RESUMO
This retrospective study examined expression of Epstein-Barr virus (EBV) latent genes in oral epithelium from human immunodeficiency virus-seropositive subjects, to identify genes associated with the pathogenesis of oral hairy leukoplakia (HLP). Transcription of EBV latent genes was detected in tissues with productive EBV replication and, also, in normal oral epithelial tissues without EBV replication. Expression of the EBV EBNA-2 open-reading frame in oral epithelium was identified as an important cofactor associated with the pathogenesis of HLP. In vitro experiments suggested that a recombinant variant of the EBNA-2 gene may play a role in the pathogenesis of HLP, through modulation of EBNA-2 protein function.
Assuntos
Aciclovir/análogos & derivados , Antígenos Nucleares do Vírus Epstein-Barr/genética , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/genética , Leucoplasia Pilosa/virologia , Mucosa Bucal/virologia , Valina/análogos & derivados , Aciclovir/uso terapêutico , Biópsia , Epitélio/virologia , Antígenos Nucleares do Vírus Epstein-Barr/fisiologia , Genes Virais , Infecções por HIV/complicações , Herpesvirus Humano 4/patogenicidade , Humanos , Leucoplasia Pilosa/tratamento farmacológico , RNA Mensageiro/análise , RNA Viral/genética , Estudos Retrospectivos , Transcrição Gênica , Valaciclovir , Valina/uso terapêutico , Proteínas da Matriz Viral/genética , Proteínas Virais , Replicação ViralRESUMO
Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2), a direct transcriptional activator of viral and cellular genes, is required for EBV-induced B-cell transformation. The functional role of conserved regions within the amino terminus of the protein preceding the poly-proline region has yet to be fully characterized. Thus, we tested whether the EBNA2 amino-terminal 30 amino acid residues, containing evolutionarily conserved region 1, are required for stimulating viral and cellular gene expression necessary for B-cell transformation in a viral transcomplementation assay. We found that these residues are required for its ability to induce LMP-1 expression in lymphoblastoid cell lines (LCLs), to stimulate LMP-1 promoter reporter plasmids in transient-cotransfection assays, and to rescue LCL growth following inactivation of endogenous wild-type EBNA2 protein. Deletion of amino acid residues 3 to 30 also impaired its ability to self-associate in coimmunoprecipitation assays. These data indicate that EBNA2 residues 3 to 30 comprise an essential domain required for induction of LMP-1 expression and, consequently, for maintenance of the immortalized phenotype of LCLs. The ability to self-associate into dimers or multimers conferred by this domain may be an important mechanism for these effects.
Assuntos
Proteínas de Transporte/biossíntese , Transformação Celular Viral/fisiologia , Antígenos Nucleares do Vírus Epstein-Barr/química , Antígenos Nucleares do Vírus Epstein-Barr/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Linfócitos B/citologia , Linfócitos B/virologia , Sequência de Bases , Sítios de Ligação/genética , Linfoma de Burkitt/genética , Linfoma de Burkitt/fisiopatologia , Linfoma de Burkitt/virologia , Proteínas de Transporte/genética , Linhagem Celular , Linhagem Celular Tumoral , Transformação Celular Viral/genética , Proteínas do Citoesqueleto , DNA Viral/genética , Proteínas de Ligação a DNA/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/genética , Expressão Gênica , Genes myc , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas com Domínio LIM , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Transfecção , Proteínas ViraisRESUMO
Epstein-Barr virus nuclear antigen 2 (EBNA2) is required for EBV-mediated immortalization of primary human B cells and is a direct transcriptional activator of viral and cellular genes. The prototype EBNA2 protein contains a unique motif in which 43 out of 45 amino acids are prolines (polyproline region [PPR]). Previous genetic analysis has shown that deletion of the PPR resulted in viruses unable to immortalize B cells, although the protein did appear transcriptionally functional (R. Yalamanchili, S. Harada, and E. Kieff, J. Virol. 70:2468-2473, 1996). The PPR's uniqueness and requirement for immortalization make it an attractive therapeutic target. However, the role of this highly unusual motif for immortalization remains enigmatic. We have recently developed a transcomplementation assay that allows both genetic and functional analyses of EBNA2 in EBV-mediated immortalization maintenance (A. V. Gordadze, R. Peng, J. Tan, G. Liu, R. Sutton, B. Kempkes, G. W. Bornkamm, and P. D. Ling, J. Virol. 75:5899-5912, 2001). Surprisingly, we found that DeltaPPR-EBNA2 was able to support B-cell proliferation similar to that of wild-type EBNA2 in this assay, indicating that deletion of the PPR from EBNA2 does not result in a loss of function required for immortalization maintenance. Further analysis of this mutant EBNA2 revealed that it consistently activated the viral LMP1 and LMP2A promoters severalfold better than wild-type EBNA2 in transient cotransfection assays. In addition, one striking difference between lymphoblastoid cell lines expressing wild-type EBNA2 from those expressing DeltaPPR-EBNA2 is that the latter cells have significantly reduced EBV genomic levels. The data are consistent with a model in which lower EBNA2 target gene dosage may be selected for in DeltaPPR-EBNA2-dependent cell lines to compensate for hyperactive stimulation of viral genes, such as LMP-1, which is cytostatic for B cells when overexpressed. It is conceivable that the hyperactivity rather than the loss of function, as hypothesized previously, could be responsible for the inability of recombinant DeltaPPR-EBNA2 EBVs to immortalize B cells.
