MCAF1 and Rta-activated BZLF1 transcription in Epstein-Barr virus

Abstract

Epstein-Barr virus (EBV) expresses two transcription factors, Rta and Zta, which are involved in the transcriptional activation of EBV lytic genes. This study sought to elucidate the mechanism by which Rta activates transcription of the Zta-encoding gene, BZLF1, through the ZII element in the gene promoter. In a DNA affinity precipitation assay, ATF2 was found to associate with an Rta-interacting protein, MCAF1, at the ZII element. The interaction between Rta, MCAF1, and ATF2 at the same site in the ZII region was further verified in vivo by chromatin immunoprecipitation assay. The complex appears to be crucial for the activation of BZLF1 transcription, as the overexpression of two ATF2-dominant negative mutants, or the introduction of MCAF1 siRNA into 293T cells, were both found to substantially reduce Rta-mediated transcription levels of BZLF1. Moreover, this study also found that the Rta-MCAF1-ATF2 complex binds to a typical AP-1 binding sequence on the promoter of BMRF2, a key viral gene for EBV infection. Mutation of this sequence decreased Rta-mediated promoter activity significantly. Taken together, these results indicate a critical role for MCAF1 in AP-1-dependent Rta activation of BZLF1 transcription.

Figure 1. Transcriptional activation of Zp by Rta.

(A) Three reporter plasmids, pNS1-ZII, pNS1-ZIIM, and pNS3, were used in this transient transfection experiment. (B) 293T cells were cotransfected with pCMV-R, and the respective reporter plasmids. Luciferase activities were determined at 24 h after transfection. Each transfection experiment was performed three times, and all samples were prepared in duplicate. The p value from each experiment was derived using Student's t-test. Luc: luciferase gene.

Figure 2. Interaction between Rta, MCAF1 and ATF2.

P3HR1 cells were treated with TPA and sodium butyrate for 24(A) Proteins in the lysate were immunoprecipitated with anti-ATF2 antibody (lanes 3, 8), anti-Rta antibody (lanes 4, 7) or anti-IgG antibody (lanes 2, 6). Immunoblot analysis was performed using anti-Rta (lanes 1–4) and anti-ATF2 antibodies (lanes 5–8). (B) Proteins in the lysate from P3HR1 cells were immunoprecipitated with anti-IgG antibody (lanes 2, 6), anti-ATF2 antibody (lanes 3, 8), or anti-MCAF1 antibody (lanes 4, 7). Immunoprecipitated proteins were detected by immunoblotting, using anti-MCAF1 antibody (lanes 1–4) or anti-ATF2 antibody (lanes 5–8). Lanes 1 and 5 in (A) and (B) were loaded with 1% of total protein from the cell lysate. (C) GST-ATF2 (lane 5) or GST (lane 4) was added to the lysate prepared from E. coli BL21(DE3)(pET-MCAF1) (lane 1). Proteins bound to GST-ATF2 were pulled down by glutathione-Sepharose beads, and analyzed by immunoblotting (IB) with anti-His antibody (lanes 1–3). Lane 1 was loaded with 1% of cell lysate. GST or GST-ATF2 bound to glutathione-Sepharose beads were analyzed by immunoblotting (IB) with anti-GST antibody (lanes 4, 5).

Figure 3. Binding of the Rta-MCAF1-ATF2 complex to the ATF2-binding site in Zp.

(A) A biotin-labeled double-stranded ZII probe, containing the ATF2-binding sequences in Zp, was added to a lysate prepared from P3HR1 cells treated with TPA and sodium butyrate. A mutant probe, mZII, which contains a mutated ATF2-binding sequence, was used as a negative control. Proteins bound to the probes were captured using streptavidin magnetic beads, and were then extracted and detected by immunoblotting with anti-Rta, anti-MCAF1, and anti-ATF2 antibodies. Input lanes were loaded with 5% of the cell lysate. DAPA: DNA-affinity precipitation assay. (B) P3HR1 cells treated with TPA and sodium butyrate for 48 h (lytic), or DMSO (latent), were analyzed by ChIP assay using anti-Rta, anti-MCAF1 and anti-ATF2 antibodies, with anti-IgG antibody used as a negative control. The binding capabilities of ATF2, MCAF1, and Rta to the ZII region in Zp were examined by qPCR. qPCR with primers specific for the BcLF1 intergenic region was used as a negative control. The error bar represents standard error. The p values from each experiment were derived using the Student's t- test. * p<0.05.

