RESUMO
EBV, a member of the herpes virus family, is a paradigm for human tumor viruses and a model of viral latency amenable for study in vitro. It induces resting human B lymphocytes to proliferate indefinitely in vitro and initially establishes a strictly latent infection in these cells. BZLF1, related to the cellular activating protein 1 (AP-1) family of transcription factors, is the viral master gene essential and sufficient to mediate the switch to induce the EBV lytic phase in latently infected B cells. Enigmatically, after infection BZLF1 is expressed very early in the majority of primary B cells, but its early expression fails to induce the EBV lytic phase. We show that the early expression of BZLF1 has a critical role in driving the proliferation of quiescent naïve and memory B cells but not of activated germinal center B cells. BZLF1's initial failure to induce the EBV lytic phase relies on the viral DNA at first being unmethylated. We have found that the eventual and inevitable methylation of viral DNA is a prerequisite for productive infection in stably, latently infected B cells which then yield progeny virus lacking cytosine-phosphatidyl-guanosine (CpG) methylation. This progeny virus then can repeat EBV's epigenetically regulated, biphasic life cycle. Our data indicate that the viral BZLF1 protein is crucial both to establish latency and to escape from it. Our data also indicate that EBV has evolved to appropriate its host's mode of methylating DNA for its own epigenetic regulation.
Assuntos
Epigênese Genética/genética , Genoma Viral , Herpesvirus Humano 4/genética , Transativadores/genética , Fator de Transcrição AP-1/metabolismo , Linfócitos B/patologia , Linfócitos B/virologia , Divisão Celular , Metilação de DNA , DNA Viral/genética , Infecções por Vírus Epstein-Barr/patologia , Genes Essenciais , Genes Precoces , Genes Virais , Humanos , Linfócitos/patologia , Linfócitos/virologia , Transativadores/metabolismo , Transfecção , Vírion/genética , Replicação Viral/genéticaRESUMO
DNA methylation is the major modification of eukaryotic genomes and plays an essential role in mammalian gene regulation. In general, cytosine-phosphatidyl-guanosine (CpG)-methylated promoters are transcriptionally repressed and nuclear proteins such as MECP2, MBD1, MBD2, and MBD4 bind CpG-methylated DNA and contribute to epigenetic silencing. Methylation of viral DNA also regulates gene expression of Epstein-Barr virus (EBV), which is a model of herpes virus latency. In latently infected human B cells, the viral DNA is CpG-methylated, the majority of viral genes is repressed and virus synthesis is therefore abrogated. EBV's BZLF1 encodes a transcription factor of the AP-1 family (Zta) and is the master gene to overcome viral gene repression. In a genome-wide screen, we now identify and characterize those viral genes, which Zta regulates. Among them are genes essential for EBV's lytic phase, which paradoxically depend on strictly CpG-methylated promoters for their Zta-induced expression. We identified novel DNA recognition motifs, termed meZRE (methyl-Zta-responsive element), which Zta selectively binds in order to 'read' DNA in a methylation- and sequence-dependent manner unlike any other known protein. Zta is a homodimer but its binding characteristics to meZREs suggest a sequential, non-palindromic and bipartite DNA recognition element, which confers superior DNA binding compared to CpG-free ZREs. Our findings indicate that Zta has evolved to transactivate cytosine-methylated, hence repressed, silent promoters as a rule to overcome epigenetic silencing.
Assuntos
Ilhas de CpG/genética , Metilação de DNA , Epigênese Genética , Regulação Viral da Expressão Gênica , Regiões Promotoras Genéticas/genética , Transativadores/genética , Latência Viral/genética , Linfócitos B/patologia , Linfócitos B/virologia , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , DNA Viral/genética , DNA Viral/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/patologia , Infecções por Vírus Epstein-Barr/virologia , Genes Virais , Herpesvirus Humano 4/fisiologia , Humanos , Imunoprecipitação , Rim/citologia , Rim/metabolismo , Rim/virologia , Luciferases/metabolismo , RNA Mensageiro/genética , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/metabolismo , Fator de Transcrição AP-1/metabolismo , Replicação ViralRESUMO
In Fusarium fujikuroi, bikaverin (BIK) biosynthesis is subject to repression by nitrogen. Unlike most genes subject to nitrogen metabolite repression, it has been shown that transcription of bik biosynthetic genes is not AreA dependent. Searching for additional transcription factors that may be involved in nitrogen regulation, we cloned and characterized the orthologue of Aspergillus nidulans meaB, which encodes a bZIP transcription factor. Two transcripts are derived from F. fujikuroi meaB: the large transcript (meaB(L)) predominates under nitrogen-sufficient conditions and the smaller transcript (meaB(S)) under nitrogen limitation, in an AreA-dependent manner. MeaB is specifically translocated to the nucleus under nitrogen-sufficient conditions in both F. fujikuroi and A. nidulans. Deletion of meaB resulted in partial upregulation of several nitrogen-regulated genes, but only in the ΔmeaB ΔareA double mutant were the bikaverin genes significantly upregulated in the presence of glutamine. These data demonstrate that MeaB and AreA coordinately mediate nitrogen metabolite repression and, importantly, that independently of AreA, MeaB can mediate nitrogen metabolite repression at specific loci in F. fujikuroi.