RESUMO
Multiple sclerosis (MS) is a demyelinating disease of the CNS. Epstein-Barr virus (EBV) contributes to the MS pathogenesis because high levels of EBV EBNA386-405-specific antibodies cross react with the CNS-derived GlialCAM370-389. However, it is unclear why only some individuals with such high autoreactive antibody titers develop MS. Here, we show that autoreactive cells are eliminated by distinct immune responses, which are determined by genetic variations of the host, as well as of the infecting EBV and human cytomegalovirus (HCMV). We demonstrate that potent cytotoxic NKG2C+ and NKG2D+ natural killer (NK) cells and distinct EBV-specific T cell responses kill autoreactive GlialCAM370-389-specific cells. Furthermore, immune evasion of these autoreactive cells was induced by EBV-variant-specific upregulation of the immunomodulatory HLA-E. These defined virus and host genetic pre-dispositions are associated with an up to 260-fold increased risk of MS. Our findings thus allow the early identification of patients at risk for MS and suggest additional therapeutic options against MS.
Assuntos
Autoimunidade , Infecções por Vírus Epstein-Barr , Esclerose Múltipla , Humanos , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/genética , Antígenos de Histocompatibilidade Classe I , Esclerose Múltipla/imunologia , Células Matadoras Naturais/imunologiaRESUMO
Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that persists as a multicopy episome in proliferating host cells. Episome maintenance is strictly dependent on EBNA1, a sequence-specific DNA-binding protein with no known enzymatic activities. Here, we show that EBNA1 forms a cell cycle-dependent DNA crosslink with the EBV origin of plasmid replication oriP. EBNA1 tyrosine 518 (Y518) is essential for crosslinking to oriP and functionally required for episome maintenance and generation of EBV-transformed lymphoblastoid cell lines (LCLs). Mechanistically, Y518 is required for replication fork termination at oriP in vivo and for formation of SDS-resistant complexes in vitro. EBNA1-DNA crosslinking corresponds to single-strand endonuclease activity specific to DNA structures enriched at replication-termination sites, such as 4-way junctions. These findings reveal that EBNA1 forms tyrosine-dependent DNA-protein crosslinks and single-strand cleavage at oriP required for replication termination and viral episome maintenance.
Assuntos
Ciclo Celular , Reagentes de Ligações Cruzadas/química , DNA Viral/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Plasmídeos/metabolismo , Origem de Replicação , Replicação Viral/fisiologia , Sequência de Aminoácidos , Linfócitos B/metabolismo , Linhagem Celular , Adutos de DNA/metabolismo , Replicação do DNA , Endonucleases/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/química , Antígenos Nucleares do Vírus Epstein-Barr/genética , Humanos , Mutação/genética , Ligação Proteica , Recombinação Genética/genética , Tirosina/metabolismoRESUMO
Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with B cell lymphomas. EBV glycoprotein 42 (gp42) binds HLA class II and activates membrane fusion with B cells. We isolated gp42-specific monoclonal antibodies (mAbs), A10 and 4C12, which use distinct mechanisms to neutralize virus infection. mAb A10 was more potent than the only known neutralizing gp42 mAb, F-2-1, in neutralizing EBV infection and blocking binding to HLA class II. mAb 4C12 was similar to mAb A10 in inhibiting glycoprotein-mediated B cell fusion but did not block receptor binding, and it was less effective in neutralizing infection. Crystallographic structures of gH/gL/gp42/A10 and gp42/4C12 complexes revealed two distinct sites of vulnerability on gp42 for receptor binding and B cell fusion. Passive transfer of mAb A10 into humanized mice conferred nearly 100% protection from viremia and EBV lymphomas after EBV challenge. These findings identify vulnerable sites on EBV that may facilitate therapeutics and vaccines.
