title: The SWI/SNF Chromatin Regulator BRG1 Modulates the Transcriptional Regulatory Activity of the Epstein-Barr Virus DNA Polymerase Processivity Factor BMRF1.
abstract: During the lytic phase of Epstein-Barr virus (EBV), binding of the transactivator Zta to the origin of lytic replication (oriLyt) and the BHLF1 transcript, forming a stable RNA-DNA hybrid, is required to initiate viral DNA replication. EBV-encoded viral DNA replication proteins form complexes to amplify viral DNA. BMRF1, the viral DNA polymerase accessory factor, is essential for lytic DNA replication and also known as a transcriptional regulator of the expression of BHLF1 and BALF2 (single-stranded DNA [ssDNA]-binding protein). In order to determine systematically how BMRF1 regulates viral transcription, a BMRF1 knockout bacmid was generated to analyze viral gene expression using a viral DNA microarray. We found that a subset of Rta-responsive late genes, including BcLF1, BLLF1, BLLF2, and BDLF3, were downregulated in cells harboring a BMRF1 knockout EBV bacmid (p2089ΔBMRF1). In reporter assays, BMRF1 appears to transactivate a subset of viral late promoters through distinct pathways. BMRF1 activates the BDLF3 promoter in an SP1-dependent manner. Notably, BMRF1 associates with the transcriptional regulator BRG1 in EBV-reactivated cells. BMRF1-mediated transactivation activities on the BcLF1 and BLLF1 promoters were attenuated by knockdown of BRG1. In BRG1-depleted EBV-reactivated cells, BcLF1 and BLLF1 transcripts were reduced in number, resulting in reduced virion secretion. BMRF1 and BRG1 bound to the adjacent upstream regions of the BcLF1 and BLLF1 promoters, and depletion of BRG1 attenuated the recruitment of BMRF1 onto both promoters, suggesting that BRG1 is involved in BMRF1-mediated regulation of these two genes. Overall, we reveal a novel pathway by which BMRF1 can regulate viral promoters through interaction with BRG1.IMPORTANCEThe cascade of viral gene expression during Epstein-Barr virus (EBV) replication is exquisitely regulated by the coordination of the viral DNA replication machinery and cellular factors. Upon lytic replication, the EBV immediate early proteins Zta and Rta turn on the expression of early proteins that assemble into viral DNA replication complexes. The DNA polymerase accessory factor, BMRF1, also is known to transactivate early gene expression through its interaction with SP1 or Zta on specific promoters. Through a global analysis, we demonstrate that BMRF1 also turns on a subset of Rta-regulated, late structural gene promoters. Searching for BMRF1-interacting cellular partners revealed that the SWI/SNF chromatin modifier BRG1 contributes to BMRF1-mediated transactivation of a subset of late promoters through protein-protein interaction and viral chromatin binding. Our findings indicate that BMRF1 regulates the expression of more viral genes than thought previously through distinct viral DNA replication-independent mechanisms.
authors.affiliation: Graduate Institute and Department of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Mei-Tzu
authors.initials: MT
authors.lastname: Su
authors.affiliation: Graduate Institute and Department of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Ya-Ting
authors.initials: YT
authors.lastname: Wang
authors.affiliation: Graduate Institute and Department of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan.|National Institute of Cancer Research, National Health Research Institutes, Miaoli County, Taiwan.
authors.forename: Yen-Ju
authors.initials: YJ
authors.lastname: Chen
authors.affiliation: National Institute of Cancer Research, National Health Research Institutes, Miaoli County, Taiwan.
authors.forename: Su-Fang
authors.identifier: http://orcid.org/0000-0001-8706-2439
authors.initials: SF
authors.lastname: Lin
authors.affiliation: Graduate Institute and Department of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Ching-Hwa
authors.initials: CH
authors.lastname: Tsai
authors.affiliation: Graduate Institute and Department of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan mrc@ntu.edu.tw.
authors.forename: Mei-Ru
authors.initials: MR
authors.lastname: Chen
mesh_terms: D000956:Antigens, Viral / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D055786:Gene Knockout Techniques; D057809:HEK293 Cells; D006367:HeLa Cells; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics
chemical_list: D000956:Antigens, Viral; C109483:BDLF-3 protein, Human herpesvirus 4; C063676:BZLF1 protein, Herpesvirus 4, Human; D002843:Chromatin; D004279:DNA, Viral; C083204:Epstein-Barr virus early antigen diffuse component; C079185:IE2 protein, bovine herpesvirus type 4; D017874:Immediate-Early Proteins; D008562:Membrane Glycoproteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D015534:Trans-Activators; D014157:Transcription Factors; D014764:Viral Proteins; C084108:SMARCA4 protein, human; D004265:DNA Helicases
keywords: BMRF1; BRG1; DNA polymerase accessory factor; Epstein-Barr virus; chromatin regulator; transcriptional regulation