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Methylation-dependent binding of the epstein-barr virus BZLF1 protein to viral promoters.

Dickerson SJ, Xing Y, Robinson AR, Seaman WT, Gruffat H, Kenney SC - PLoS Pathog. (2009)

Bottom Line: Z serine residue 186, which was previously shown to be required for Z binding to methylated ZREs in Rp, but not for Z binding to the AP1 site, is required for Z binding to methylated Nap ZREs.The Z(S186A) mutant cannot activate methylated Nap in reporter gene assays and does not induce Na expression in cells with latent EBV infection.Molecular modeling studies of Z bound to the methylated Nap ZREs help to explain why methylation is required for Z binding, and the role of the Z Ser186 residue.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory, Departments of Oncology and Medicine, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.

ABSTRACT
The switch between latent and lytic Epstein-Barr virus (EBV) infection is mediated by the viral immediate-early (IE) protein, BZLF1 (Z). Z, a homologue of c-jun that binds to AP1-like motifs (ZREs), induces expression of the BRLF1 (R) and BRRF1 (Na) viral proteins, which cooperatively activate transcription of the Z promoter and thereby establish a positive autoregulatory loop. A unique feature of Z is its ability to preferentially bind to, and activate, the methylated form of the BRLF1 promoter (Rp). To date, however, Rp is the only EBV promoter known to be regulated in this unusual manner. We now demonstrate that the promoter driving transcription of the early BRRF1 gene (Nap) has two CpG-containing ZREs (ACGCTCA and TCGCCCG) that are only bound by Z in the methylated state. Both Nap ZREs are highly methylated in cells with latent EBV infection. Z efficiently activates the methylated, but not unmethylated, form of Nap in reporter gene assays, and both ZREs are required. Z serine residue 186, which was previously shown to be required for Z binding to methylated ZREs in Rp, but not for Z binding to the AP1 site, is required for Z binding to methylated Nap ZREs. The Z(S186A) mutant cannot activate methylated Nap in reporter gene assays and does not induce Na expression in cells with latent EBV infection. Molecular modeling studies of Z bound to the methylated Nap ZREs help to explain why methylation is required for Z binding, and the role of the Z Ser186 residue. Methylation-dependent Z binding to critical viral promoters may enhance lytic reactivation in latently infected cells, where the viral genome is heavily methylated. Conversely, since the incoming viral genome is initially unmethylated, methylation-dependent Z activation may also help the virus to establish latency following infection.

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Methylation of the Nap promotes Z activation.HeLa (A) and DG75 (B) cells were transfected with a promoterless luciferase vector, or luciferase vectors driven by the Na, R, or BHLF1 lytic EBV promoters, in the presence or absence of a co-transfected Z expression vector. Luciferase vectors were either mock-methylated or methylated in vitro using M.Sss1 prior to transfection as indicted and the amount of luciferase activity determined 48 hours after transfection. The fold-increase in promoter activity in the presence of co-transfected Z (versus a vector control) is shown (obtained from duplicate transfections).
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ppat-1000356-g003: Methylation of the Nap promotes Z activation.HeLa (A) and DG75 (B) cells were transfected with a promoterless luciferase vector, or luciferase vectors driven by the Na, R, or BHLF1 lytic EBV promoters, in the presence or absence of a co-transfected Z expression vector. Luciferase vectors were either mock-methylated or methylated in vitro using M.Sss1 prior to transfection as indicted and the amount of luciferase activity determined 48 hours after transfection. The fold-increase in promoter activity in the presence of co-transfected Z (versus a vector control) is shown (obtained from duplicate transfections).

Mentions: To examine how methylation of the Nap affects its ability to be activated by Z, we transfected HeLa cells with methylated or mock-methylated forms of the Nap-LUC vector in the presence or absence of a Z expression vector. We also examined the effect of methylation on Z activation of Rp-LUC, BHLF1p-LUC (a luciferase vector driven by the early viral BHLF1 promoter, in which the known ZREs cannot be methylated), or the promoterless control luciferase vector (pGL3-basic). Z clearly activated the methylated form of Nap-LUC much more efficiently than the unmethylated form (140-fold activation versus 13-fold) in HeLa cells (Fig. 3A). Similar results were obtained in the EBV-negative B cell line, DG75 (Fig. 3B). As previously described [34], methylation of the Rp also enhanced its ability to be activated by Z (Fig. 3A). In contrast to its effects on the Rp and Nap, methylation of the BHLF1p (which contains CpG-free ZREs) strongly inhibited its ability to be activated by Z (Fig. 3A). Z also induced detectable activation of the promoterless luciferase vector in HeLa and DG75 cells (perhaps reflecting the presence of a previously described cryptic AP1 motif in the luciferase vector) [43], and this activation was inhibited by methylation (Fig. 3A and 3B). These results indicate that both the Nap and Rp are preferentially activated by Z in the methylated form, consistent with the presence of methylation-dependent ZREs in their promoters.


