RESUMO
Antigenic peptides derived from introns are presented on major histocompatibility (MHC) class I molecules, but how these peptides are produced is poorly understood. Here, we show that an MHC class I epitope (SL8) sequence inserted in the second intron of the ß-globin gene in a C57BL/6 mouse (HBB) generates immune tolerance. Introduction of SL8-specific CD8+ T cells derived from OT-1 transgenic mice resulted in a threefold increase in OT-1 T cell proliferation in HBB animals, as compared to wild-type animals. The growth of MCA sarcoma cells expressing the intron-derived SL8 epitope was suppressed in wild-type animals compared to HBB mice. The ß-globin pre-mRNA was detected in the light polysomal fraction, and introducing stop codons identified a non-AUG initiation site between +228 and +255 nts upstream of the SL8. Isolation of ribosome footprints confirmed translation initiation within this 27 nt sequence. Furthermore, treatment with splicing inhibitor shifts the translation of the pre-mRNA to monosomal fractions and results in an increase of intron-derived peptide substrate as shown by polysome profiling and cell imaging. These results show that non-AUG-initiated translation of pre-mRNAs generates peptides for MHC class I immune tolerance and helps explain why alternative tissue-specific splicing is tolerated by the immune system.
Assuntos
Antígenos de Histocompatibilidade Classe I , Precursores de RNA , Animais , Camundongos , Antígenos de Histocompatibilidade Classe I/genética , Precursores de RNA/genética , Linfócitos T CD8-Positivos , Biossíntese de Proteínas , Apresentação de Antígeno , Camundongos Endogâmicos C57BL , Peptídeos/metabolismo , Tolerância Imunológica/genética , Epitopos , Antígenos de Histocompatibilidade Classe II/genéticaRESUMO
Protein aggregates and abnormal proteins are toxic and associated with neurodegenerative diseases. There are several mechanisms to help cells get rid of aggregates but little is known on how cells prevent aggregate-prone proteins from being synthesised. The EBNA1 of the Epstein-Barr virus (EBV) evades the immune system by suppressing its own mRNA translation initiation in order to minimize the production of antigenic peptides for the major histocompatibility (MHC) class I pathway. Here we show that the emerging peptide of the disordered glycine-alanine repeat (GAr) within EBNA1 dislodges the nascent polypeptide-associated complex (NAC) from the ribosome. This results in the recruitment of nucleolin to the GAr-encoding mRNA and suppression of mRNA translation initiation in cis. Suppressing NAC alpha (NACA) expression prevents nucleolin from binding to the GAr mRNA and overcomes GAr-mediated translation inhibition. Taken together, these observations suggest that EBNA1 exploits a nascent protein quality control pathway to regulate its own rate of synthesis that is based on sensing the nascent GAr peptide by NAC followed by the recruitment of nucleolin to the GAr-encoding RNA sequence.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Proteínas de Ligação a RNA/metabolismo , Alanina , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Glicina , Herpesvirus Humano 4/genética , Humanos , Peptídeos/genética , Fosfoproteínas , Agregados Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , NucleolinaRESUMO
The role of G-quadruplex (G4) RNA structures is multifaceted and controversial. Here, we have used as a model the EBV-encoded EBNA1 and the Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded LANA1 mRNAs. We have compared the G4s in these two messages in terms of nucleolin binding, nuclear mRNA retention, and mRNA translation inhibition and their effects on immune evasion. The G4s in the EBNA1 message are clustered in one repeat sequence and the G4 ligand PhenDH2 prevents all G4-associated activities. The RNA G4s in the LANA1 message take part in similar multiple mRNA functions but are spread throughout the message. The different G4 activities depend on flanking coding and non-coding sequences and, interestingly, can be separated individually. Together, the results illustrate the multifunctional, dynamic and context-dependent nature of G4 RNAs and highlight the possibility to develop ligands targeting specific RNA G4 functions. The data also suggest a common multifunctional repertoire of viral G4 RNA activities for immune evasion.
