Lytic cycle gene regulation of Epstein-Barr virus

Abstract

Episomal reporter plasmids containing the Epstein-Barr virus (EBV) oriP sequence stably transfected into Akata Burkitt's lymphoma cells were used to analyze EBV lytic cycle gene regulation. First, we found that the Zp promoter of EBV, but not the Rp promoter, can be activated in the absence of protein synthesis in these oriP plasmids, casting doubt on the immediate early status of Rp. An additional level of regulation of Zp was implied by analysis of a mutation of the ZV element. Second, our analysis of late lytic cycle promoters revealed that the correct relative timing, dependence on ori lyt in cis, and sensitivity to inhibitors of DNA replication were reconstituted on the oriP plasmids. Late promoter luciferase activity from oriP plasmids also incorporating replication-competent ori lyt was phosphonoacetic acid sensitive, a hallmark of EBV late genes. A minimal ori lyt, which only replicates weakly, was sufficient to confer late timing of expression specifically on late promoters. Finally, deletion analysis of EBV late promoter sequences upstream of the transcription start site confirmed that sequences between -49 and +30 are sufficient for late gene expression, which is dependent on ori lyt in cis. However, the TATT version of the TATA box found in many late genes was not essential for late expression.

FIG. 1.

(A) Structure of the 2.8-kb RNA encoding BRLF1; coordinates of the first exon are numbered. The small rightward arrow shows the position of the 5′ RACE primers. The BRLF1 open reading frame is shaded. (B) EBV genome sequence up to position 93893, the start of the 2.8-kb mRNA. The additional 5′ sequence (TTTTTA in two clones and TTTTTTA in one clone sequenced) present in the 5′ RACE cDNAs is shown. (C) Polyacrylamide gel electrophoresis of the 5′ RACE products, showing the 192- and 162-nucleotide products. Akata cells were treated with anti-Ig with (An/Ig) or without (Ig) anisomycin to inhibit protein synthesis. The values on the left are nucleotides.

FIG. 2.

(A) Schematic diagram of the pHEBo:Rp-Luc reporter plasmid and the EBNA1 expression plasmid puro/oriP-EBNA1. (B) Luciferase reporter assays following induction with anti-Ig. Rp+EBV and Zp+EBV were in AK2003 cells, and Rp and Zp were in EBV-negative AK31 cells. Co is the pHEBo-luc vector control in AK2003 cells. (C) Western blotting assay to detect induction of BZLF1 (BZ-1 antibody) or BRLF1 (8C12 antibody, 1:100) in EBV-negative AK31 cell lines containing p294:ZRp-254, p294:Zp-1276, or p294:ZRp-7101 compared to the positive control AK6 cells.

FIG. 3.

Western blot assays of BZLF1 and PCNA at the indicated times after induction with anti-Ig in AK31 cells containing Zp-82LF1 plasmids. The upper panels are wild-type Zp, and the lower panels are the MV mutant form of Zp. Protein samples were electrophoresed on 12.5% SDS gels, blotted to nitrocellulose, and probed with either BZ-1 (1:500 dilution) or PCNA antibody PC10 (1:1,000), followed by enhanced-chemiluminescence detection (Amersham).

FIG. 4.

(A) Schematic diagram of EBV promoter in pHEBo-luc, with or without ori lyt. Late promoters were those for BcLF1, BdRF1, and BFRFC) Similar plasmids were made with the promoter for BMRF1 (delayed early) and the Cp latent promoter. (B) Luciferase reporter assays following anti-Ig induction of late promoter luciferase plasmids in AK2003 cells, with or without PAA treatment (0.85 mM). The Western blot assay of the BdRF1 protein demonstrates inhibition of the late lytic cycle by PAA and timing of BdRF1 expression. PCNA was also blotted as a loading control. kRLU, 10³ relative light units; p.i., postinfection. (C) Southern blotting of 2 μg of EcoRI-digested total DNA from AK2003 cells containing the promoter for BdRF1 with no ori lyt or with the core ori lyt or the big ori lyt. The probe was an equal mixture of the B95-8 EcoRI I fragment (for EBV) and the pGL2 vector (Promega) for the luciferase plasmid, labeled by random priming. Restriction fragments corresponding to the endogenous (endog.) EBV or the pHEBo plasmid are shown above an ethidium bromide stain of the DNA on the gel as a loading control.

FIG. 5.

(A) Luciferase reporter assays for different classes of EBV promoter in AK2000 cells to show the relative timing after anti-Ig treatment and dependence of the late promoter on ori lyt. BZLF1, immediate early; BMRF1, delayed early; BcLF1, late; Cp, latent. Data are expressed as fold induction above the level of activity without anti-Ig treatment because of the different inherent strengths of the promoters. (B) All three of the late promoters tested in AK2000 cells require ori lyt for induction in response to anti-Ig and show late timing. (C) Core ori lyt reduces the lytic cycle activity of the delayed early promoter (BMRF1) in AK2000 cell lines and has no effect on the latent cycle promoter (Cp) in luciferase reporter assays after anti-Ig treatment. (D) Luciferase reporter assays with AK2000 cell lines for the BFRF3 and BcLF1 promoters with or without core ori lyt, compared to empty vector pHEBo-luc without ori lyt. Relative light unit (kRLU, 10³ relative light units) data are shown as averages of duplicate determinations.

FIG. 6.

(A) Schematic diagram of the conserved −73 to −81 sequence present upstream of some late promoters and the mutation introduced. For the TATA box mutation, TATT was changed to TATA. (B) Luciferase reporter assays of the late promoter for BcLF1 in response to anti-Ig treatment in AK2000 cells. 5′ deletions were to position −430, −109, or −49, and −109m has the −81 to −73 sequence mutated in the −109 background. (C) Similar to panel B but with the late promoter for BdRF1. (D) TATT-to-TATA mutations of the TATA box in promoters for BdRF1 and BcLF1 in the −109 plasmids. kRLU, 10³ relative light units; wt, wild type.