologia , Compostos Alílicos/farmacologia , Animais , Antivirais/farmacologia , Autofagia/efeitos dos fármacos , Células Cultivadas , Drosophila , Evolução Molecular , Hepatócitos/efeitos dos fármacos , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Controle de Infecções/métodos , Mamíferos , Camundongos , Fator 88 de Diferenciação Mieloide/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/imunologia , Neurônios/virologia , Quinazolinas/farmacologia , Ratos , Febre do Vale de Rift/tratamento farmacológico , Receptor 7 Toll-Like/metabolismo , Replicação ViralRESUMO
Epstein-Barr virus (EBV) is a human gammaherpesvirus that is causally associated with various lymphomas and carcinomas. Although EBV is not typically associated with multiple myeloma (MM), it can be found in some B-cell lines derived from MM patients. Here, we analyzed two EBV-positive MM-patient-derived cell lines, IM9 and ARH77, and found defective viral genomes and atypical viral gene expression patterns. We performed transcriptome sequencing to characterize the viral and cellular properties of the two EBV-positive cell lines, compared to the canonical MM cell line 8226. Principal-component analyses indicated that IM9 and ARH77 clustered together and distinct from 8226. Immunological Genome Project analysis designated these cells as stem cell and bone marrow derived. IM9 and ARH77 displayed atypical viral gene expression, including leaky lytic cycle gene expression with an absence of lytic DNA amplification. Genome sequencing revealed that the EBV genomes in ARH77 contain large deletions, while IM9 has copy number losses in multiple EBV loci. Both IM9 and ARH77 showed EBV genome heterogeneity, suggesting cells harboring multiple and variant viral genomes. We identified atypical high-level expression of lytic genes BLRF1 and BLRF2. We demonstrated that short hairpin RNA (shRNA) depletion of BLRF2 altered viral and host gene expression, including a reduction in lytic gene activation and DNA amplification. These findings demonstrate that aberrant viral genomes and lytic gene expression persist in rare B cells derived from MM tumors, and they suggest that EBV may contribute to the etiology of MM. IMPORTANCE EBV is an oncogenic herpesvirus, but its mechanisms of oncogenesis are not fully understood. A role for EBV in MM has not yet been established. We analyzed EBV-positive B-cell lines derived from MM patients and found that the cells harbored defective viral genomes with aberrant viral gene expression patterns and cell gene signatures for bone marrow-derived lymphoid stem cells. These findings suggest that aberrant EBV latent infection may contribute to the etiology of MM.
Assuntos
Linfócitos B/virologia , Vírus Defeituosos/genética , Regulação Viral da Expressão Gênica/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/isolamento & purificação , Mieloma Múltiplo/virologia , Animais , Células Cultivadas , Variações do Número de Cópias de DNA/genética , Modelos Animais de Doenças , Deleção de Genes , Genoma Viral/genética , Humanos , Camundongos , Camundongos SCID , Estresse Oxidativo/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , Transcriptoma/genética , Ativação Viral/genéticaRESUMO
BACKGROUND AND AIMS: Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is the most common EBV-associated cancer and accounts for ~ 10% of all gastric cancers (GC). Epstein-Barr virus nuclear antigen 1 (EBNA1), which is critical for the replication and maintenance of the EBV latent genome, is consistently expressed in all EBVaGC tumors. We previously developed small molecule inhibitors of EBNA1. In this study, we investigated the efficacy and selectivity of an EBNA1 inhibitor in cell-based and animal xenograft models of EBV-positive and EBV-negative gastric carcinoma. METHODS: We tested the potency of an EBNA1 inhibitor, VK-1727, in vitro and in xenograft studies, using EBV-positive (SNU719 and YCCEL1) and EBV-negative (AGS and MKN74) GC cell lines. After treatment, we analyzed cell viability, proliferation, and RNA expression of EBV genes by RT-qPCR. RESULTS: Treatment with VK-1727 selectively inhibits cell cycle progression and proliferation in vitro. In animal studies, treatment with an EBNA1 inhibitor resulted in a significant dose-dependent decrease in tumor growth in EBVaGC xenograft models, but not in EBV-negative GC xenograft studies. Gene expression analysis revealed that short term treatment in cell culture tended towards viral gene activation, while long-term treatment in animal xenografts showed a significant decrease in viral gene expression. CONCLUSIONS: EBNA1 inhibitors are potent and selective inhibitors of cell growth in tissue culture and animal models of EBV-positive GC. Long-term treatment with EBNA1 inhibitors may lead to loss of EBV in mouse xenografts. These results suggest that pharmacological targeting of EBNA1 may be an effective strategy to treat patients with EBVaGC.