Assuntos
DNA Intergênico/química , DNA Intergênico/genética , Quadruplex G , RNA/química , RNA/genética , Antígenos Nucleares do Vírus Epstein-Barr/química , Antígenos Nucleares do Vírus Epstein-Barr/genética , Regulação da Expressão Gênica , Humanos , Transporte de RNA , RNA ViralRESUMO
Cell growth requires a high level of protein synthesis and oncogenic pathways stimulate cell proliferation and ribosome biogenesis. Less is known about how cells respond to dysfunctional mRNA translation and how this feeds back into growth regulatory pathways. The Epstein-Barr virus (EBV)-encoded EBNA1 causes mRNA translation stress in cis that activates PI3Kδ. This leads to the stabilization of MDM2, induces MDM2's binding to the E2F1 mRNA and promotes E2F1 translation. The MDM2 serine 166 regulates the interaction with the E2F1 mRNA and deletion of MDM2 C-terminal RING domain results in a constitutive E2F1 mRNA binding. Phosphorylation on serine 395 following DNA damage instead regulates p53 mRNA binding to its RING domain and prevents the E2F1 mRNA interaction. The p14Arf tumour suppressor binds MDM2 and in addition to preventing degradation of the p53 protein it also prevents the E2F1 mRNA interaction. The data illustrate how two MDM2 domains selectively bind specific mRNAs in response to cellular conditions to promote, or suppress, cell growth and how p14Arf coordinates MDM2's activity towards p53 and E2F1. The data also show how EBV via EBNA1-induced mRNA translation stress targets the E2F1 and the MDM2 - p53 pathway.
Assuntos
Fator de Transcrição E2F1/genética , Neoplasias/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética , Carcinogênese/genética , Ciclo Celular/genética , Proliferação de Células/genética , Dano ao DNA/genética , Genes Supressores de Tumor , Herpesvirus Humano 4/genética , Humanos , Neoplasias/virologia , Oncogenes/genética , Fosforilação/genética , Domínios Proteicos/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , RNA Mensageiro/genética , Proteína Supressora de Tumor p14ARF/genéticaRESUMO
Many pathogens (virus, bacteria, fungi, or parasites) have developed a wide variety of mechanisms to evade their host immune system. The budding yeast Saccharomyces cerevisiae has successfully been used to decipher some of these immune evasion strategies. This includes the cis-acting mechanism that limits the expression of the oncogenic Epstein-Barr virus (EBV)-encoded EBNA1 and thus of antigenic peptides derived from this essential but highly antigenic viral protein. Studies based on budding yeast have also revealed the molecular bases of epigenetic switching or recombination underlying the silencing of all except one members of extended families of genes that encode closely related and highly antigenic surface proteins. This mechanism is exploited by several parasites (that include pathogens such as Plasmodium, Trypanosoma, Candida, or Pneumocystis) to alternate their surface antigens, thereby evading the immune system. Yeast can itself be a pathogen, and pathogenic fungi such as Candida albicans, which is phylogenetically very close to S. cerevisiae, have developed stealthiness strategies that include changes in their cell wall composition, or epitope-masking, to control production or exposure of highly antigenic but essential polysaccharides in their cell wall. Finally, due to the high antigenicity of its cell wall, yeast has been opportunistically exploited to create adjuvants and vectors for vaccination.
Assuntos
Evasão da Resposta Imune/genética , Imunidade/genética , Saccharomyces cerevisiae/genética , Transgenes , Animais , Doenças Transmissíveis/genética , Doenças Transmissíveis/imunologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Humanos , Saccharomyces cerevisiae/imunologiaRESUMO
The oncogenic Epstein-Barr virus (EBV) evades the immune system through limiting the expression of its highly antigenic and essential genome maintenance protein, EBNA1, to the minimal level to ensure viral genome replication, thereby also minimizing the production of EBNA1-derived antigenic peptides. This regulation is based on inhibition of translation of the virally-encoded EBNA1 mRNA, and involves the interaction of host protein nucleolin (NCL) with G-quadruplex (G4) structures that form in the glycine-alanine repeat (GAr)-encoding sequence of the EBNA1 mRNA. Ligands that bind to these G4-RNA can prevent their interaction with NCL, leading to disinhibition of EBNA1 expression and antigen presentation, thereby interfering with the immune evasion of EBNA1 and therefore of EBV (M.J. Lista et al., Nature Commun., 2017, 8, 16043). In this work, we synthesized and studied a series of 20 cationic bis(acylhydrazone) derivatives designed as G4 ligands. The in vitro evaluation showed that most derivatives based on central pyridine (Py), naphthyridine (Naph) or phenanthroline (Phen) units were efficient G4 binders, in contrast to their pyrimidine (Pym) counterparts, which were poor G4 binders due to a significantly different molecular geometry. The influence of lateral heterocyclic units (N-substituted pyridinium or quinolinium residues) on G4-binding properties was also investigated. Two novel compounds, namely PyDH2 and PhenDH2, used at a 5⯵M concentration, were able to significantly enhance EBNA1 expression in H1299â¯cells in a GAr-dependent manner, while being significantly less toxic than the prototype drug PhenDC3 (GI50â¯>â¯50⯵M). Antigen presentation, RNA pull-down and proximity ligation assays confirmed that the effect of both drugs was related to the disruption of NCL-EBNA1 mRNA interaction and the subsequent promotion of GAr-restricted antigen presentation. Our work provides a novel modular scaffold for the development of G-quadruplex-targeting drugs acting through interference with G4-protein interaction.
