RESUMEN
Epstein-Barr virus (EBV), which is ubiquitous in the adult population, is causally associated with human malignancies. Like many infectious agents, EBV has evolved strategies to block host cell death, including through expression of viral homologues of cellular BCL-2 pro-survival proteins (vBCL-2s), such as BHRF1. Small molecule inhibitors of the cellular pro-survival BCL-2 family proteins, termed 'BH3-mimetics', have entered clinical trials for blood cancers with the BCL-2 inhibitor venetoclax already approved for treatment of therapy refractory chronic lymphocytic leukaemia and acute myeloid leukaemia in the elderly. The generation of BH3-mimetics that could specifically target vBCL-2 proteins may be an attractive therapeutic option for virus-associated cancers, since these drugs would be expected to only kill virally infected cells with only minimal side effects on normal healthy tissues. To achieve this, a better understanding of the contribution of vBCL-2 proteins to tumorigenesis and insights into their biochemical functions is needed. In the context of Burkitt lymphoma (BL), BHRF1 expression conferred strong resistance to diverse apoptotic stimuli. Furthermore, BHRF1 expression in mouse haematopoietic stem and progenitor cells accelerated MYC-induced lymphoma development in a model of BL. BHRF1 interacts with the cellular pro-apoptotic BCL-2 proteins, BIM, BID, PUMA and BAK, but its capability to inhibit apoptosis could not be mapped solely to one of these interactions, suggesting plasticity is a key feature of BHRF1. Site-directed mutagenesis revealed a site in BHRF1 that was critical for its interaction with PUMA and blocking DNA-damage-induced apoptosis, identifying a potentially therapeutically targetable vulnerability in BHRF1.
Asunto(s)
Apoptosis , Linfoma de Burkitt/patología , Carcinogénesis/patología , Resistencia a Antineoplásicos , Proteínas Proto-Oncogénicas c-bcl-2/química , Homología de Secuencia de Aminoácido , Proteínas Virales/metabolismo , Animales , Apoptosis/genética , Proteína 11 Similar a Bcl2/metabolismo , Linfoma de Burkitt/virología , Muerte Celular , Línea Celular Tumoral , Citoprotección , Resistencia a Antineoplásicos/genética , Humanos , Mutación con Pérdida de Función , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Latencia del VirusRESUMEN
Although the over-expression of angiogenic factors is reported in diffuse large B-cell lymphoma (DLBCL), the poor response to anti-VEGF drugs observed in clinical trials suggests that angiogenesis in these tumours might be driven by VEGF-independent pathways. We show that sphingosine kinase-1 (SPHK1), which generates the potent bioactive sphingolipid sphingosine-1-phosphate (S1P), is over-expressed in DLBCL. A meta-analysis of over 2000 cases revealed that genes correlated with SPHK1 mRNA expression in DLBCL were significantly enriched for tumour angiogenesis meta-signature genes; an effect evident in both major cell of origin (COO) and stromal subtypes. Moreover, we found that S1P induces angiogenic signalling and a gene expression programme that is present within the tumour vasculature of SPHK1-expressing DLBCL. Importantly, S1PR1 functional antagonists, including Siponimod, and the S1P neutralising antibody, Sphingomab, inhibited S1P signalling in DLBCL cells in vitro. Furthermore, Siponimod, also reduced angiogenesis and tumour growth in an S1P-producing mouse model of angiogenic DLBCL. Our data define a potential role for S1P signalling in driving an angiogenic gene expression programme in the tumour vasculature of DLBCL and suggest novel opportunities to target S1P-mediated angiogenesis in patients with DLBCL.