Assuntos
Infecções por Vírus Epstein-Barr , Neoplasias Gástricas , Animais , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Antígenos Nucleares do Vírus Epstein-Barr/genética , Herpesvirus Humano 4 , Xenoenxertos , Humanos , Camundongos , Neoplasias Gástricas/tratamento farmacológicoRESUMO
Epstein-Barr Virus (EBV) is a ubiquitous human herpesvirus that contributes to the etiology of diverse human cancers and auto-immune diseases. EBV establishes a relatively benign, long-term latent infection in over 90 percent of the adult population. Yet, it also increases risk for certain cancers and auto-immune disorders depending on complex viral, host, and environmental factors that are only partly understood. EBV latent infection is found predominantly in memory B-cells, but the natural infection cycle and pathological aberrations enable EBV to infect numerous other cell types, including oral, nasopharyngeal, and gastric epithelia, B-, T-, and NK-lymphoid cells, myocytes, adipocytes, astrocytes, and neurons. EBV infected cells, free virus, and gene products can also be found in the CNS. In addition to the direct effects of EBV on infected cells and tissue, the effect of chronic EBV infection on the immune system is also thought to contribute to pathogenesis, especially auto-immune disease. Here, we review properties of EBV infection that may shed light on its potential pathogenic role in neurological disorders.
RESUMO
Neonatal seizures disrupt normal synaptic maturation and often lead to later-life epilepsy and cognitive deficits. During early life, the brain exhibits heightened synaptic plasticity, in part due to a developmental overabundance of CaV1.2 L-type voltage gated calcium (Ca2+) channels (LT-VGCCs) and Ca2+-permeable AMPARs (CP-AMPARs) lacking GluA2 subunits. We hypothesized that early-life seizures overactivate these channels, in turn dysregulating Ca2+-dependent signaling pathways including that of methyl CPG binding protein 2 (MeCP2), a transcription factor implicated in the autism spectrum disorder (ASD) Rett Syndrome. Here, we show that in vivo hypoxia-induced seizures (HS) in postnatal day (P)10 rats acutely induced phosphorylation of the neuronal-specific target of activity-dependent MeCP2 phosphorylation, S421, as well as its upstream activator CaMKII T286. We next identified mechanisms by which activity-dependent Ca2+ influx induced MeCP2 phosphorylation using in vitro cortical and hippocampal neuronal cultures at embryonic day (E)18â¯+â¯10â¯days in vitro (DIV). In contrast to the prevalent role of NMDARs in the adult brain, we found that both CP-AMPARs and LT-VGCCs mediated MeCP2 S421 and CaMKII T286 phosphorylation induced by kainic acid (KA) or high potassium chloride (KCl) stimulation. Furthermore, in vivo post-seizure treatment with the broad-spectrum AMPAR antagonist NBQX, the CP-AMPAR blocker IEM-1460, or the LT-VGCC antagonist nimodipine blocked seizure-induced MeCP2 phosphorylation. Collectively, these results demonstrate that early-life seizures dysregulate critical activity-dependent developmental signaling pathways, in part via CP-AMPAR and LT-VGCC activation, providing novel age-specific therapeutic targets for convergent pathways underlying epilepsy and ASDs.