Assuntos
Benzenoacetamidas , Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Piperidonas , Animais , Camundongos , Proteínas Virais/metabolismo , Glicoproteínas/metabolismo , Anticorpos AntiviraisRESUMO
Human immune responses to viral infections are highly variable, but the genetic factors that contribute to this variability are not well characterized. We used VirScan, a high-throughput epitope scanning technology, to analyze pan-viral antibody reactivity profiles of twins and SNP-genotyped individuals. Using these data, we determined the heritability and genomic loci associated with antibody epitope selection, response breadth, and control of Epstein-Barr virus (EBV) viral load. 107 EBV peptide reactivities were heritable and at least two Epstein-Barr nuclear antigen 2 (EBNA-2) reactivities were associated with variants in the MHC class II locus. We identified an EBV serosignature that predicted viral load in peripheral blood mononuclear cells and was associated with variants in the MHC class I locus. Our study illustrates the utility of epitope profiling to investigate the genetics of pathogen immunity, reports heritable features of the antibody response to viruses, and identifies specific HLA loci important for EBV epitope selection.
Assuntos
Anticorpos Antivirais/metabolismo , Epitopos/metabolismo , Infecções por Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Genótipo , Herpesvirus Humano 4/fisiologia , Epitopos Imunodominantes/metabolismo , Proteínas Virais/metabolismo , Adolescente , Adulto , Idoso , Estudos de Coortes , Mapeamento de Epitopos , Epitopos/genética , Infecções por Vírus Epstein-Barr/imunologia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Feminino , Antígenos HLA/genética , Antígenos HLA/metabolismo , Humanos , Imunidade Humoral , Epitopos Imunodominantes/genética , Masculino , Pessoa de Meia-Idade , Peptídeos/genética , Polimorfismo de Nucleotídeo Único , Característica Quantitativa Herdável , Estudos Soroepidemiológicos , Carga Viral , Proteínas Virais/genética , Adulto JovemRESUMO
Epstein-Barr virus (EBV) is nearly ubiquitous in adults. EBV causes infectious mononucleosis and is associated with B cell lymphomas, epithelial cell malignancies, and multiple sclerosis. The EBV gH/gL glycoprotein complex facilitates fusion of virus membrane with host cells and is a target of neutralizing antibodies. Here, we examined the sites of vulnerability for virus neutralization and fusion inhibition within EBV gH/gL. We developed a panel of human monoclonal antibodies (mAbs) that targeted five distinct antigenic sites on EBV gH/gL and prevented infection of epithelial and B cells. Structural analyses using X-ray crystallography and electron microscopy revealed multiple sites of vulnerability and defined the antigenic landscape of EBV gH/gL. One mAb provided near-complete protection against viremia and lymphoma in a humanized mouse EBV challenge model. Our findings provide structural and antigenic knowledge of the viral fusion machinery, yield a potential therapeutic antibody to prevent EBV disease, and emphasize gH/gL as a target for herpesvirus vaccines and therapeutics.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Cricetinae , Camundongos , Animais , Humanos , Proteínas do Envelope Viral , Cricetulus , Glicoproteínas de Membrana , Células CHORESUMO
Epstein-Barr virus (EBV) encodes a G protein-coupled receptor (GPCR) termed BILF1 that is essential for EBV-mediated immunosuppression and oncogenesis. BILF1 couples with inhibitory G protein (Gi), the major intracellular signaling effector for human chemokine receptors, and exhibits constitutive signaling activity; the ligand(s) for BILF1 are unknown. We studied the origins of BILF1's constitutive activity through structure determination of BILF1 bound to the inhibitory G protein (Gi) heterotrimer. The 3.2-Å resolution cryo-electron microscopy structure revealed an extracellular loop within BILF1 that blocked the typical chemokine binding site, suggesting ligand-autonomous receptor activation. Rather, amino acid substitutions within BILF1 transmembrane regions at hallmark ligand-activated class A GPCR "microswitches" stabilized a constitutively active BILF1 conformation for Gi coupling in a ligand-independent fashion. Thus, the constitutive activity of BILF1 promotes immunosuppression and virulence independent of ligand availability, with implications for the function of GPCRs encoded by related viruses and for therapeutic targeting of EBV.
