The Epstein-Barr virus BcRF1 gene product is a TBP-like protein with an essential role in late gene expression

Abstract

That the expression of late genes is coupled to viral genome replication is well established for all herpesviruses, but the exact mechanisms of their regulation, especially by viral proteins, are poorly understood. Here, we report the identification of the Epstein-Barr virus (EBV) early protein BcRF1 as a viral factor crucial for the activation of late gene transcription following viral DNA replication during the productive cycle. In order to study the function of the BcRF1 protein, we constructed a recombinant EBV lacking this gene. In HEK293 cells, this recombinant virus underwent normal DNA replication during the productive cycle but failed to express high levels of late gene transcripts or proteins, resulting in a nonproductive infection. Interestingly, a TATT motif is present in the promoter of most EBV late genes, at the position of the TATA box. We show here that BcRF1 forms a complex with the TATT motif and that this interaction is required for activation of late viral gene expression. Moreover, our results suggest that BcRF1 acts via interaction with other viral proteins.

Fig 1

The BcRF1 protein is a TATT-binding protein. BcRF1 protein was produced as a GST fusion protein in bacteria and used in a gel shift assay with the ³²P-labeled TATT_BcLF1 probe. Competition was carried out with a 1-, 2.5-, 5-, or 10-fold excess of unlabeled TATT_BcLF1 or TATA_BMLF1 double-stranded oligonucleotide or with the unrelated ZRE-DR oligonucleotide. The GST-BcRF1–DNA complex is indicated by an asterisk.

Fig 2

Construction of recombinant BacEBV_ΔBcRF1. (A) Homologous recombination between the wild-type EBV genome (p2089) and a PCR fragment carrying the kanamycin resistance gene (Kana) flanked by 50 bp of BcRF1 sequences was performed in E. coli DH10B containing the pKD46 plasmid. Clones carrying the recombinant EBV were selected for chloramphenicol and kanamycin resistance. The resulting BAC-EBV_ΔBcRF1 recombinant has the kanamycin gene inserted between positions 126120 and 126596 in the BcRF1 gene. The putative structural homology domain between the TATA-binding protein and BcRF1 is labeled TBP and was partially deleted in the recombinant. (B) Restriction enzyme analysis of the BAC-EBV_ΔBcRF1 plasmid DNA. The BamHI restriction pattern of BAC-EBV_ΔBcRF1 mutant DNA was compared to that of the wild-type BAC-EBV DNA. The modified DNA pattern is indicated by a plus sign and a minus sign. (C) PCR amplification analysis of the BAC-EBV_ΔBcRF1 clone compared to the BAC-EBV clone carrying the wild-type EBV genome. The PCR was made with the two primers indicated by arrowheads in panel A. As expected, the PCR product is shifted due to the insertion-deletion in the BcRF1 gene.

Fig 3

BcRF1 is necessary for virion production. (A) Raji cells were incubated with supernatants from (a) untransfected HEK293_EBVΔBcRF1 cells, (b) HEK293_EBVΔBcRF1 cells transfected with an expression plasmid encoding the EB1 protein in order to induce the productive cycle, and (c) HEK293 _EBVΔBcRF1 cells transfected with an expression plasmid encoding the EB1 protein and an expression plasmid encoding the BcRF1 protein in order to complement BcRF1 deficiency. GFP fluorescence of Raji cells was analyzed 48 h after infection. Corresponding images obtained with phase-contrast light microscopy were merged with the images showing UV fluorescence. (B) Immunoblot analysis of Flag-BcRF1, EB2, and EB1 proteins expressed in HEK293_EBVΔBcRF1 cells that were either not transfected (lane 1), transfected with an EB1 expression plasmid (lane 2), or cotransfected with expression plasmids for both EB1 and BcRF1 (lane 3).

Fig 4

BcRF1 is not required for viral DNA replication. Viral DNA replication in uninduced and induced (transfected with the EB1 expression plasmid) cells was assessed by semiquantitative PCR. Different dilutions of the purified DNA (50-fold to 5000-fold) were used for the PCR in order to demonstrate that the PCR is in a linear range. The PCR products were loaded onto a 2% agarose gel. The amount of viral DNA in the induced cells was compared to the amount of viral DNA present in the uninduced cells.