Assuntos
Córtex Cerebral/metabolismo , Hipocampo/metabolismo , Proteína 2 de Ligação a Metil-CpG/metabolismo , Convulsões/metabolismo , Serina/metabolismo , Animais , Células Cultivadas , Córtex Cerebral/crescimento & desenvolvimento , Hipocampo/crescimento & desenvolvimento , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Fosforilação/fisiologia , Ratos , Convulsões/genética , Serina/genéticaRESUMO
Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel bunyavirus that recently emerged in China. Infection with SFTSV is associated with case-fatality rates of up to 30%, and neither antivirals nor vaccines are available at present. Development of antiviral strategies requires the elucidation of virus-host cell interactions. Here, we analyzed host cell entry of SFTSV. Employing lentiviral and rhabdoviral vectors, we found that the Gn/Gc glycoproteins (Gn/Gc) of SFTSV mediate entry into a broad range of human and animal cell lines, as well as human macrophages and dendritic cells. The Gn/Gc proteins of La Crosse virus (LACV) and Rift Valley Fever Virus (RVFV), other members of the bunyavirus family, facilitated entry into an overlapping but not identical range of cell lines, suggesting that SFTSV, LACV, and RVFV might differ in their receptor requirements. Entry driven by SFTSV Gn/Gc was dependent on low pH but did not require the activity of the pH-dependent endosomal/lysosomal cysteine proteases cathepsins B and L. Instead, the activity of a cellular serine protease was required for infection driven by SFTSV and LACV Gn/Gc. Sera from convalescent SFTS patients inhibited SFTSV Gn/Gc-driven host cell entry in a dose-dependent fashion, demonstrating that the vector system employed is suitable to detect neutralizing antibodies. Finally, the C-type lectin DC-SIGN was found to serve as a receptor for SFTSV Gn/Gc-driven entry into cell lines and dendritic cells. Our results provide initial insights into cell tropism, receptor usage, and proteolytic activation of SFTSV and will aid in the understanding of viral spread and pathogenesis.
Assuntos
Moléculas de Adesão Celular/metabolismo , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Orthobunyavirus/fisiologia , Receptores de Superfície Celular/metabolismo , Receptores Virais/metabolismo , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Células Dendríticas/virologia , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/virologia , Glicoproteínas de Membrana/imunologia , Orthobunyavirus/imunologia , Serina Proteases/metabolismo , Proteínas do Envelope Viral/imunologia , Tropismo ViralRESUMO
Epstein-Barr virus (EBV) is an aetiologic risk factor for the development of multiple sclerosis (MS). However, the role of EBV-infected B cells in the immunopathology of MS is not well understood. Here we characterized spontaneous lymphoblastoid cell lines (SLCLs) isolated from MS patients and healthy controls (HC) ex vivo to study EBV and host gene expression in the context of an individual's endogenous EBV. SLCLs derived from MS patient B cells during active disease had higher EBV lytic gene expression than SLCLs from MS patients with stable disease or HCs. Host gene expression analysis revealed activation of pathways associated with hypercytokinemia and interferon signalling in MS SLCLs and upregulation of forkhead box protein 1 (FOXP1), which contributes to EBV lytic gene expression. We demonstrate that antiviral approaches targeting EBV replication decreased cytokine production and autologous CD4+ T cell responses in this ex vivo model. These data suggest that dysregulation of intrinsic B cell control of EBV gene expression drives a pro-inflammatory, pathogenic B cell phenotype that can be attenuated by suppressing EBV lytic gene expression.