Asunto(s)
Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Lisofosfolípidos/metabolismo , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Transducción de Señal , Esfingosina/análogos & derivados , Transcriptoma , Animales , Línea Celular Tumoral , Biología Computacional/métodos , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Linfoma de Células B Grandes Difuso/patología , Lisofosfolípidos/genética , Ratones , ARN Mensajero/genética , Esfingosina/genética , Esfingosina/metabolismoRESUMEN
While the association of Epstein-Barr virus (EBV) with Burkitt lymphoma (BL) has long been recognised, the precise role of the virus in BL pathogenesis is not fully resolved. EBV can be lost spontaneously from some BL cell lines, and these EBV-loss lymphoma cells reportedly have a survival disadvantage. Here we have generated an extensive panel of EBV-loss clones from multiple BL backgrounds and examined their phenotype comparing them to their isogenic EBV-positive counterparts. We report that, while loss of EBV from BL cells is rare, it is consistently associated with an enhanced predisposition to undergo apoptosis and reduced tumorigenicity in vivo. Importantly, reinfection of EBV-loss clones with EBV, but surprisingly not transduction with individual BL-associated latent viral genes, restored protection from apoptosis. Expression profiling and functional analysis of apoptosis-related proteins and transcripts in BL cells revealed that EBV inhibits the upregulation of the proapoptotic BH3-only proteins, BIM and PUMA. We conclude that latent EBV genes cooperatively enhance the survival of BL cells by suppression of the intrinsic apoptosis pathway signalling via inhibition of the potent apoptosis initiators, BIM and PUMA.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2/metabolismo , Linfoma de Burkitt/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteína 11 Similar a Bcl2/antagonistas & inhibidores , Linfoma de Burkitt/patología , Línea Celular , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/crecimiento & desarrollo , Humanos , Proteínas Proto-Oncogénicas/antagonistas & inhibidoresRESUMEN
UNLABELLED: The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8(+) cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8(+) cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8(+) cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4(+) cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8(+) and CD4(+) T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. IMPORTANCE: Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8(+) T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8(+) T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8(+) T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4(+) T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways.
Asunto(s)
Herpesvirus Humano 4/inmunología , Herpesvirus Humano 4/fisiología , Antígenos de Histocompatibilidad Clase II/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Evasión Inmune , Glicoproteínas de Membrana/metabolismo , Ubiquitinación , Proteínas Virales/metabolismo , Línea Celular , Regulación hacia Abajo , Humanos , Complejo Mayor de Histocompatibilidad , Proteínas de la Membrana/metabolismo , Linfocitos T/inmunologíaRESUMEN
UNLABELLED: Epstein-Barr Virus (EBV) persists for the lifetime of the infected host despite eliciting strong immune responses. This persistence requires a fine balance between the host immune system and EBV immune evasion. Accumulating evidence suggests an important role for natural killer (NK) cells in this balance. NK cells can kill EBV-infected cells undergoing lytic replication in vitro, and studies in both humans and mice with reconstituted human immune systems have shown that NK cells can limit EBV replication and prevent infectious mononucleosis. We now show that NK cells, via NKG2D and DNAM-1 interactions, recognize and kill EBV-infected cells undergoing lytic replication and that expression of a single EBV lytic gene, BZLF1, is sufficient to trigger sensitization to NK cell killing. We also present evidence suggesting the possibility of the existence of an as-yet-unidentified DNAM-1 ligand which may be particularly important for killing lytically infected normal B cells. Furthermore, while cells entering the lytic cycle become sensitized to NK cell killing, we observed that cells in the late lytic cycle are highly resistant. We identified expression of the vBcl-2 protein, BHRF1, as one effective mechanism by which EBV mediates this protection. Thus, contrary to the view expressed in some reports, EBV has evolved the ability to evade NK cell responses. IMPORTANCE: This report extends our understanding of the interaction between EBV and host innate responses. It provides the first evidence that the susceptibility to NK cell lysis of EBV-infected B cells undergoing lytic replication is dependent upon the phase of the lytic cycle. Induction of the lytic cycle is associated with acquired sensitization to NK cell killing, while progress through the late lytic cycle is associated with acquired resistance to killing. We provide mechanistic explanations for this novel observation, indicating important roles for the BZLF1 immediate early transactivator, the BHRF1 vBcl-2 homologue, and a novel ligand for the DNAM-1 NK cell receptor.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/virología , Herpesvirus Humano 4/fisiología , Evasión Inmune , Células Asesinas Naturales/inmunología , Transactivadores/metabolismo , Activación Viral , Células Cultivadas , Herpesvirus Humano 4/inmunología , Humanos , Replicación ViralRESUMEN
The working model to describe the mechanisms used to replicate the cancer-associated virus Epstein-Barr virus (EBV) is partly derived from comparisons with other members of the Herpes virus family. Many genes within the EBV genome are homologous across the herpes virus family. Published transcriptome data for the EBV genome during its lytic replication cycle show extensive transcription, but the identification of the proteins is limited. We have taken a global proteomics approach to identify viral proteins that are expressed during the EBV lytic replication cycle. We combined an enrichment method to isolate cells undergoing EBV lytic replication with SILAC-labeling coupled to mass-spectrometry and identified viral and host proteins expressed during the OPEN ACCESS Pathogens 2015, 4 740 EBV lytic replication cycle. Amongst the most frequently identified viral proteins are two components of the DNA replication machinery, the single strand DNA binding protein BALF2, DNA polymerase accessory protein BMRF1 and both subunits of the viral ribonucleoside-diphosphate reductase enzyme (BORF2 and BaRF1). An additional 42 EBV lytic cycle proteins were also detected. This provides proteomic identification for many EBV lytic replication cycle proteins and also identifies post-translational modifications.