Methylation-dependent binding of the epstein-barr virus BZLF1 protein to viral promoters.

Dickerson SJ, Xing Y, Robinson AR, Seaman WT, Gruffat H, Kenney SC - PLoS Pathog. (2009)

Methylation of the Nap promotes Z activation.HeLa (A) and DG75 (B) cells were transfected with a promoterless luciferase vector, or luciferase vectors driven by the Na, R, or BHLF1 lytic EBV promoters, in the presence or absence of a co-transfected Z expression vector. Luciferase vectors were either mock-methylated or methylated in vitro using M.Sss1 prior to transfection as indicted and the amount of luciferase activity determined 48 hours after transfection. The fold-increase in promoter activity in the presence of co-transfected Z (versus a vector control) is shown (obtained from duplicate transfections).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654727&req=5

ppat-1000356-g003: Methylation of the Nap promotes Z activation.HeLa (A) and DG75 (B) cells were transfected with a promoterless luciferase vector, or luciferase vectors driven by the Na, R, or BHLF1 lytic EBV promoters, in the presence or absence of a co-transfected Z expression vector. Luciferase vectors were either mock-methylated or methylated in vitro using M.Sss1 prior to transfection as indicted and the amount of luciferase activity determined 48 hours after transfection. The fold-increase in promoter activity in the presence of co-transfected Z (versus a vector control) is shown (obtained from duplicate transfections).
Mentions: To examine how methylation of the Nap affects its ability to be activated by Z, we transfected HeLa cells with methylated or mock-methylated forms of the Nap-LUC vector in the presence or absence of a Z expression vector. We also examined the effect of methylation on Z activation of Rp-LUC, BHLF1p-LUC (a luciferase vector driven by the early viral BHLF1 promoter, in which the known ZREs cannot be methylated), or the promoterless control luciferase vector (pGL3-basic). Z clearly activated the methylated form of Nap-LUC much more efficiently than the unmethylated form (140-fold activation versus 13-fold) in HeLa cells (Fig. 3A). Similar results were obtained in the EBV-negative B cell line, DG75 (Fig. 3B). As previously described [34], methylation of the Rp also enhanced its ability to be activated by Z (Fig. 3A). In contrast to its effects on the Rp and Nap, methylation of the BHLF1p (which contains CpG-free ZREs) strongly inhibited its ability to be activated by Z (Fig. 3A). Z also induced detectable activation of the promoterless luciferase vector in HeLa and DG75 cells (perhaps reflecting the presence of a previously described cryptic AP1 motif in the luciferase vector) [43], and this activation was inhibited by methylation (Fig. 3A and 3B). These results indicate that both the Nap and Rp are preferentially activated by Z in the methylated form, consistent with the presence of methylation-dependent ZREs in their promoters.

Bottom Line: Z serine residue 186, which was previously shown to be required for Z binding to methylated ZREs in Rp, but not for Z binding to the AP1 site, is required for Z binding to methylated Nap ZREs.The Z(S186A) mutant cannot activate methylated Nap in reporter gene assays and does not induce Na expression in cells with latent EBV infection.Molecular modeling studies of Z bound to the methylated Nap ZREs help to explain why methylation is required for Z binding, and the role of the Z Ser186 residue.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory, Departments of Oncology and Medicine, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.

ABSTRACT
The switch between latent and lytic Epstein-Barr virus (EBV) infection is mediated by the viral immediate-early (IE) protein, BZLF1 (Z). Z, a homologue of c-jun that binds to AP1-like motifs (ZREs), induces expression of the BRLF1 (R) and BRRF1 (Na) viral proteins, which cooperatively activate transcription of the Z promoter and thereby establish a positive autoregulatory loop. A unique feature of Z is its ability to preferentially bind to, and activate, the methylated form of the BRLF1 promoter (Rp). To date, however, Rp is the only EBV promoter known to be regulated in this unusual manner. We now demonstrate that the promoter driving transcription of the early BRRF1 gene (Nap) has two CpG-containing ZREs (ACGCTCA and TCGCCCG) that are only bound by Z in the methylated state. Both Nap ZREs are highly methylated in cells with latent EBV infection. Z efficiently activates the methylated, but not unmethylated, form of Nap in reporter gene assays, and both ZREs are required. Z serine residue 186, which was previously shown to be required for Z binding to methylated ZREs in Rp, but not for Z binding to the AP1 site, is required for Z binding to methylated Nap ZREs. The Z(S186A) mutant cannot activate methylated Nap in reporter gene assays and does not induce Na expression in cells with latent EBV infection. Molecular modeling studies of Z bound to the methylated Nap ZREs help to explain why methylation is required for Z binding, and the role of the Z Ser186 residue. Methylation-dependent Z binding to critical viral promoters may enhance lytic reactivation in latently infected cells, where the viral genome is heavily methylated. Conversely, since the incoming viral genome is initially unmethylated, methylation-dependent Z activation may also help the virus to establish latency following infection.

Show MeSH
Related in: MedlinePlus