Assuntos
Linfócitos B , Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Esclerose Múltipla , Humanos , Herpesvirus Humano 4/genética , Esclerose Múltipla/virologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/virologia , Infecções por Vírus Epstein-Barr/virologia , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/complicações , Citocinas/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD4-Positivos/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Transcriptoma , Replicação Viral , Regulação Viral da Expressão Gênica , Linhagem Celular , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Perfilação da Expressão Gênica , Adulto , Feminino , MasculinoRESUMO
La Crosse virus (LACV) is a leading cause of pediatric encephalitis and aseptic meningitis in the midwestern and southern United States, where it is considered an emerging human pathogen. No specific therapies or vaccines are available for LACV or any other orthobunyaviruses. Inhibition of LACV entry into cells is a potential target for therapeutic intervention, but this approach is limited by our current knowledge of the entry process. Here, we determined that clathrin-mediated endocytosis is the primary mechanism of orthobunyavirus entry and identified key cellular factors in this process. First, we demonstrated that LACV colocalized with clathrin shortly after infection in HeLa cells; we then confirmed the functional requirement of dynamin- and clathrin-mediated endocytosis for orthobunyavirus entry using several independent assays and, importantly, extended these findings to primary neuronal cultures. We also determined that macropinocytosis and caveolar endocytosis, both established routes of virus entry, are not critical for cellular entry of LACV. Moreover, we demonstrated that LACV infection is dependent on Rab5, which plays an important regulatory role in early endosomes, but not on Rab7, which is associated with late endosomes. These findings provide the first description of bunyavirus entry into cells of the central nervous system, where infection can cause severe neurological disease, and will aid in the design and development of antivirals and therapeutics that may be useful in the treatment of LACV and, more broadly, arboviral infections of the central nervous system.
Assuntos
Clatrina/metabolismo , Encefalite da Califórnia/metabolismo , Endocitose , Endossomos/metabolismo , Vírus La Crosse/metabolismo , Internalização do Vírus , Animais , Chlorocebus aethiops , Clatrina/genética , Cricetinae , Encefalite da Califórnia/tratamento farmacológico , Encefalite da Califórnia/genética , Endossomos/genética , Endossomos/virologia , Células HeLa , Humanos , Vírus La Crosse/genética , Células Vero , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Epstein-Barr virus (EBV) is a ubiquitous human lymphotropic herpesvirus with a well-established causal role in several cancers. Recent studies have provided compelling epidemiological and mechanistic evidence for a causal role of EBV in multiple sclerosis (MS). MS is the most prevalent chronic inflammatory and neurodegenerative disease of the central nervous system and is thought to be triggered in genetically predisposed individuals by an infectious agent, with EBV as the lead candidate. How a ubiquitous virus that typically leads to benign latent infections can promote cancer and autoimmune disease in at-risk populations is not fully understood. Here we review the evidence that EBV is a causal agent for MS and how various risk factors may affect EBV infection and immune control. We focus on EBV contributing to MS through reprogramming of latently infected B lymphocytes and the chronic presentation of viral antigens as a potential source of autoreactivity through molecular mimicry. We consider how knowledge of EBV-associated cancers may be instructive for understanding the role of EBV in MS and discuss the potential for therapies that target EBV to treat MS.
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Infecções por Vírus Epstein-Barr , Esclerose Múltipla , Neoplasias , Doenças Neurodegenerativas , Humanos , Herpesvirus Humano 4 , Infecções por Vírus Epstein-Barr/complicaçõesRESUMO
Kaposi's Sarcoma (KS) is a heterogenous, multifocal vascular malignancy caused by the human herpesvirus 8 (HHV8), also known as Kaposi's Sarcoma-Associated Herpesvirus (KSHV). Here, we show that KS lesions express iNOS/NOS2 broadly throughout KS lesions, with enrichment in LANA positive spindle cells. The iNOS byproduct 3-nitrotyrosine is also enriched in LANA positive tumor cells and colocalizes with a fraction of LANA-nuclear bodies. We show that iNOS is highly expressed in the L1T3/mSLK tumor model of KS. iNOS expression correlated with KSHV lytic cycle gene expression, which was elevated in late-stage tumors (>4 weeks) but to a lesser degree in early stage (1 week) xenografts. Further, we show that L1T3/mSLK tumor growth is sensitive to an inhibitor of nitric oxide, L-NMMA. L-NMMA treatment reduced KSHV gene expression and perturbed cellular gene pathways relating to oxidative phosphorylation and mitochondrial dysfunction. These finding suggest that iNOS is expressed in KSHV infected endothelial-transformed tumor cells in KS, that iNOS expression depends on tumor microenvironment stress conditions, and that iNOS enzymatic activity contributes to KS tumor growth.