RESUMEN
Allogeneic stem cell transplantation (allo-HSCT) provides a unique opportunity to track Epstein-Barr virus (EBV) infection in the context of the reconstituting B-cell system. Although many allo-HSCT recipients maintain low or undetectable levels of EBV DNA posttransplant, a significant proportion exhibit elevated and rapidly increasing EBV loads which, if left untreated, may lead to potentially fatal EBV-associated posttransplant lymphoproliferative disease. Intriguingly, this high-level EBV reactivation typically arises in the first 3 months posttransplant, at a time when the peripheral blood contains low numbers of CD27+ memory cells which are the site of EBV persistence in healthy immunocompetent donors. To investigate this apparent paradox, we prospectively monitored EBV levels and B-cell reconstitution in a cohort of allo-HSCT patients for up to 12 months posttransplant. In patients with low or undetectable levels of EBV, the circulating B-cell pool consisted predominantly of transitional and naive cells, with a marked deficiency of CD27+ memory cells which lasted >12 months. However, among patients with high EBV loads, there was a significant increase in both the proportion and number of CD27+ memory B cells. Analysis of sorted CD27+ memory B cells from these patients revealed that this population was preferentially infected with EBV, expressed EBV latent transcripts associated with B-cell growth transformation, had a plasmablastic phenotype, and frequently expressed the proliferation marker Ki-67. These findings suggest that high-level EBV reactivation following allo-HSCT may drive the expansion of latently infected CD27+ B lymphoblasts in the peripheral blood.
Asunto(s)
Linfocitos B/virología , Transformación Celular Viral/fisiología , Infecciones por Virus de Epstein-Barr/complicaciones , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Herpesvirus Humano 4/fisiología , Activación Viral/inmunología , Adulto , Anciano , Subgrupos de Linfocitos B/virología , ADN Viral/sangre , Femenino , Humanos , Memoria Inmunológica/inmunología , Trastornos Linfoproliferativos/virología , Masculino , Persona de Mediana Edad , Trasplante Homólogo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Carga Viral/inmunologíaRESUMEN
The ability of Epstein-Barr virus (EBV) to establish latency despite specific immune responses and to successfully persist lifelong in the human host shows that EBV has developed powerful strategies and mechanisms to exploit, evade, abolish, or downsize otherwise effective immune responses to ensure its own survival. This chapter focuses on current knowledge on innate immune responses against EBV and its evasion strategies for own benefit and summarizes the questions that remain to be tackled. Innate immune reactions against EBV originate both from the main target cells of EBV and from nontarget cells, which are elements of the innate immune system. Thus, we structured our review accordingly but with a particular focus on the innate recognition of EBV in its two stages in its life cycle, latent state and lytic replication. Specifically, we discuss (I) innate sensing and resulting innate immune responses against EBV by its main target cells, focusing on (i) EBV transmission between epithelial cells and B cells and their life cycle stages; and (ii) elements of innate immunity in EBV's target cells. Further, we debate (II) the innate recognition and resulting innate immune responses against EBV by cells other than the main target cells, focusing on (iii) myeloid cells: dendritic cells, monocytes, macrophages, and neutrophil granulocytes; and (iv) natural killer cells. Finally, we address (III) how EBV counteracts or exploits innate immunity in its latent and lytic life cycle stages, concentrating on (v) TLRs; (vi) EBERs; and (vii) microRNAs.