Fig 5

BcRF1 enhances expression of the late viral genes. (A) Total RNA from HEK293_EBV or HEK293_EBVΔBcRF1 cells that were either not transfected (lanes 1 and 5), transfected with an EB1 expression plasmid (lanes 2 and 6), or cotransfected with expression plasmids for both EB1 and BcRF1 (lanes 3 and 7) was purified, reverse transcribed, and analyzed by PCR using specific primers for actin (used to monitor the quality of mRNA purification and to normalize the amount of mRNA used under each condition), the BMRF1 early gene, and the BDLF1 and BcLF1 late genes. Lanes 4 and 8 show the results of a PCR amplification made with RNA used in lanes 3 and 7 in the absence of reverse transcriptase. (B) Immunoblot analysis of gp350/220, EB2, and EB1 proteins expressed from HEK293_EBV and HEK293_EBVΔBcRF1 cells that were either not transfected (lanes 1 and 3), transfected with an EB1 expression plasmid (lanes 2 and 4), or cotransfected with expression plasmids for both EB1 and BcRF1 (lane 5).

Fig 6

Binding of BcRF1 to the TATT box is required for its function. (A) Characterization of BcRF1 mutant proteins that do not bind DNA. Two BcRF1 mutants in which Asn at positions 420 and 422 (BcRF1_MutN1-N2) or Asn at positions 512 and 514 (BcRF1_MutN8-N9) was mutated to Ala residues were produced as GST fusion proteins. Increasing amounts of the GST (lanes 2 and 3), GST-BcRF1 (lanes 4 and 5), GST-BcRF1_MutN1-N2 (lanes 6 and 7), and GST-BcRF1_MutN8-N9 (lanes 8 and 9) proteins were used in a gel shift assay with the ³²P-labeled TATT_BcLF1 probe. The GST-BcRF1–DNA complexes are indicated with an asterisk. (B) Comparison of transcomplementation efficiency by BcRF1 and BcRF1_MutN1-N2 or BcRF1_MutN8-N9 proteins. Raji cells were infected with supernatant from HEK293_EBVΔBcRF1 cells that had been either not transfected (lane 1), transfected with an EB1 expression plasmid (lane 2), or cotransfected with expression plasmids for both EB1 and BcRF1 (lane 3), BcRF1_MutN1-N2 (lane 4), or BcRF1_MutN8-N9 (lane 5). The amount of GFP-expressing Raji cells was determined by FACS analysis. The experiment was done three times, and the error bars represent standard deviations. (B) Immunoblot analysis for one representative experiment.

Fig 7

A TATT box element is necessary for the function of BcRF1. (A) Schematic representation of the luciferase reporter vectors used. (B) HEK293_EBVΔBcRF1 cells were either transfected with the reporter vectors alone (columns 1 and 5), cotransfected with the reporter vectors and an EB1 expression plasmid (columns 2 and 6) or with a BcRF1 expression plasmid (columns 3 and 7), or cotransfected with expression plasmids for both EB1 and BcRF1 (lanes 4 and 8). The amount of luciferase expressed was measured 24 h after transfection. Luciferase activation was calculated relative to the value obtained with the pTATT-BcLF1-Luc reporter transfected alone. The experiment was done three times, and the error bars represent standard deviations. (C) Immunoblot analysis for one representative experiment.

Fig 8

BcRF1 is also required for late gene expression in B cells (A) Primary B cells were immortalized with the recombinant ΔBcRF1 virus and then were either transfected with the reporter luciferase vectors alone as indicated (columns 1 and 5), cotransfected with the reporter vectors and with an EB1 expression plasmid (columns 2 and 6) or with a BcRF1 expression plasmid (columns 3 and 7), or cotransfected with expression plasmids for both EB1 and BcRF1 (columns 4 and 8). The amount of luciferase expressed was measured 24 h posttransfection. Luciferase activation was calculated relative to the value obtained with the pTATT-BcLF1-Luc reporter transfected alone. The experiment was done three times, and the error bars represent standard deviations. (B) Immunoblot analysis for one representative experiment.