Assuntos
Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Fatores Imunológicos/imunologia , Receptores Acoplados a Proteínas G/imunologia , Proteínas Virais/imunologia , Animais , Sítios de Ligação/imunologia , Linhagem Celular , Quimiocinas/imunologia , Microscopia Crioeletrônica/métodos , Infecções por Vírus Epstein-Barr/virologia , Células HEK293 , Humanos , Ligação Proteica/imunologia , Células Sf9 , Transdução de Sinais/imunologiaRESUMO
Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with epithelial-cell cancers and B cell lymphomas. An effective EBV vaccine is not available. We found that antibodies to the EBV glycoprotein gH/gL complex were the principal components in human plasma that neutralized infection of epithelial cells and that antibodies to gH/gL and gp42 contributed to B cell neutralization. Immunization of mice and nonhuman primates with nanoparticle vaccines that displayed components of the viral-fusion machinery EBV gH/gL or gH/gL/gp42 elicited antibodies that potently neutralized both epithelial-cell and B cell infection. Immune serum from nonhuman primates inhibited EBV-glycoprotein-mediated fusion of epithelial cells and B cells and targeted an epitope critical for virus-cell fusion. Therefore, unlike the leading EBV gp350 vaccine candidate, which only protects B cells from infection, these EBV nanoparticle vaccines elicit antibodies that inhibit the virus-fusion apparatus and provide cell-type-independent protection from virus infection.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Células Epiteliais/imunologia , Infecções por Vírus Epstein-Barr/prevenção & controle , Herpesvirus Humano 4/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Linfócitos B/virologia , Células CHO , Fusão Celular , Linhagem Celular Tumoral , Cricetulus , Células Epiteliais/virologia , Infecções por Vírus Epstein-Barr/imunologia , Feminino , Células HEK293 , Células HeLa , Humanos , Soros Imunes/administração & dosagem , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/imunologia , Ligação ViralRESUMO
Epstein-Barr virus (EBV) infection has long been associated with multiple sclerosis (MS), but the role of EBV in the pathogenesis of MS is not clear. Our hypothesis is that a major fraction of the expanded clones of T lymphocytes in the cerebrospinal fluid (CSF) are specific for autologous EBV-infected B cells. We obtained blood and CSF samples from eight relapsing-remitting patients in the process of diagnosis. We stimulated cells from the blood with autologous EBV-infected lymphoblastoid cell lines (LCL), EBV, varicella zoster virus, influenza, and candida and sorted the responding cells with flow cytometry after 6 d. We sequenced the RNA for T cell receptors (TCR) from CSF, unselected blood cells, and the antigen-specific cells. We used the TCR Vß CDR3 sequences from the antigen-specific cells to assign antigen specificity to the sequences from the CSF and blood. LCL-specific cells comprised 13.0 ± 4.3% (mean ± SD) of the total reads present in CSF and 13.3 ± 7.5% of the reads present in blood. The next most abundant antigen specificity was flu, which was 4.7 ± 1.7% of the reads in the CSF and 9.3 ± 6.6% in the blood. The prominence of LCL-specific reads was even more marked in the top 1% most abundant CSF clones with statistically significant 47% mean overlap with LCL. We conclude that LCL-specific sequences form a major portion of the TCR repertoire in both CSF and blood and that expanded clones specific for LCL are present in MS CSF. This has important implications for the pathogenesis of MS.