Assuntos
Hidrazonas/farmacologia , Evasão da Resposta Imune/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Fosfoproteínas/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular Tumoral , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Quadruplex G , Herpesvirus Humano 4/genética , Humanos , Hidrazonas/síntese química , Hidrazonas/química , Fatores Imunológicos/síntese química , Fatores Imunológicos/química , Ligantes , Camundongos , RNA Mensageiro/genética , NucleolinaRESUMO
Peptides presented on major histocompatibility (MHC) class I molecules form an essential part of the immune system's capacity to detect virus-infected or transformed cells. Earlier works have shown that pioneer translation peptides (PTPs) for the MHC class I pathway are as efficiently produced from introns as from exons, or from mRNAs targeted for the nonsense-mediated decay pathway. The production of PTPs is a target for viral immune evasion but the underlying molecular mechanisms that govern this non-canonical translation are unknown. Here, we have used different approaches to show how events taking place on the nascent transcript control the synthesis of PTPs and full-length proteins. By controlling the subcellular interaction between the G-quadruplex structure (G4) of a gly-ala encoding mRNA and nucleolin (NCL) and by interfering with mRNA maturation using multiple approaches, we demonstrate that antigenic peptides derive from a nuclear non-canonical translation event that is independently regulated from the synthesis of full-length proteins. Moreover, we show that G4 are exploited to control mRNA localization and translation by distinguishable mechanisms that are targets for viral immune evasion.
Assuntos
Antígenos/genética , Antígenos de Histocompatibilidade Classe I/genética , Peptídeos/genética , Biossíntese de Proteínas/genética , Antígenos/imunologia , Núcleo Celular/genética , Núcleo Celular/imunologia , Quadruplex G , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/imunologia , Degradação do RNAm Mediada por Códon sem Sentido/genética , Degradação do RNAm Mediada por Códon sem Sentido/imunologia , Peptídeos/imunologia , Biossíntese de Proteínas/imunologia , RNA Mensageiro/genética , RNA Mensageiro/imunologiaRESUMO
Protein-RNA interactions (PRIs) control pivotal steps in RNA biogenesis, regulate multiple physiological and pathological cellular networks, and are emerging as important drug targets. However, targeting of specific protein-RNA interactions for therapeutic developments is still poorly advanced. Studies and manipulation of these interactions are technically challenging and in vitro drug screening assays are often hampered due to the complexity of RNA structures. The binding of nucleolin (NCL) to a G-quadruplex (G4) structure in the messenger RNA (mRNA) of the Epstein-Barr virus (EBV)-encoded EBNA1 has emerged as an interesting therapeutic target to interfere with immune evasion of EBV-associated cancers. Using the NCL-EBNA1 mRNA interaction as a model, we describe a quantitative proximity ligation assay (PLA)-based in cellulo approach to determine the structure activity relationship of small chemical G4 ligands. Our results show how different G4 ligands have different effects on NCL binding to G4 of the EBNA1 mRNA and highlight the importance of in-cellulo screening assays for targeting RNA structure-dependent interactions.