Asunto(s)
Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Inmunidad Innata , Animales , Linfocitos B/inmunología , Linfocitos B/virología , Células Dendríticas/inmunología , Células Dendríticas/virología , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/genética , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/virología , Macrófagos/inmunología , Macrófagos/virologíaRESUMEN
Lytic replication of the human gamma herpes virus Epstein-Barr virus (EBV) is an essential prerequisite for the spread of the virus. Differential regulation of a limited number of cellular genes has been reported in B-cells during the viral lytic replication cycle. We asked whether a viral bZIP transcription factor, Zta (BZLF1, ZEBRA, EB1), drives some of these changes. Using genome-wide chromatin immunoprecipitation coupled to next-generation DNA sequencing (ChIP-seq) we established a map of Zta interactions across the human genome. Using sensitive transcriptome analyses we identified 2263 cellular genes whose expression is significantly changed during the EBV lytic replication cycle. Zta binds 278 of the regulated genes and the distribution of binding sites shows that Zta binds mostly to sites that are distal to transcription start sites. This differs from the prevailing view that Zta activates viral genes by binding exclusively at promoter elements. We show that a synthetic Zta binding element confers Zta regulation at a distance and that distal Zta binding sites from cellular genes can confer Zta-mediated regulation on a heterologous promoter. This leads us to propose that Zta directly reprograms the expression of cellular genes through distal elements.
Asunto(s)
Regulación Viral de la Expresión Génica/fisiología , Herpesvirus Humano 4/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Transactivadores/fisiología , Secuencia de Bases , Línea Celular , Inmunoprecipitación de Cromatina , Cartilla de ADN , Humanos , Reacción en Cadena de la Polimerasa , TranscriptomaRESUMEN
UNLABELLED: Epstein-Barr virus (EBV) infection of B cells leads to the sequential activation of two viral promoters, Wp and Cp, resulting in the expression of six EBV nuclear antigens (EBNAs) and the viral Bcl2 homologue BHRF1. The viral transactivator EBNA2 is required for this switch from Wp to Cp usage during the initial stages of infection. EBNA2-dependent Cp transcription is mediated by the EBNA2 response element (E2RE), a region that contains at least two binding sites for cellular factors; one of these sites, CBF1, interacts with RBP-JK, which then recruits EBNA2 to the transcription initiation complex. Here we demonstrate that the B cell-specific transcription factor BSAP/Pax5 binds to a second site, CBF2, in the E2RE. Deletion of the E2RE in the context of a recombinant virus greatly diminished levels of Cp-initiated transcripts during the initial stages of infection but did not affect the levels of Wp-initiated transcripts or EBNA mRNAs. Consistent with this finding, viruses deleted for the E2RE were not markedly impaired in their ability to induce B cell transformation in vitro. In contrast, a larger deletion of the entire Cp region did reduce EBNA mRNA levels early after infection and subsequently almost completely ablated lymphoblastoid cell line (LCL) outgrowth. Notably, however, rare LCLs could be established following infection with Cp-deleted viruses, and these were indistinguishable from wild-type-derived LCLs in terms of steady-state EBV gene transcription. These data indicate that, unlike Wp, Cp is dispensable for the virus' growth-transforming activity. IMPORTANCE: Epstein-Barr virus (EBV), a B lymphotropic herpesvirus etiologically linked to several B cell malignancies, efficiently induces B cell proliferation leading to the outgrowth of lymphoblastoid cell lines (LCLs). The initial stages of this growth-transforming infection are characterized by the sequential activation of two viral promoters, Wp and Cp, both of which appear to be preferentially active in target B cells. In this work, we have investigated the importance of Cp activity in initiating B cell proliferation and maintaining LCL growth. Using recombinant viruses, we demonstrate that while Cp is not essential for LCL outgrowth in vitro, it enhances transformation efficiency by >100-fold. We also show that Cp, like Wp, interacts with the B cell-specific activator protein BSAP/Pax5. We suggest that EBV has evolved this two-promoter system to ensure efficient colonization of the host B cell system in vivo.
