Activation of the Epstein-Barr virus DNA polymerase promoter by the BRLF1 immediate-early protein is mediated through USF and E2F

Abstract

The Epstein-Barr virus (EBV) DNA polymerase (pol) is essential for the replication of viral genomes during productive EBV infection. We have previously reported that the EBV DNA pol promoter, which is TATA-less and constitutively inactive, is activated by a genomic clone expressing both immediate-early viral transactivators, BZLF1Z and BRLF1 (R), in EBV-infected lymphoid cells. Here we demonstrate that R alone is sufficient to activate the pol promoter in EBV-negative B cells. Unlike other early promoters to which the R protein binds directly, its effect on the pol promoter does not appear to involve a direct DNA-binding mechanism. Instead, we found that two cellular transcription factors, an upstream stimulatory factor USF, and a member of the E2F family of proteins, bind directly to the pol promoter at positions ؊795 to ؊786 and ؊186 to ؊170, respectively, regions previously identified as important for activation of the pol promoter. These two sites contribute to or are essential for transactivation of the pol promoter by R in EBV-noninfected B cells. These data suggest that the R immediate-early protein may activate a key early EBV promoter (pol) through both USF and E2F. * Corresponding author. Phone: (919) 966-3036. Fax: (919) 966-3015.on July 10, 2020 by guest http://jvi.asm.org/ Downloaded from BMRF1, and BHRF1/BHLF1, R appears to act indirectly either together with or through E2F and USF. MATERIALS AND METHODSCell lines. Raji and Akata are latently EBV-infected B-lymphocyte lines. DG75 is an EBV-negative Burkitt's lymphoma B-cell line. NPC-KT is an EBVpositive epithelial cell line, and ADAH is an EBV-negative epithelial cell line. All lymphoid cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum. Epithelial cells were grown in Dulbecco's modified Eagle's medium plus 10% fetal calf serum.Plasmid constructs. The construction of plasmid pPOLCAT has been described previously (28). The internal deletion mutants were constructed by restriction digestion and religation. Site-directed mutants of pPOLCAT were made with the Bio-Rad Muta-Gene phagemid in vitro mutagenesis kit on the basis of the Kunkle method (53). The expression constructs of EBV immediate-early proteins have been described elsewhere (68). The pCMV-Z plasmid contains the BZLF1 cDNA fragment under the control of the cytomegalovirus immediateearly promoter in the pGem2-based vector (pHD1013). The pCMV-R plasmid is derived from a HindIII-HindIII fragment from a genomic expression construct, pCMV-RZ, such that the BRLF1 gene is linked to the cytomegalovirus immediate-early promoter in a pUC-18 vector.Transfections and chloramphenicol acetyltransferase assays. Plasmid DNA was purified through Qiagen columns. For each transfection, 10 7 cells were electroporated at 1,500 V with the University of Wisconsin Zapper electroporation unit. Cells were then suspended in 10 ml of RPMI 1640 medium supplemented with 10% fetal calf serum and incubated for 48 h at 37ЊC in 5% CO 2 . Cell extracts were prepared by washing cell pellets t...

CD8+ immunodominance among Epstein-Barr virus lytic cycle antigens directly reflects the efficiency of antigen presentation in lytically infected cells

“…Nevertheless, the consistent pattern of results obtained from different patients with different HLA backgrounds, added to more limited data from an earlier study (18), suggests that the hierarchy of immunodominance observed is indeed real. The relationship between immunodominance and time of antigen expression in the lytic cycle is further emphasized by the fact that, of the four most commonly recognized antigens within the E protein group, BMLF1 and BMRF1 are proteins whose synthesis is directly induced by the BZLF1 and BRLF1 IE transactivators (24)(25)(26)(27), whereas BALF5, and potentially also BALF2, synthesis is induced by BRLF1 acting in concert with cellular transcription factors (28). Therefore, the expression of such proteins would be among the first markers of progression into early phase.…”

The Epstein-Barr Virus Immediate-Early Gene Product, BRLF1, Interacts with the Retinoblastoma Protein during the Viral Lytic Cycle

“…Previous data from our laboratory suggested that Rb may be a target for the R protein. R activation of the promoter for the EBV pol gene has been mapped to elements which bind the transcription factors USF and E2F; mutation at either site reduced R-mediated activation of pol (42). Additional promoters are activated by R in an indirect manner (26,52,57,82).…”

“…The 605-amino-acid (aa) R protein contains two transactivation domains and an N-terminal DNA-binding and dimerization domain (29,44). R can activate promoters both by binding a specific DNA sequence, an R-responsive element (29,44), and indirectly, possibly by activating or recruiting cellular transcription factors (42,81,82). Promoters activated in the latter manner include BRLF1 itself (57,82), the human immunodeficiency virus long terminal repeat (52), and c-myc (26).…”

“…Promoters activated in the latter manner include BRLF1 itself (57,82), the human immunodeficiency virus long terminal repeat (52), and c-myc (26). Recent data show that the promoter for the EBV DNA polymerase, which is responsible for lytic viral DNA replication, is also activated by R indirectly through the transcription factors USF and E2F (42).…”

Strategies in subversion: de‐regulation of the mammalian cell cycle by viral gene products

“…De-regulation of the mammalian cell cycle Q 2001 Blackwell Science Ltd, International Journal of Experimental Pathology, 82, 3±13 through transcription factors USF and E2F (Liu et al 1996). EBNALP is also reported to interact with Rb in vitro and localize with Rb in the cell (Szekely et al 1993).…”