Limits...
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.

Show MeSH

Related in: MedlinePlus

Z binds to the Na promoter, and binding is enhanced by Nap methylation.(A) Latently infected EBV+ 293 Z-KO cells were transfected with a BZLF1 expression vector (+) or an empty vector control (−). Chromatin immunoprecipitation assay was performed 20 hours after transfection using anti-Z and control goat IgG antibodies as indicated to examine Z binding to the Na promoter (Nap), ZREs within the EBV oriLyt (positive control) and EBV sequences in the 3′ end of the EBV BALF5 gene (negative control). (B) The ability of in vitro translated Z to bind to 32P end-labeled probes (in either the methylated, or unmethylated forms) containing the BRRF1 promoter (Nap) sequences from −280 to −470, −108 to −301, and +77 to −128 (relative to the BRRF1 mRNA start site) was examined by EMSA. A probe containing the BRLF1 promoter (Rp) sequences from −1 to −270 (in the methylated form) served as a positive control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2654727&req=5

ppat-1000356-g001: Z binds to the Na promoter, and binding is enhanced by Nap methylation.(A) Latently infected EBV+ 293 Z-KO cells were transfected with a BZLF1 expression vector (+) or an empty vector control (−). Chromatin immunoprecipitation assay was performed 20 hours after transfection using anti-Z and control goat IgG antibodies as indicated to examine Z binding to the Na promoter (Nap), ZREs within the EBV oriLyt (positive control) and EBV sequences in the 3′ end of the EBV BALF5 gene (negative control). (B) The ability of in vitro translated Z to bind to 32P end-labeled probes (in either the methylated, or unmethylated forms) containing the BRRF1 promoter (Nap) sequences from −280 to −470, −108 to −301, and +77 to −128 (relative to the BRRF1 mRNA start site) was examined by EMSA. A probe containing the BRLF1 promoter (Rp) sequences from −1 to −270 (in the methylated form) served as a positive control.

Mentions: Regulation of the Nap by viral and cellular factors has not been well studied. Although the Nap was previously reported to be activated by Z, but not R, in reporter gene assays, specific ZRE sites required for Z activation of the Nap were not defined [18]. Furthermore, although the Nap has a number of potential CpG-containing ZRE motifs, the effect of methylation on Z binding to the Nap has not previously been examined. To determine if Z binds to the Nap in the context of the intact viral genome, latently EBV infected 293 cells were transfected with a Z expression vector or a control vector, and ChIP assays were performed 20 hours later to detect Z binding. As shown in Fig. 1A, the ChIP assays confirmed that Z binds to the Nap in vivo.


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)

Z binds to the Na promoter, and binding is enhanced by Nap methylation.(A) Latently infected EBV+ 293 Z-KO cells were transfected with a BZLF1 expression vector (+) or an empty vector control (−). Chromatin immunoprecipitation assay was performed 20 hours after transfection using anti-Z and control goat IgG antibodies as indicated to examine Z binding to the Na promoter (Nap), ZREs within the EBV oriLyt (positive control) and EBV sequences in the 3′ end of the EBV BALF5 gene (negative control). (B) The ability of in vitro translated Z to bind to 32P end-labeled probes (in either the methylated, or unmethylated forms) containing the BRRF1 promoter (Nap) sequences from −280 to −470, −108 to −301, and +77 to −128 (relative to the BRRF1 mRNA start site) was examined by EMSA. A probe containing the BRLF1 promoter (Rp) sequences from −1 to −270 (in the methylated form) served as a positive control.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000356-g001: Z binds to the Na promoter, and binding is enhanced by Nap methylation.(A) Latently infected EBV+ 293 Z-KO cells were transfected with a BZLF1 expression vector (+) or an empty vector control (−). Chromatin immunoprecipitation assay was performed 20 hours after transfection using anti-Z and control goat IgG antibodies as indicated to examine Z binding to the Na promoter (Nap), ZREs within the EBV oriLyt (positive control) and EBV sequences in the 3′ end of the EBV BALF5 gene (negative control). (B) The ability of in vitro translated Z to bind to 32P end-labeled probes (in either the methylated, or unmethylated forms) containing the BRRF1 promoter (Nap) sequences from −280 to −470, −108 to −301, and +77 to −128 (relative to the BRRF1 mRNA start site) was examined by EMSA. A probe containing the BRLF1 promoter (Rp) sequences from −1 to −270 (in the methylated form) served as a positive control.
Mentions: Regulation of the Nap by viral and cellular factors has not been well studied. Although the Nap was previously reported to be activated by Z, but not R, in reporter gene assays, specific ZRE sites required for Z activation of the Nap were not defined [18]. Furthermore, although the Nap has a number of potential CpG-containing ZRE motifs, the effect of methylation on Z binding to the Nap has not previously been examined. To determine if Z binds to the Nap in the context of the intact viral genome, latently EBV infected 293 cells were transfected with a Z expression vector or a control vector, and ChIP assays were performed 20 hours later to detect Z binding. As shown in Fig. 1A, the ChIP assays confirmed that Z binds to the Nap in vivo.

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