Asunto(s)
Linfocitos B/fisiología , Linfocitos B/virología , Transformación Celular Viral , Herpesvirus Humano 4/fisiología , Interacciones Huésped-Patógeno , Regiones Promotoras Genéticas , Proliferación Celular , Humanos , Unión Proteica , Transcripción Genética , Proteínas Virales/metabolismoRESUMEN
We have validated a flexible, high-throughput and relatively inexpensive RT-QPCR array platform for absolute quantification of Epstein-Barr virus transcripts in different latent and lytic infection states. Several novel observations are reported. First, during infection of normal B cells, Wp-initiated latent gene transcripts remain far more abundant following activation of the Cp promoter than was hitherto suspected. Second, EBNA1 transcript levels are remarkably low in all forms of latency, typically ranging from 1 to 10 transcripts per cell. EBNA3A, -3B and -3C transcripts are likewise very low in Latency III, typically at levels similar to or less than EBNA1 transcripts. Thirdly, a subset of lytic gene transcripts is detectable in Burkitt lymphoma lines at low levels, including: BILF1, which has oncogenic properties, and the poorly characterized LF1, LF2 and LF3 genes. Analysis of seven African BL biopsies confirmed this transcription profile but additionally revealed significant expression of LMP2 transcripts.
Asunto(s)
Herpesvirus Humano 4/genética , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Viral/análisis , ARN Viral/genética , Linfocitos B/virología , Linfoma de Burkitt/virología , Línea Celular Tumoral , Antígenos Nucleares del Virus de Epstein-Barr/genética , Regulación Viral de la Expresión Génica , Genes Virales , Humanos , Receptores Acoplados a Proteínas G/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transcripción Genética , Proteínas Virales/genética , Virión/genética , Latencia del Virus/genéticaRESUMEN
In 1964, a new herpesvirus, Epstein-Barr virus (EBV), was discovered in cultured tumor cells derived from a Burkitt lymphoma (BL) biopsy taken from an African patient. This was a momentous event that reinvigorated research into viruses as a possible cause of human cancers. Subsequent studies demonstrated that EBV was a potent growth-transforming agent for primary B cells, and that all cases of BL carried characteristic chromosomal translocations resulting in constitutive activation of the c-MYC oncogene. These results hinted at simple oncogenic mechanisms that would make Burkitt lymphoma paradigmatic for cancers with viral etiology. In reality, the pathogenesis of this tumor is rather complicated with regard to both the contribution of the virus and the involvement of cellular oncogenes. Here, we review the current understanding of the roles of EBV and c-MYC in the pathogenesis of BL and the implications for new therapeutic strategies to treat this lymphoma.