Assuntos
Infecções por Vírus Epstein-Barr , Influenza Humana , Esclerose Múltipla , Humanos , Linfócitos T , Herpesvirus Humano 4 , Receptores de Antígenos de Linfócitos TRESUMO
Epstein-Barr Virus (EBV) infects more than 90% of the adult population worldwide. EBV infection is associated with Burkitt lymphoma (BL) though alone is not sufficient to induce carcinogenesis implying the involvement of co-factors. BL is endemic in African regions faced with mycotoxins exposure. Exposure to mycotoxins and oncogenic viruses has been shown to increase cancer risks partly through the deregulation of the immune response. A recent transcriptome profiling of B cells exposed to aflatoxin B1 (AFB1) revealed an upregulation of the Chemokine ligand 22 (CCL22) expression although the underlying mechanisms were not investigated. Here, we tested whether mycotoxins and EBV exposure may together contribute to endemic BL (eBL) carcinogenesis via immunomodulatory mechanisms involving CCL22. Our results revealed that B cells exposure to AFB1 and EBV synergistically stimulated CCL22 secretion via the activation of Nuclear Factor-kappa B pathway. By expressing EBV latent genes in B cells, we revealed that elevated levels of CCL22 result not only from the expression of the latent membrane protein LMP1 as previously reported but also from the expression of other viral latent genes. Importantly, CCL22 overexpression resulting from AFB1-exposure in vitro increased EBV infection through the activation of phosphoinositide-3-kinase pathway. Moreover, inhibiting CCL22 in vitro and in humanized mice in vivo limited EBV infection and decreased viral genes expression, supporting the notion that CCL22 overexpression plays an important role in B cell infection. These findings unravel new mechanisms that may underpin eBL development and identify novel pathways that can be targeted in drug development.
Assuntos
Linfoma de Burkitt , Infecções por Vírus Epstein-Barr , Animais , Camundongos , Herpesvirus Humano 4/genética , Infecções por Vírus Epstein-Barr/complicações , Aflatoxina B1/toxicidade , Ligantes , Linfoma de Burkitt/metabolismo , Quimiocinas , CarcinogêneseRESUMO
Polyomaviruses are small, circular dsDNA viruses that can cause cancer. Alternative splicing of polyomavirus early transcripts generates large and small tumor antigens (LT, ST) that play essential roles in viral replication and tumorigenesis. Some polyomaviruses also express middle tumor antigens (MTs) or alternate LT open reading frames (ALTOs), which are evolutionarily related but have distinct gene structures. MTs are a splice variant of the early transcript whereas ALTOs are overprinted on the second exon of the LT transcript in an alternate reading frame and are translated via an alternative start codon. Merkel cell polyomavirus (MCPyV), the only human polyomavirus that causes cancer, encodes an ALTO but its role in the viral lifecycle and tumorigenesis has remained elusive. Here, we show MCPyV ALTO acts as a tumor suppressor and is silenced in Merkel cell carcinoma (MCC). Rescuing ALTO in MCC cells induces growth arrest and activates NF-κB signaling. ALTO activates NF-κB by binding SQSTM1 and TRAF2&3 via two N-Terminal Activating Regions (NTAR1+2), resembling Epstein-Barr virus (EBV) Latent Membrane Protein 1 (LMP1). Following activation, NF-κB dimers bind the MCPyV noncoding control region (NCCR) and downregulate early transcription. Beyond MCPyV, NTAR motifs are conserved in other polyomavirus ALTOs, which activate NF-κB signaling, but are lacking in MTs that do not. Furthermore, polyomavirus ALTOs downregulate their respective viral early transcription in an NF-κB- and NTAR-dependent manner. Our findings suggest that ALTOs evolved to suppress viral replication and promote viral latency and that MCPyV ALTO must be silenced for MCC to develop.
Assuntos
Regulação Viral da Expressão Gênica , NF-kappa B , Transdução de Sinais , Humanos , NF-kappa B/metabolismo , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismo , Poliomavírus das Células de Merkel/genética , Infecções por Polyomavirus/virologia , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/metabolismo , Carcinoma de Célula de Merkel/virologia , Carcinoma de Célula de Merkel/genética , Carcinoma de Célula de Merkel/metabolismo , Fases de Leitura Aberta/genética , Linhagem Celular Tumoral , Regulação para Baixo , Processamento AlternativoRESUMO
The two γ-herpesviruses Epstein Barr virus (EBV) and Kaposi sarcoma associated herpesvirus (KSHV) are each associated with more than 1% of all tumors in humans. While EBV establishes persistent infection in nearly all adult individuals, KSHV benefits from this widespread EBV prevalence for its own persistence. Interestingly, EBV infection expands early differentiated NKG2A+KIR- NK cells that protect against lytic EBV infection, while KSHV co-infection drives accumulation of poorly functional CD56-CD16+ NK cells. Thus persistent γ-herpesvirus infections are sculptors of human NK cell repertoires and the respectively stimulated NK cell subsets should be considered for immunotherapies of EBV and KSHV associated malignancies.
