RESUMO
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
Transforming growth factor beta 1 (TGF-ß1) signal transduction has been implicated in many second-messenger pathways, including the NF-κB pathway. We provide evidence of a novel TGF-ß1-mediated pathway that leads to extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, which in turn induces expression of an Epstein-Barr virus (EBV) protein, ZEBRA, that is responsible for the induction of the viral lytic cycle. This pathway includes two unexpected steps, both of which are required to control ERK 1/2 phosphorylation: first, a quick and transient activation of NF-κB, and second, downregulation of inducible nitric oxide synthase (iNOS) activity that requires the participation of NF-κB activity. Although necessary, NF-κB alone is not sufficient to produce downregulation of iNOS, suggesting that another uncharacterized event(s) is involved in this pathway. Dissection of the steps involved in the switch from the EBV latent cycle to the lytic cycle will be important to understand how virus-host relationships modulate the innate immune system.
Assuntos
Regulação para Baixo , Herpesvirus Humano 4/fisiologia , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Ativação Viral , Linfócitos B/virologia , Linhagem Celular , Linhagem Celular Transformada , Regulação da Expressão Gênica , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/genética , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Transativadores/genética , Transativadores/metabolismoRESUMO
The Epstein-Barr virus (EBV) has been detected in subsets of breast cancers. In order to elaborate on these observations, we quantified by real-time PCR (Q-PCR) the EBV genome in biopsy specimens of breast cancer tissue as well as in tumor cells isolated by microdissection. Our findings show that EBV genomes can be detected by Q-PCR in about half of tumor specimens, usually in low copy numbers. However, we also found that the viral load is highly variable from tumor to tumor. Moreover, EBV genomes are heterogeneously distributed in morphologically identical tumor cells, with some clusters of isolated tumor cells containing relatively high genome numbers while other tumor cells isolated from the same specimen may be negative for EBV DNA. Using reverse transcription-PCR, we detected EBV gene transcripts: EBNA-1 in almost all of the EBV-positive tumors and RNA of the EBV oncoprotein LMP-1 in a smaller subset of the tissues analyzed. Moreover, BARF-1 RNA was detected in half of the cases studied. Furthermore, we observed that in vitro EBV infection of breast carcinoma cells confers resistance to paclitaxel (taxol) and provokes overexpression of a multidrug resistance gene (MDR1). Consequently, even if a small number of breast cancer cells are EBV infected, the impact of EBV infection on the efficiency of anticancer treatment might be of importance.