Asunto(s)
Linfoma de Burkitt/virología , Herpesvirus Humano 4 , Linfocitos B , Humanos , Translocación GenéticaRESUMEN
CD8+ T cell responses to Epstein-Barr virus (EBV) lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE) and some early (E) antigens are more frequently observed than responses to epitopes of late (L) expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs) which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE)- and early (E)-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L)-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L), interference by BILF1 increases with progression through lytic cycle (IEAsunto(s)
Presentación de Antígeno/inmunología
, Linfocitos T CD8-positivos/inmunología
, Infecciones por Virus de Epstein-Barr/inmunología
, Evasión Inmune/inmunología
, Western Blotting
, Desoxirribonucleasas/inmunología
, Técnicas de Silenciamiento del Gen
, Herpesvirus Humano 4/inmunología
, Humanos
, Reacción en Cadena en Tiempo Real de la Polimerasa
, Receptores Acoplados a Proteínas G/inmunología
, Proteínas de la Matriz Viral/inmunología
, Proteínas Virales/inmunología
RESUMEN
A number of diverse environmental cues have been linked to B lymphocyte differentiation and activation. One such cue, Notch-2, may be particularly relevant to the biology of infection with Epstein-Barr virus (EBV), which colonizes the B cell compartment. Activated Notch and EBV nuclear antigen 2 (EBNA2) both function as transcriptional activators by virtue of their interactions with the transcription factor RBP-Jκ. Although EBNA2 and activated Notch appear to have partially overlapping functions, we now report that activated Notch counteracts a crucial EBNA2 function both in newly infected primary B cells and in lymphoblastoid cell lines (LCLs). EBNA2 is directly responsible for the initiation of transcription of the majority of EBV proteins associated with type III latency, leading to the outgrowth of LCLs. One of the key proteins driving this outgrowth is latent membrane protein 1 (LMP1), which is regulated by an EBNA2-responsive element within its ED-L1 promoter. Activation of Notch-2 via Delta-like ligand 1 inhibits EBNA2-mediated initiation of LMP1 transcription. Furthermore, ligated Notch-2 also efficiently turns off LMP1 expression from the ED-L1 promoter in LCLs already expressing LMP1. Modulation of EBV gene expression by Notch was not confined to EBNA2-dependent events. Activated Notch-2 also inhibited EBV entry into the lytic cycle in a B cell non-Hodgkin's lymphoma line by upregulating the cellular transcription factor Zeb2, which represses the transcription of BZLF1. These results support the concept that in vivo, cumulative signals from the microenvironment downregulate EBV gene expression in B cells to the latency 0 gene expression profile observed in B cells entering the peripheral blood. Importance: Experimental infection of resting B cells by Epstein-Barr virus leads to the growth transformation program of virus gene expression and the outgrowth of lymphoblastoid cell lines. Previous studies at the single-cell level revealed complex cellular and viral signaling networks regulating transcription of the viral genome. This study demonstrates that viral gene expression can also be radically altered by molecules expressed on stromal cells in the microenvironment of lymphoid tissue, specifically, Delta-like ligand 1 on stromal cells ligating Notch-2 on infected B cells. Activation of Notch interferes with the transactivation function of EBNA2, downregulates the expression of LMP1 and LMP2a, and inhibits the activation of lytic virus replication in a B cell non-Hodgkin's lymphoma line by preventing expression of BZLF1. The significance of these observations is that they indicate new mechanisms whereby the microenvironment in normal lymphoid tissue may facilitate the repression of viral gene expression, enabling establishment of true latency in memory B cells.
Asunto(s)
Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/fisiología , Interacciones Huésped-Patógeno , Receptores Notch/metabolismo , Proteínas Virales/metabolismo , Animales , Secuencia de Bases , Línea Celular , Células Cultivadas , Cartilla de ADN , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/genética , Proteínas de Homeodominio/fisiología , Humanos , Ratones , Reacción en Cadena de la Polimerasa , Proteínas Represoras/fisiología , Transactivadores/genética , Caja Homeótica 2 de Unión a E-Box con Dedos de ZincRESUMEN
Epstein Barr virus (EBV) is a highly prevalent human gamma 1 lymphocryptovirus which infects both B lymphocytes and epithelial cells. In the healthy host, infection of these different cell lineages broadly reflects the different phases of the virus lifecycle. Memory B cells are the reservoir for latent EBV, in which viral gene expression is highly restricted to maintain an asymptomatic lifelong infection. In contrast, epithelial cells may be a major site of the virus lytic cycle, where infectious virus is propagated and transmitted via saliva to uninfected hosts. To achieve this dual tropism, EBV has evolved a unique set of glycoproteins in addition to a highly conserved set, which interact with cell lineage-specific receptors and switch cellular tropism during infection.