Assuntos
Infecções por Vírus Epstein-Barr , Infecções por Herpesviridae , Herpesvirus Humano 8 , Neoplasias , Adulto , Humanos , Herpesvirus Humano 4/fisiologia , Herpesvirus Humano 8/fisiologia , Células Matadoras NaturaisRESUMO
Epstein-Barr virus (EBV) is a human tumor virus associated with a variety of malignancies, including nasopharyngeal carcinoma, gastric cancers, and B-cell lymphomas. N6-methyladenosine (m6A) modifications modulate a wide range of cellular processes and participate in the regulation of virus-host cell interactions. Here, we discovered that EBV infection downregulates toll-like receptor 9 (TLR9) m6A modification levels and thus inhibits TLR9 expression. TLR9 has multiple m6A modification sites. Knockdown of METTL3, an m6A "writer", decreases TLR9 protein expression by inhibiting its mRNA stability. Mechanistically, Epstein-Barr nuclear antigen 1 increases METTL3 protein degradation via K48-linked ubiquitin-proteasome pathway. Additionally, YTHDF1 was identified as an m6A "reader" of TLR9, enhancing TLR9 expression by promoting mRNA translation in an m6A -dependent manner, which suggests that EBV inhibits TLR9 translation by "hijacking" host m6A modification mechanism. Using the METTL3 inhibitor STM2457 inhibits TLR9-induced B cell proliferation and immunoglobulin secretion, and opposes TLR9-induced immune responses to assist tumor cell immune escape. In clinical lymphoma samples, the expression of METTL3, YTHDF1, and TLR9 was highly correlated with immune cells infiltration. This study reveals a novel mechanism that EBV represses the important innate immunity molecule TLR9 through modulating the host m6A modification system.
Assuntos
Adenosina , Herpesvirus Humano 4 , Metiltransferases , Proteínas de Ligação a RNA , Receptor Toll-Like 9 , Humanos , Adenosina/análogos & derivados , Adenosina/metabolismo , Linfócitos B/metabolismo , Linfócitos B/imunologia , Linfócitos B/virologia , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Infecções por Vírus Epstein-Barr/genética , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Evasão da Resposta Imune , Metiltransferases/metabolismo , Metiltransferases/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Receptor Toll-Like 9/metabolismo , Receptor Toll-Like 9/genética , Linhagem Celular TumoralRESUMO
Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, persistently infects over 90% of the human adult population and is associated with several human cancers. To establish life-long infection, EBV tampers with the induction of type I interferon (IFN I)-dependent antiviral immunity in the host. How various EBV genes help orchestrate this crucial strategy is incompletely defined. Here, we reveal a mechanism by which the EBV nuclear antigen 3A (EBNA3A) may inhibit IFNß induction. Using proximity biotinylation we identify the histone acetyltransferase P300, a member of the IFNß transcriptional complex, as a binding partner of EBNA3A. We further show that EBNA3A also interacts with the activated IFN-inducing transcription factor IRF3 that collaborates with P300 in the nucleus. Both events are mediated by the N-terminal domain of EBNA3A. We propose that EBNA3A limits binding of IRF3 to the IFNß promoter, thereby hampering downstream IFN I signaling. Collectively, our findings suggest a new mechanism of immune evasion by EBV, affected by its latency gene EBNA3A.