Figure 4. Mapping the interaction domains in MCAF1 and ATF2.

Plasmids expressing GFP-MCAF1-N (lanes 2, 7), GFP-MCAF1-DM1 (lanes 3, 8), GFP-MCAF1-M (lanes 4, 9), or GFP-MCAF1-DM2 (lanes 5, 10) were transfected into 293T cells. Plasmid pEGFP-C1 (lanes 1, 6) transfectants were used as a control (A). After transfection, cells were treated with TPA, and proteins in the lysates were immunoprecipitated (IP) using anti-ATF2 antibody. Detection by immunoblotting (IB), using anti-GFP or anti-ATF2 antibodies, was subsequently performed (B). Additionally, lysates prepared from E. coli expressing either His-ATF2 (lanes 2, 7), His-ATF2-N1 (lanes 3, 8), His-ATF2-N2 (lanes 4, 9), or His-ATF2-C (lanes 5, 10) (C) were mixed with the lysates prepared from 293T cells transfected with pcDNA-MCAF1 (lane 11). Proteins immunoprecipitated with anti-Flag antibody (lanes 12–16) were detected by immunoblotting with anti-His (lanes 6–10) or anti-Flag (lanes 12–16) antibodies. E. coli BL21 (pET32a) lysate was used as a negative control (lanes 1, 6) (D). Input lanes in 4B and 4D were loaded with 1% of the cell lysate.

Figure 5. Involvement of MCAF1 in the interaction between ATF2 and Rta.

(A) His-tagged proteins, including Rta, ATF2, and MCAF1, were expressed in E. coli BL21(DE3). Lysates were mixed in different combinations for immunoprecipitation (IP) using anti-ATF2 antibody, and proteins bound to protein A/G agarose beads were then detected by immunoblotting (IB) using anti-Rta or anti-ATF2 antibodies. Lane 1 was loaded with 1% His-Rta. The reaction was also conducted using anti-IgG as a control. (B) 293T cells were cotransfected with pCMV-R and either MCAF1 siRNA or control siRNA. After transfection, cells were immunoprecipitated (IP) by anti-ATF2 (lanes 4, 6) or anti-IgG antibody (lanes 3, 5), and detected by immunoblotting (IB) using anti-Rta antibody. The effect of MCAF1 siRNA on the expression of MCAF1 was examined by immunoblotting using anti-MCAF1 and anti-ATF2 antibodies (lanes 1, 2). Lanes 1 and 2 were loaded with 1% of total protein from cell lysates.

Figure 6. Involvement of MCAF1 in Rta-activated BZLF1 transcription.

(A) 293T cells were cotransfected with pCMV-R and a reporter plasmid, pNS1-ZII, pNS1-ZIIM, or pNS3, as well as control siRNA or MCAF1 siRNA. Luciferase activity exhibited by the cells was examined at 48 h after transfection. The reduction of MCAF1 by siRNA was examined by immunoblotting with anti-MCAF1 antibody. Anti-α-tubulin antibody was used as a control. In addition, 293T cells were cotransfected with pCMV-R and a reporter plasmid, pNS1-ZII, pNS1-ZIIM, or pNS3, as well as varying amounts (µg) of pEGFP-ATF2-C (B) or pEGFP-ATF2 (69A/71A) (C). The expression of GFP-ATF2-C (B) or GFP-ATF2 (69A/71A) (C) was examined by immunoblotting, using anti-GFP antibody. Promoter activities were determined at 48 h after transfection. Each transfection experiment was performed at least three times, and all samples were prepared in duplicate. The p values from each experiment were derived using the Student's t-test.

Figure 7. Activation of the BMRF2 promoter by Rta via the AP-1 binding site.

(A) The BMRF2 reporter plasmid and a mutant version. 293T cells were cotransfected with pCMV-R and a reporter plasmid, pBMRF2-AP1, pBMRF2-mAP1, or pGL2-Basic. Luciferase activities were examined at 24 h after transfection. (B) P3HR1 cells that had been treated with TPA and sodium butyrate for 48 h (lytic) or DMSO (latent) were analyzed by ChIP assay using anti-Rta, anti-MCAF1 and anti-ATF2 antibodies. Anti-IgG antibody was used as a control. The binding of ATF2, MCAF1 and Rta to the AP-1 sequence in the BMRF2 promoter was examined by qPCR. Error bars represent standard error. The p values from each experiment were derived using the Student's t-test. * p<0.05, ** p<0.001. Luc: luciferase gene.