Assuntos
Bioensaio/métodos , Quadruplex G , Fosfoproteínas/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Aminoquinolinas/química , Linhagem Celular Tumoral , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Humanos , Ácidos Picolínicos/química , NucleolinaRESUMO
The c-myc oncogene stimulates ribosomal biogenesis and protein synthesis to promote cellular growth. However, the pathway by which cells sense and restore dysfunctional mRNA translation and how this is linked to cell proliferation and growth is not known. We here show that mRNA translation stress in cis triggered by the gly-ala repeat sequence of Epstein-Barr virus (EBV)-encoded EBNA1, results in PI3Kδ-dependent induction of E2F1 mRNA translation with the consequent activation of c-Myc and cell proliferation. Treatment with a specific PI3Kδ inhibitor Idelalisib (CAL-101) suppresses E2F1 and c-Myc levels and causes cell death in EBNA1-induced B cell lymphomas. Suppression of PI3Kδ prevents E2F1 activation also in non-EBV-infected cells. These data illustrate an mRNA translation stress-response pathway for E2F1 activation that is exploited by EBV to promote cell growth and proliferation, offering new strategies to treat EBV-carrying cancers.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Fator de Transcrição E2F1/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Fator de Transcrição E2F1/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Regulação Neoplásica da Expressão Gênica , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno/genética , Humanos , Camundongos , Neoplasias/genética , Neoplasias/patologia , Neoplasias/virologiaRESUMO
The oncogenic Epstein-Barr virus (EBV) evades the immune system but has an Achilles heel: its genome maintenance protein EBNA1. Indeed, EBNA1 is essential for viral genome replication and maintenance but also highly antigenic. Hence, EBV evolved a system in which the glycine-alanine repeat (GAr) of EBNA1 limits the translation of its own mRNA at a minimal level to ensure its essential function thereby, at the same time, minimizing immune recognition. Defining intervention points where to interfere with EBNA1 immune evasion is an important step to trigger an immune response against EBV-carrying cancers. Thanks to a yeast-based assay that recapitulates all the aspects of EBNA1 self-limitation of expression, a recent study by Lista et al. [Nature Communications (2017) 7, 435-444] has uncovered the role of the host cell nucleolin (NCL) in this process via a direct interaction of this protein with G-quadruplexes (G4) formed in GAr-encoding sequence of EBNA1 mRNA. In addition, the G4 ligand PhenDC3 prevents NCL binding on EBNA1 mRNA and reverses GAr-mediated repression of translation and antigen presentation. This shows that the NCL-EBNA1 mRNA interaction is a relevant therapeutic target to unveil EBV-carrying cancers to the immune system and that the yeast model can be successfully used for uncovering drugs and host factors that interfere with EBV stealthiness.
RESUMO
The oncogenic Epstein-Barr virus (EBV) evades the immune system but has an Achilles heel: its genome maintenance protein EBNA1, which is essential for viral genome maintenance but highly antigenic. EBV has seemingly evolved a system in which the mRNA sequence encoding the glycine-alanine repeats (GAr) of the EBNA1 protein limits its expression to the minimal level necessary for function while minimizing immune recognition. Here, we identify nucleolin (NCL) as a host factor required for this process via a direct interaction with G-quadruplexes formed in GAr-encoding mRNA sequence. Overexpression of NCL enhances GAr-based inhibition of EBNA1 protein expression, whereas its downregulation relieves the suppression of both expression and antigen presentation. Moreover, the G-quadruplex ligand PhenDC3 prevents NCL binding to EBNA1 mRNA and reverses GAr-mediated repression of EBNA1 expression and antigen presentation. Hence the NCL-EBNA1 mRNA interaction is a relevant therapeutic target to trigger an immune response against EBV-carrying cancers.
Assuntos
Linfócitos B/imunologia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune/genética , Fosfoproteínas/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Aminoquinolinas/farmacologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/virologia , Linhagem Celular Tumoral , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Quadruplex G , Células HCT116 , Herpesvirus Humano 4/efeitos dos fármacos , Herpesvirus Humano 4/imunologia , Humanos , Leontopithecus , Ligantes , Fosfoproteínas/imunologia , Ácidos Picolínicos/farmacologia , Quinolinas/farmacologia , RNA Mensageiro/imunologia , Proteínas de Ligação a RNA/imunologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , NucleolinaRESUMO
Cellular immune reactions against non-self-epitopes require activation of cytotoxic CD8+ T-cells via cross-presentation of MHC class I-restricted peptides by professional antigen presenting cells (pAPCs), with the consequent detection and elimination of cells expressing the same antigens via the endogenous (direct) pathway. The source of peptides for the endogenous pathway is constituted of alternative mRNA translation products; however, it is still unclear which source of peptides is used for cross-presentation. Furthermore, the presentation of non-canonical translation products, produced during a non-conventional translation event, on class I molecules of tumor cells has been reported but how these peptides are generated, presented to pAPCs, and their capacity to stimulate CD8+ T cells is still not known. Here, we report that pioneer translation peptides (PTPs) derived from intron or exon pre-mRNAs can serve as tumor-associated antigens (TA-PTPs) and are delivered from the producing tumor cells to pAPCs via exosomes where they are processed by the cytosolic pathway. Injection of TA-PTPs and tumor-derived exosomes efficiently induce CD8+ T-cell proliferation and prevent tumor growth in mice. Our results show that TA-PTPs represent an efficient source of antigenic peptides for CD8+ T cell activation and that full-length proteins are not required for cross-presentation. These findings can have interesting implications for generating tolerance and for designing vectors to generate vaccines.