RESUMO
Glioma is a highly fatal cancer with prognostically significant molecular subtypes and few known risk factors. Multiple studies have implicated infections in glioma susceptibility, but evidence remains inconsistent. Genetic variants in the human leukocyte antigen (HLA) region modulate host response to infection and have been linked to glioma risk. In this study, we leveraged genetic predictors of antibody response to 12 viral antigens to investigate the relationship with glioma risk and survival. Genetic reactivity scores (GRSs) for each antigen were derived from genome-wide-significant (p < 5 × 10-8) variants associated with immunoglobulin G antibody response in the UK Biobank cohort. We conducted parallel analyses of glioma risk and survival for each GRS and HLA alleles imputed at two-field resolution by using data from 3,418 glioma-affected individuals subtyped by somatic mutations and 8,156 controls. Genetic reactivity scores to Epstein-Barr virus (EBV) ZEBRA and EBNA antigens and Merkel cell polyomavirus (MCV) VP1 antigen were associated with glioma risk and survival (Bonferroni-corrected p < 0.01). GRSZEBRA and GRSMCV were associated in opposite directions with risk of IDH wild-type gliomas (ORZEBRA = 0.91, p = 0.0099/ORMCV = 1.11, p = 0.0054). GRSEBNA was associated with both increased risk for IDH mutated gliomas (OR = 1.09, p = 0.040) and improved survival (HR = 0.86, p = 0.010). HLA-DQA1∗03:01 was significantly associated with decreased risk of glioma overall (OR = 0.85, p = 3.96 × 10-4) after multiple testing adjustment. This systematic investigation of the role of genetic determinants of viral antigen reactivity in glioma risk and survival provides insight into complex immunogenomic mechanisms of glioma pathogenesis. These results may inform applications of antiviral-based therapies in glioma treatment.
Assuntos
Infecções por Vírus Epstein-Barr , Glioma , Esclerose Múltipla , Antígenos Virais , Infecções por Vírus Epstein-Barr/complicações , Glioma/complicações , Glioma/genética , Herpesvirus Humano 4/genética , Humanos , Imunogenética , Esclerose Múltipla/genéticaRESUMO
We demonstrate an optimized oligonucleotide library-based approach for the identification of virus-reactive T-cell receptors using Epstein-Barr virus as an example. HEK293T served as antigen-presenting cells and were co-cultured with human T cells that were transduced with T-cell receptors in question. T-cell activation was detected by CD137 expression.
Assuntos
Biblioteca Gênica , Herpesvirus Humano 4 , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T , Humanos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/genética , Células HEK293 , Herpesvirus Humano 4/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Técnicas de CoculturaRESUMO
Treatment options for Epstein-Barr virus (EBV)-cancers are limited, underscoring the need for new therapeutic approaches. We have previously shown that EBV-transformed cells and cancers lack homologous recombination (HR) repair, a prominent error-free pathway that repairs double-stranded DNA breaks; instead, EBV-transformed cells demonstrate genome-wide scars of the error-prone microhomology-mediated end joining (MMEJ) repair pathway. This suggests that EBV-cancers are vulnerable to synthetic lethal therapeutic approaches that target MMEJ repair. Indeed, we have previously found that targeting PARP, an enzyme that contributes to MMEJ, results in the death of EBV-lymphoma cells. With the emergence of clinical resistance to PARP inhibitors and the recent discovery of inhibitors of Polymerase theta (POLθ), the polymerase essential for MMEJ, we investigated the role of POLθ in EBV-lymphoma cells. We report that EBV-transformed cell lines, EBV-lymphoma cell lines, and EBV-lymphomas in AIDS patients demonstrate greater abundance of POLθ, driven by the EBV protein EBNA1, compared to EBV-uninfected primary lymphocytes and EBV-negative lymphomas from AIDS patients (a group that also abundantly expresses POLθ). We also find POLθ enriched at cellular DNA replication forks and exposure to the POLθ inhibitor Novobiocin impedes replication fork progress, impairs MMEJ-mediated repair of DNA double-stranded breaks, and kills EBV-lymphoma cells. Notably, cell killing is not due to Novobiocin-induced activation of the lytic/replicative phase of EBV. These findings support a role for POLθ not just in DNA repair but also DNA replication and as a therapeutic target in EBV-lymphomas and potentially other EBV-cancers as EBNA1 is expressed in all EBV-cancers.IMPORTANCEEpstein-Barr virus (EBV) contributes to ~2% of the global cancer burden. With a recent estimate of >200,000 deaths a year, identifying molecular vulnerabilities will be key to the management of these frequently aggressive and treatment-resistant cancers. Building on our earlier work demonstrating reliance of EBV-cancers on microhomology-mediated end-joining repair, we now report that EBV lymphomas and transformed B cell lines abundantly express the MMEJ enzyme POLθ that likely protects cellular replication forks and repairs replication-related cellular DNA breaks. Importantly also, we show that a newly identified POLθ inhibitor kills EBV-cancer cells, revealing a novel strategy to block DNA replication and repair of these aggressive cancers.