Asunto(s)
Linfocitos B/virología , Células Epiteliales/virología , Herpesvirus Humano 4/fisiología , Tropismo Viral , Internalización del Virus , HumanosRESUMEN
The transcriptional regulator c-MYC is abnormally overexpressed in many human cancers. Evasion from apoptosis is critical for cancer development, particularly c-MYC-driven cancers. We explored which anti-apoptotic BCL-2 family member (expressed under endogenous regulation) is essential to sustain c-MYC-driven lymphoma growth to reveal which should be targeted for cancer therapy. Remarkably, inducible Cre-mediated deletion of even a single Mcl-1 allele substantially impaired the growth of c-MYC-driven mouse lymphomas. Mutations in p53 could diminish but not obviate the dependency of c-MYC-driven mouse lymphomas on MCL-1. Importantly, targeting of MCL-1 killed c-MYC-driven human Burkitt lymphoma cells, even those bearing mutations in p53. Given that loss of one allele of Mcl-1 is well tolerated in healthy tissues, our results suggest that therapeutic targeting of MCL-1 would be an attractive therapeutic strategy for MYC-driven cancers.
Asunto(s)
Linfoma/genética , Linfoma/terapia , Mutación , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteína p53 Supresora de Tumor/genética , Animales , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Linfoma/patología , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
Epstein-Barr virus (EBV) is an oncogenic virus that is associated with the pathogenesis of several human lymphoid malignancies, including Hodgkin's lymphoma. Infection of normal resting B cells with EBV results in activation to lymphoblasts that are phenotypically similar to those generated by physiological stimulation with CD40L plus IL-4. One important difference is that infection leads to the establishment of permanently growing lymphoblastoid cell lines, whereas CD40L/IL-4 blasts have finite proliferation lifespans. To identify early events which might later determine why EBV infected blasts go on to establish transformed cell lines, we performed global transcriptome analyses on resting B cells and on EBV and CD40L/IL-4 blasts after 7 days culture. As anticipated there was considerable overlap in the transcriptomes of the two types of lymphoblasts when compared to the original resting B cells, reflecting common changes associated with lymphocyte activation and proliferation. Of interest to us was a subset of 255 genes that were differentially expressed between EBV and CD40L/IL-4 blasts. Genes which were more highly expressed in EBV blasts were substantially and significantly enriched for a set of interferon-stimulated genes which on further in silico analyses were found to be repressed by IL-4 in other cell contexts and to be up-regulated in micro-dissected malignant cells from Hodgkin's lymphoma biopsies when compared to their normal germinal center cell counterparts. We hypothesized that EBV and IL-4 were targeting and discordantly regulating a common set of genes. This was supported experimentally in our B cell model where IL-4 stimulation partially reversed transcriptional changes which follow EBV infection and it impaired the efficiency of EBV-induced B cell transformation. Taken together, these data suggest that the discordant regulation of interferon and IL-4 pathway genes by EBV that occurs early following infection of B cells has relevance to the development or maintenance of an EBV-associated malignancy.
Asunto(s)
Linfocitos B/metabolismo , Linfocitos B/virología , Transformación Celular Viral/genética , Regulación de la Expresión Génica , Herpesvirus Humano 4/fisiología , Factores Reguladores del Interferón/genética , Interleucina-4/metabolismo , Linfocitos B/patología , Ligando de CD40/inmunología , Ligando de CD40/farmacología , Línea Celular Tumoral , Transformación Celular Viral/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/metabolismo , Humanos , Interferones/metabolismo , Interleucina-4/farmacología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Transducción de Señal , Transcriptoma , Proteínas de la Matriz Viral/metabolismoRESUMEN