RESUMO
The Epstein-Barr gammaherpesvirus (EBV) is the first oncogenic virus discovered in human. Indeed, EBV has been known for more than 50 years to be tightly associated with certain human cancers. As such, EBV has been the subject of extensive studies aiming at deciphering various aspects of its biological cycle, ranging from the regulation of its genome replication and maintenance to the induction of its lytic cycle, including the mechanisms that allow its immune evasion or that are related to its tumorogenicity. For more than 30 years the budding yeast Saccharomyces cerevisiae has fruitfully contributed to a number of these studies. The aim of this article is to review the various aspects of EBV biology for which yeast has been instrumental, and to propose new possible applications for these yeast-based assays, as well as the creation of further yeast models dedicated to EBV. This review article illustrates the tremendous potential of S. cerevisiae in integrated chemobiological approaches for the biomedical research.
Assuntos
Pesquisa Biomédica/métodos , Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Interações Hospedeiro-Patógeno , Modelos Imunológicos , Saccharomyces cerevisiae , Bioensaio , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Genoma Viral , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/patogenicidade , Humanos , Saccharomyces cerevisiae/imunologia , Saccharomyces cerevisiae/virologiaRESUMO
The notion that alternative peptide substrates can be processed and presented to the MHC class I pathway has opened for new aspects on how the immune system detects infected or damaged cells. Recent works show that antigenic peptides are derived from intron sequences in pre-mRNAs target for the nonsense-mediated degradation pathway. Introns are spliced out co-transcriptionally suggesting that such pioneer translation products (PTPs) are synthesized on the nascent RNAs in the nuclear compartment to ensure that the first peptides to emerge from an mRNA are destined for the class I pathway. This illustrates an independent translation event during mRNA maturation that give rise to specific peptide products with a specific function in the immune system. The characterization of the translation apparatus responsible for PTP synthesis will pave the way for understanding how PTP production is regulated in different tissues under different conditions and will help designing new vaccine strategies.
Assuntos
Apresentação de Antígeno/genética , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos/imunologia , Biossíntese de Proteínas/imunologia , Splicing de RNA/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Núcleo Celular/genética , Núcleo Celular/imunologia , Citosol/imunologia , Citosol/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Íntrons , Peptídeos/genética , Fagossomos/genética , Fagossomos/imunologia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/imunologia , Precursores de RNA/genética , Precursores de RNA/imunologiaRESUMO
The EBV-encoded EBNA1 was first discovered 40 years ago, approximately 10 years after the presence of EBV had been demonstrated in Burkitt's lymphoma cells. It took another 10 years before the functions of EBNA1 in maintaining the viral genome were revealed, and it has since been shown to be an essential viral factor expressed in all EBV-carrying cells. Apart from serving to maintain the viral episome and to control viral replication and gene expression, EBNA1 also harbours a cis-acting mechanism that allows virus-carrying host cells to evade the immune system. This relates to a particular glycine-alanine repeat (GAr) within EBNA1 that has the capacity to suppress antigen presentation to the major histocompatibility complex (MHC) class I pathway. We discuss the role of the GAr sequence at the level of mRNA translation initiation, rather than at the protein level, as at least part of the mechanism to avoid MHC presentation. Interfering with this mechanism has become the focus of the development of immune-based therapies against EBV-carrying cancers, and some lead compounds that affect translation of GAr-carrying mRNAs have been identified. In addition, we describe the EBV-encoded ZEBRA factor and the switch from the latent to the lytic cycle as an alternative virus-specific target for treating EBV-carrying cancers. Understanding the molecular mechanisms of how EBNA1 and ZEBRA interfere with cellular pathways not only opens new therapeutic approaches but continues to reveal new cell-biological insights on the interplay between host and virus. This review is a tale of discoveries relating to how EBNA1 and ZEBRA have emerged as targets for specific cancer therapies against EBV-carrying diseases, and serves as an illustration of how mRNA translation can play roles in future immune-based strategies to target viral disease.