Assuntos
DNA Polimerase teta , DNA Polimerase Dirigida por DNA , Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Humanos , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Infecções por Vírus Epstein-Barr/virologia , Linhagem Celular Tumoral , Reparo do DNA por Junção de Extremidades , Linfoma/virologia , Linfoma/tratamento farmacológico , Linfoma/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/genética , Quebras de DNA de Cadeia Dupla , Mutações Sintéticas Letais , Replicação do DNA/efeitos dos fármacosRESUMO
Most mature B-cell malignancies originate from the malignant transformation of germinal center (GC) B cells. The GC reaction appears to have a role in malignant transformation, in which a major player of the GC reaction is BCL6, a key regulator of this process. We now demonstrate that BCL6 protein levels were dramatically decreased in Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines and Burkitt's lymphoma cell lines. Notably, BCL6 degradation was significantly enhanced in the presence of both EBNA3C and FBXO11. Furthermore, the amino-terminal domain of EBNA3C, which contains residues 50-100, interacts directly with FBXO11. The expression of EBNA3C and FBXO11 resulted in a significant induction of cell proliferation. Furthermore, BCL6 protein expression levels were regulated by EBNA3C via the Skp Cullin Fbox (SCF)FBXO11 complex, which mediated its ubiquitylation, and knockdown of FBXO11 suppressed the transformation of lymphoblastoid cell lines. These data provide new insights into the function of EBNA3C in B-cell transformation during GC reaction and raise the possibility of developing new targeted therapies against EBV-associated cancers. IMPORTANCE: The novel revelation in our study involves the suppression of BCL6 expression by the essential Epstein-Barr virus (EBV) antigen EBNA3C, shedding new light on our current comprehension of how EBV contributes to lymphomagenesis by impeding the germinal center reaction. It is crucial to note that while several EBV latent proteins are expressed in infected cells, the collaborative mechanisms among these proteins in regulating B-cell development or inducing B-cell lymphoma require additional investigation. Nonetheless, our findings carry significance for the development of emerging strategies aimed at addressing EBV-associated cancers.
Assuntos
Antígenos Nucleares do Vírus Epstein-Barr , Proteínas F-Box , Herpesvirus Humano 4 , Proteínas Proto-Oncogênicas c-bcl-6 , Humanos , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 4/genética , Linhagem Celular Tumoral , Linfócitos B/metabolismo , Linfócitos B/virologia , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Proteólise , Proliferação de Células , Ubiquitinação , Linfoma de Burkitt/virologia , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , Antígenos Virais/metabolismo , Antígenos Virais/genética , Centro Germinativo/metabolismo , Centro Germinativo/virologia , Proteína-Arginina N-MetiltransferasesRESUMO
Epstein-Barr virus (EBV) has a lifelong latency period after initial infection. Rarely, however, when the EBV immediate early gene BZLF1 is expressed by a specific stimulus, the virus switches to the lytic cycle to produce progeny viruses. We found that EBV infection reduced levels of various ceramide species in gastric cancer cells. As ceramide is a bioactive lipid implicated in the infection of various viruses, we assessed the effect of ceramide on the EBV lytic cycle. Treatment with C6-ceramide (C6-Cer) induced an increase in the endogenous ceramide pool and increased production of the viral product as well as BZLF1 expression. Treatment with the ceramidase inhibitor ceranib-2 induced EBV lytic replication with an increase in the endogenous ceramide pool. The glucosylceramide synthase inhibitor Genz-123346 inhibited C6-Cer-induced lytic replication. C6-Cer induced extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB phosphorylation, c-JUN expression, and accumulation of the autophagosome marker LC3B. Treatment with MEK1/2 inhibitor U0126, siERK1&2, or siCREB suppressed C6-Cer-induced EBV lytic replication and autophagy initiation. In contrast, siJUN transfection had no impact on BZLF1 