Assuntos
Herpesvirus Humano 8 , Sarcoma de Kaposi , Animais , Humanos , Camundongos , Antígenos Virais/genética , Herpesvirus Humano 8/genética , ômega-N-Metilarginina , Sarcoma de Kaposi/genética , Microambiente TumoralRESUMO
Epidemiological studies have demonstrated that Epstein-Barr virus (EBV) is a known etiologic risk factor, and perhaps prerequisite, for the development of MS. EBV establishes life-long latent infection in a subpopulation of memory B cells. Although the role of memory B cells in the pathobiology of MS is well established, studies characterizing EBV-associated mechanisms of B cell inflammation and disease pathogenesis in EBV (+) B cells from MS patients are limited. Accordingly, we analyzed spontaneous lymphoblastoid cell lines (SLCLs) from multiple sclerosis patients and healthy controls to study host-virus interactions in B cells, in the context of an individual's endogenous EBV. We identify differences in EBV gene expression and regulation of both viral and cellular genes in SLCLs. Our data suggest that EBV latency is dysregulated in MS SLCLs with increased lytic gene expression observed in MS patient B cells, especially those generated from samples obtained during "active" disease. Moreover, we show increased inflammatory gene expression and cytokine production in MS patient SLCLs and demonstrate that tenofovir alafenamide, an antiviral that targets EBV replication, decreases EBV viral loads, EBV lytic gene expression, and EBV-mediated inflammation in both SLCLs and in a mixed lymphocyte assay. Collectively, these data suggest that dysregulation of EBV latency in MS drives a pro-inflammatory, pathogenic phenotype in memory B cells and that this response can be attenuated by suppressing EBV lytic activation. This study provides further support for the development of antiviral agents that target EBV-infection for use in MS.
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PARP1 has been shown to regulate EBV latency. However, the therapeutic effect of PARP1 inhibitors on EBV+ lymphomagenesis has not yet been explored. Here, we show that PARPi BMN-673 has a potent anti-tumor effect on EBV-driven LCL in a mouse xenograft model. We found that PARP1 inhibition induces a dramatic transcriptional reprogramming of LCLs driven largely by the reduction of the MYC oncogene expression and dysregulation of MYC targets, both in vivo and in vitro. PARP1 inhibition also reduced the expression of viral oncoprotein EBNA2, which we previously demonstrated depends on PARP1 for activation of MYC. Further, we show that PARP1 inhibition blocks the chromatin association of MYC, EBNA2, and tumor suppressor p53. Overall, our study strengthens the central role of PARP1 in EBV malignant transformation and identifies the EBNA2/MYC pathway as a target of PARP1 inhibitors and its utility for the treatment of EBNA2-driven EBV-associated cancers.
RESUMO
IMPORTANCE: Epstein-Barr virus (EBV) latency is controlled by epigenetic silencing by DNA methylation [5-methyl cytosine (5mC)], histone modifications, and chromatin looping. However, how they dictate the transcriptional program in EBV-associated gastric cancers remains incompletely understood. EBV-associated gastric cancer displays a 5mC hypermethylated phenotype. A potential treatment for this cancer subtype is the DNA hypomethylating agent, which induces EBV lytic reactivation and targets hypermethylation of the cellular DNA. In this study, we identified a heterogeneous pool of EBV epialleles within two tumor-derived gastric cancer cell lines that are disrupted with a hypomethylating agent. Stochastic DNA methylation patterning at critical regulatory regions may be an underlying mechanism for spontaneous reactivation. Our results highlight the critical role of epigenetic modulation on EBV latency and life cycle, which is maintained through the interaction between 5mC and the host protein CCCTC-binding factor.