Hodgkin's lymphoma is unusual among B cell lymphomas, in so far as the malignant Hodgkin/Reed-Sternberg (HRS) cells lack a functional B cell receptor (BCR), as well as many of the required downstream signalling components. In Epstein-Barr virus (EBV)-positive cases of Hodgkin's lymphoma, HRS cells express the viral latent membrane proteins (LMP)-1 and -2A. LMP2A is thought to contribute to the pathogenesis of Hodgkin's lymphoma by providing a surrogate BCR-like survival signal. However, LMP2A has also been shown to induce the virus-replicative cycle in B cells, an event presumably incompatible with lymphomagenesis. In an attempt to resolve this apparent paradox, we compared the transcriptional changes observed in primary HRS cells with those induced by LMP2A and by BCR activation in primary human germinal centre (GC) B cells, the presumed progenitors of HRS cells. We found a subset of genes that were up-regulated by both LMP2A expression and BCR activation but which were down-regulated in primary HRS cells. These genes included EGR1, an immediate-early gene that is required for BCR-induced entry to the virus-replicative cycle. We present data supporting a model for the pathogenesis of EBV-positive Hodgkin's lymphoma in which LMP2A-expressing HRS cells lacking BCR signalling functions cannot induce EGR1 and are consequently protected from entry to the virus lytic cycle. The primary microarray data are available from GEO (http://www.ncbi.nlm.nih.gov/geo/) under series Accession No 46143.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Herpesvirus Humano 4/metabolismo , Enfermedad de Hodgkin/metabolismo , Células de Reed-Sternberg/metabolismo , Proteínas de la Matriz Viral/metabolismo , Linfocitos B/metabolismo , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , Linfoma de Burkitt/virología , Línea Celular Tumoral , Efecto Citopatogénico Viral , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidad , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/patología , Enfermedad de Hodgkin/virología , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Antígenos de Linfocitos B/metabolismo , Células de Reed-Sternberg/patología , Células de Reed-Sternberg/virología , Transactivadores/metabolismo , Transfección , Regulación hacia Arriba , Proteínas de la Matriz Viral/genéticaRESUMEN
Evasion of immune T cell responses is crucial for persistent viruses to establish a normal carrier state. Most studies on active immune modulation mechanisms have focused on the stage of virus production in infected cells, when large numbers of viral antigens and potential immune modulators are expressed. For oncogenic viruses such as Kaposi's sarcoma-associated herpesvirus (KSHV), which is carried as a lifelong infection, usually with little harmful effect, but can cause various tumors, the immune evasion strategies can also be relevant in the context of tumorigenesis. Here we report that the virus-encoded interferon regulatory factor 3 (vIRF3) latent viral gene expressed in KSHV-related tumors functions as a potent immunevasin. Expression of vIRF3 downregulates surface major histocompatibility complex class II (MHC-II) DR expression with slow kinetics but, more importantly, can substantially inhibit recognition by KSHV-specific CD4 T cells prior to its effects on MHC-II DR downregulation in model cell systems. This property of vIRF3 is only partly due to its ability to inhibit the transcription of CIITA and, thus, MHC-II expression; CIITA-independent inhibition of MHC-II transcripts and another as yet unidentified posttranscriptional mechanism are also involved in qualitatively modulating the availability of specific peptide/MHC-II complexes at the cell surface. Consistent with these observations, the vIRF3-expressing KSHV-associated primary effusion lymphoma (PEL) lines are generally resistant to recognition by KSHV-specific CD4 T cells. Interestingly, some PEL lines exhibit small subpopulations with lower vIRF3 expression that can be recognized. These data implicate vIRF3 as a critical determinant of the MHC-II antigen presentation function in KSHV-associated PELs that is likely to be important in the pathogenesis of these tumors.
Asunto(s)
Presentación de Antígeno , Herpesvirus Humano 8/patogenicidad , Antígenos de Histocompatibilidad Clase II/metabolismo , Interacciones Huésped-Patógeno , Evasión Inmune , Factores Reguladores del Interferón/metabolismo , Transactivadores/antagonistas & inhibidores , Proteínas Virales/metabolismo , Transformación Celular Neoplásica , Regulación hacia Abajo , Herpesvirus Humano 8/inmunología , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase II/inmunología , HumanosRESUMEN
Lifelong persistent infection by herpesviruses depends on the balance between host immune responses and viral immune evasion. CD4 T cells responding to antigens presented on major histocompatibility complex class II (MHC-II) molecules are known to play an important role in controlling herpesvirus infections. Here we review, with emphasis on human herpesvirus infections, the strategies evolved to evade CD4 T cell immunity. These viruses target multiple points on the MHC class II antigen presentation pathway. The mechanisms include: suppression of CIITA to inhibit the synthesis of MHC class II molecules, diversion or degradation of HLA-DR molecules during membrane transport, and direct targeting of the invariant chain chaperone of HLA-DR.