expression. The use of 3-methyladenine (3-MA), an inhibitor targeting class III phosphoinositide 3-kinases (PI3Ks) to inhibit autophagy initiation, resulted in reduced beclin-1 expression, along with suppressed C6-Cer-induced BZLF1 expression and LC3B accumulation. Chloroquine, an inhibitor of autophagosome-lysosome fusion, increased BZLF1 protein intensity and LC3B accumulation. However, siLC3B transfection had minimal effect on BZLF1 expression. The results suggest the significance of ceramide-related sphingolipid metabolism in controlling EBV latency, highlighting the potential use of drugs targeting sphingolipid metabolism for treating EBV-positive gastric cancer.IMPORTANCEEpstein-Barr virus remains dormant in the host cell but occasionally switches to the lytic cycle when stimulated. However, the exact molecular mechanism of this lytic induction is not well understood. In this study, we demonstrate that Epstein-Barr virus infection leads to a reduction in ceramide levels. Additionally, the restoration of ceramide levels triggers lytic replication of Epstein-Barr virus with increase in phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB. Our study suggests that the Epstein-Barr virus can inhibit lytic replication and remain latent through reduction of host cell ceramide levels. This study reports the regulation of lytic replication by ceramide in Epstein-Barr virus-positive gastric cancer.
Assuntos
Carcinoma , Ceramidas , Infecções por Vírus Epstein-Barr , Neoplasias Gástricas , Humanos , Carcinoma/virologia , Linhagem Celular Tumoral , Ceramidas/farmacologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Proteína Quinase 3 Ativada por Mitógeno , Neoplasias Gástricas/virologia , Transativadores/metabolismo , Ativação ViralRESUMO
The tripartite interaction motif (TRIM) family of proteins is known for their antiviral activity through different mechanisms, such as interfering with viral components, regulating immune responses, and participating in autophagy-mediated defense pathways. In this study, we investigated the role of tripartite interaction motif 26 (TRIM26), which is encoded by a major histocompatibility complex (MHC) gene, in regulating Epstein-Barr virus (EBV) infection of nasopharyngeal epithelial cells. We found that TRIM26 expression was induced upon EBV infection and that it indirectly targeted EphA2, a crucial epithelial receptor for EBV entry. Our results showed that TRIM26 interacted with heat shock protein 90-beta (HSP-90ß) and promoted its polyubiquitination, which led to its degradation via the proteasome pathway. This, in turn, affected EphA2 integrity and suppressed EBV infection. These findings suggest that TRIM26 could be a valuable target for developing therapeutic interventions against EBV infection and its associated pathogenesis.
Assuntos
Infecções por Vírus Epstein-Barr , Humanos , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/fisiologia , Células Epiteliais/metabolismo , Ubiquitinação , Domínios Proteicos , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Hodgkin lymphoma is histologically characterised by the presence of Hodgkin (H) and Reed-Sternberg (RS) cells originating from germinal centre B-cells rearranged in the IgV gene. The formation of multinucleated RS cells is a product of telomere organisation in a process initiated by telomere aggregate accumulation in mononuclear H cells and may be mediated by latent membrane protein 1 (LMP-1) expression. LMP-1 is the main oncoprotein of EBV and supports several tumourigenic processes. LMP-1 may rescue proapoptotic B-cells through downregulation of B-cell receptor (BCR) components, mimicking and inducing multiple distinct B-cell signalling pathways to promote proliferation and survival, such as Janus kinase-signal transducer and activator of transcription (JAK-STAT), nuclear factor-kappa b (NF-кB), and cellular MYC (c-MYC), and inducing telomere instability mainly through Telomere repeat binding factor 2 (TRF2) downregulation to promote the formation of multinucleated RS cells. This review presents recent discoveries regarding the influence of LMP-1 on the surviving cellular signalling, genomic instability and mecanical formation of HRS cells.