Assuntos
Infecções por Vírus Epstein-Barr , Neoplasias Gástricas , Humanos , Cromatina , Herpesvirus Humano 4/fisiologia , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Metilação de DNA , Decitabina/metabolismo , Latência Viral/genética , DNA/metabolismo , Genômica , Sítios de LigaçãoRESUMO
BACKGROUND AND OBJECTIVES: Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that establishes lifelong latency in memory B cells and has been identified as a major risk factor of multiple sclerosis (MS). B cell depletion therapies have disease-modifying benefit in MS. However, it is unclear whether this benefit is partly attributable to the elimination of EBV+ B cells. Currently, there are no EBV-specific antiviral therapies available for targeting EBV latent infection in MS and limited experimental models to study EBV in MS. METHODS: In this study, we describe the establishment of spontaneous lymphoblastoid cell lines (SLCLs) generated ex vivo with the endogenous EBV of patients with MS and controls and treated with either an Epstein-Barr virus nuclear antigen 1 (EBNA1) inhibitor (VK-1727) or cladribine, a nucleoside analog that eliminates B cells. RESULTS: We showed that a small molecule inhibitor of EBNA1, a critical regulator of the EBV life cycle, blocks the proliferation and metabolic activity of these SLCLs. In contrast to cladribine, a highly cytotoxic B cell depleting therapy currently used in MS, the EBNA1 inhibitor VK-1727 was cytostatic rather than cytotoxic and selective for EBV+ cells, while having no discernible effects on EBV- cells. We validate that VK-1727 reduces EBNA1 DNA binding at known viral and cellular sites by ChIP-qPCR. DISCUSSION: This study shows that patient-derived SLCLs provide a useful tool for interrogating the role of EBV+ B cells in MS and suggests that a clinical trial testing the effect of EBNA1 inhibitors in MS may be warranted.
Assuntos
Infecções por Vírus Epstein-Barr , Esclerose Múltipla , Humanos , Linhagem Celular , Proliferação de Células , Cladribina/farmacologia , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Infecções por Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr , Herpesvirus Humano 4 , Estudos de Casos e ControlesRESUMO
Aberrant skipping of coding exons in CD19 and CD22 compromises responses to immunotherapy for B-cell malignancies. Here, we show that the MS4A1 gene encoding human CD20 also produces several mRNA isoforms with distinct 5' untranslated regions (5'-UTR). Four variants (V1-4) were detectable by RNA-seq in distinct stages of normal B-cell differentiation and B-lymphoid malignancies, with V1 and V3 being the most abundant by far. During B-cell activation and Epstein-Barr virus infection, redirection of splicing from V1 to V3 coincided with increased CD20 positivity. Similarly, in diffuse large B-cell lymphoma only V3, but not V1, correlated with CD20 protein levels, suggesting that V1 might be translation-deficient. Indeed, the longer V1 isoform was found to contain upstream open reading frames (uORFs) and a stem-loop structure, which cooperatively inhibited polysome recruitment. By modulating CD20 isoforms with splice-switching Morpholino oligomers, we enhanced CD20 expression and anti-CD20 antibody rituximab-mediated cytotoxicity in a panel of B-cell lines. Furthermore, reconstitution of CD20-knockout cells with V3 mRNA led to the recovery of CD20 positivity, while V1-reconstituted cells had undetectable levels of CD20 protein. Surprisingly, in vitro CD20-directed CAR T cells were able to kill both V3- and V1-expressing cells, but the bispecific T cell engager mosunetuzumab was only effective against V3-expressing cells. To determine whether CD20 splicing is involved in immunotherapy resistance, we performed RNA-seq on four post-mosunetuzumab follicular lymphoma relapses and discovered that in two of them downregulation of CD20 was accompanied by the V3-to-V1 shift. Thus, splicing-mediated mechanisms of epitope loss extend to CD20-directed immunotherapies. Key Points: In normal & malignant human B cells, CD20 mRNA is alternatively spliced into four 5'-UTR isoforms, some of which are translation-deficient.The balance between translation-deficient and -competent isoforms modulates CD20 protein levels & responses to CD20-directed immunotherapies. Explanation of Novelty: We discovered that in normal and malignant B-cells, CD20 mRNA is alternatively spliced to generate four distinct 5'-UTRs, including the longer translation-deficient V1 variant. Cells predominantly expressing V1 were still sensitive to CD20-targeting chimeric antigen receptor T-cells. However, they were resistant to the bispecific anti-CD3/CD20 antibody mosunetuzumab, and the shift to V1 were observed in CD20-negative post-mosunetuzumab relapses of follicular lymphoma.