Assuntos
Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Neoplasias/virologia , Transativadores/metabolismo , Animais , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/terapia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Regulação Viral da Expressão Gênica , Terapia Genética/métodos , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/terapia , Transativadores/genética , VirulênciaRESUMO
Epstein-Barr virus (EBV) is tightly associated with certain human cancers, but there is as yet no specific treatment against EBV-related diseases. The EBV-encoded EBNA1 protein is essential to maintain viral episomes and for viral persistence. As such, EBNA1 is expressed in all EBV-infected cells, and is highly antigenic. All infected individuals, including individuals with cancer, have CD8(+) T cells directed towards EBNA1 epitopes, yet the immune system fails to detect and destroy cells harboring the virus. EBV immune evasion depends on the capacity of the Gly-Ala repeat (GAr) domain of EBNA1 to inhibit the translation of its own mRNA in cis, thereby limiting the production of EBNA1-derived antigenic peptides presented by the major histocompatibility complex (MHC) class I pathway. Here we establish a yeast-based assay for monitoring GAr-dependent inhibition of translation. Using this assay we identify doxorubicin (DXR) as a compound that specifically interferes with the GAr effect on translation in yeast. DXR targets the topoisomerase-II-DNA complexes and thereby causes genomic damage. We show, however, that the genotoxic effect of DXR and various analogs thereof is uncoupled from the effect on GAr-mediated translation control. This is further supported by the observation that etoposide and teniposide, representing another class of topoisomerase-II-DNA targeting drugs, have no effect on GAr-mediated translation control. DXR and active analogs stimulate, in a GAr-dependent manner, EBNA1 expression in mammalian cells and overcome GAr-dependent restriction of MHC class I antigen presentation. These results validate our approach as an effective high-throughput screening assay to identify drugs that interfere with EBV immune evasion and, thus, constitute candidates for treating EBV-related diseases, in particular EBV-associated cancers.
Assuntos
Antivirais/farmacologia , Bioensaio/métodos , Herpesvirus Humano 4/efeitos dos fármacos , Herpesvirus Humano 4/imunologia , Evasão da Resposta Imune/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Apresentação de Antígeno/efeitos dos fármacos , Antivirais/análise , Antivirais/química , Dano ao DNA , Doxorrubicina/química , Doxorrubicina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Antígenos Nucleares do Vírus Epstein-Barr/química , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Células HEK293 , Humanos , Biossíntese de Proteínas/efeitos dos fármacos , Estrutura Terciária de Proteína , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
The scanning of maturing mRNAs by ribosomes plays a key role in the mRNA quality control process. When ribosomes first engage with the newly synthesized mRNA, and if peptides are produced, is unclear, however. Here we show that ribosomal scanning of prespliced mRNAs occurs in the nuclear compartment, and that this event produces peptide substrates for the MHC class I pathway. Inserting antigenic peptide sequences in introns that are spliced out before the mRNAs exit the nuclear compartment results in an equal amount of antigenic peptide products as when the peptides are encoded from the main open reading frame (ORF). Taken together with the detection of intron-encoded nascent peptides and RPS6/RPL7-carrying complexes in the perinucleolar compartment, these results show that peptides are produced by a translation event occurring before mRNA splicing. This suggests that ribosomes occupy and scan mRNAs early in the mRNA maturation process, and suggests a physiological role for nuclear mRNA translation, and also helps explain how the immune system tolerates peptides derived from tissue-specific mRNA splice variants.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos/imunologia , Biossíntese de Proteínas/imunologia , RNA Mensageiro/metabolismo , Transdução de Sinais/imunologia , Linhagem Celular , Núcleo Celular/imunologia , Humanos , Espectrometria de Massas , Peptídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribossomos/imunologia , Ribossomos/metabolismoRESUMO
The MHC class I antigen presentation pathway allows the immune system to distinguish between self and nonself. Despite extensive research on the processing of antigenic peptides, little is known about their origin. Here, we show that mRNAs carrying premature stop codons that prevent the production of full-length proteins via the nonsense-mediated decay pathway still produce a majority of peptide substrates for the MHC class I pathway by a noncanonical mRNA translation process. Blocking the interaction of the translation initiation factor eIF4E with the cap structure suppresses the synthesis of full-length proteins but has only a limited effect on the production of antigenic peptides. These results reveal an essential cell biological function for a class of translation products derived during the pioneer round of mRNA translation and will have important implications for understanding how the immune system detects cells harboring pathogens and generates tolerance.
Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/genética , RNA Mensageiro/genética , Animais , Sequência de Bases , Linhagem Celular , Fator de Iniciação 4E em Eucariotos/metabolismo , Genes MHC Classe I , Células HEK293 , Células HeLa , Humanos , Camundongos , Modelos Imunológicos , Iniciação Traducional da Cadeia Peptídica , Peptídeos/genética , Peptídeos/imunologia , Biossíntese de Proteínas , Capuzes de RNA/genética , RNA Mensageiro/metabolismo , Tolerância a Antígenos Próprios/genética , Tolerância a Antígenos Próprios/imunologiaRESUMO
Viruses are known to employ different strategies to manipulate the major histocompatibility (MHC) class I antigen presentation pathway to avoid recognition of the infected host cell by the immune system. However, viral control of antigen presentation via the processes that supply and select antigenic peptide precursors is yet relatively unknown. The Epstein-Barr virus (EBV)-encoded EBNA1 is expressed in all EBV-infected cells, but the immune system fails to detect and destroy EBV-carrying host cells. This immune evasion has been attributed to the capacity of a Gly-Ala repeat (GAr) within EBNA1 to inhibit MHC class I restricted antigen presentation. Here we demonstrate that suppression of mRNA translation initiation by the GAr in cis is sufficient and necessary to prevent presentation of antigenic peptides from mRNAs to which it is fused. Furthermore, we demonstrate a direct correlation between the rate of translation initiation and MHC class I antigen presentation from a certain mRNA. These results support the idea that mRNAs, and not the encoded full length proteins, are used for MHC class I restricted immune surveillance. This offers an additional view on the role of virus-mediated control of mRNA translation initiation and of the mechanisms that control MHC class I restricted antigen presentation in general.
Assuntos
Apresentação de Antígeno/genética , Antígenos Nucleares do Vírus Epstein-Barr/fisiologia , Herpesvirus Humano 4/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Evasão da Resposta Imune/genética , RNA Mensageiro/metabolismo , Animais , Apresentação de Antígeno/imunologia , Sequência de Bases , Linhagem Celular Tumoral , Dipeptídeos/química , Dipeptídeos/imunologia , Antígenos Nucleares do Vírus Epstein-Barr/química , Antígenos Nucleares do Vírus Epstein-Barr/genética , Fatores de Iniciação em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/fisiologia , Herpesvirus Humano 4/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Evasão da Resposta Imune/imunologia , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Camundongos , Modelos Biológicos , Conformação de Ácido Nucleico , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/imunologia , RNA Mensageiro/genética , Sequências Repetitivas de Aminoácidos/imunologia , Sequências Repetitivas de Aminoácidos/fisiologiaRESUMO
The glycine-alanine repeat (GAr) sequence of the Epstein-Barr virus-encoded EBNA-1 prevents presentation of antigenic peptides to major histocompatibility complex class I molecules. This has been attributed to its capacity to suppress mRNA translation in cis. However, the underlying mechanism of this function remains largely unknown. Here, we have further investigated the effect of the GAr as a regulator of mRNA translation. Introduction of silent mutations in each codon of a 30-amino-acid GAr sequence does not significantly affect the translation-inhibitory capacity, whereas minimal alterations in the amino acid composition have strong effects, which underscores the observation that the amino acid sequence and not the mRNA sequence mediates GAr-dependent translation suppression. The capacity of the GAr to repress translation is dose and position dependent and leads to a relative accumulation of preinitiation complexes on the mRNA. Taken together with the surprising observation that fusion of the 5' untranslated region (UTR) of the c-myc mRNA to the 5' UTR of GAr-carrying mRNAs specifically inactivates the effect of the GAr, these results indicate that the GAr targets components of the translation initiation process. We propose a model in which the nascent GAr peptide delays the assembly of the initiation complex on its own mRNA.