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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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Modeling of Z binding to methylated ZRE sites versus AP1.(A) The predicted structure and hydrogen interactions between WT Z bound to Nap ZRE2 (upper panel, stereoview) is compared to Z(S186A) bound to the AP1 site (lower panel, stereoview). Z is shown as a green ribbon; hydrogen interactions are indicated by dotted black lines and the two DNA strands are shown as orange and cyan tubes. The left half-sites of Nap ZRE2 and AP1 are shown in cyan basepair stick as indicated. (B) Stereoview illustration of Z binding to the right half-site of methylated Nap ZRE1 is shown. A purple ball-and-stick model highlights the key difference of the Z/AP1 structure (at position −3' thymine and +3 adenine) compared to the Z/Nap ZRE1 structure, as well as the different sidechain conformations of Asn182'. (C) Z Cys189 forms van der Waals contacts with the −2' thymine unique to Rp ZRE3. Van der Waals contacts between the −2' thymine position unique to the Rp ZRE3 DNA molecule and Cys189 are highlighted (yellow contour). This interaction is greatly reduced with a guanine present at the −2' position (purple contour). Additionally, mutation of Cys189 to a serine residue greatly reduces the van der Waals interaction (red contour) with the −2' Rp ZRE3 thymine.
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ppat-1000356-g010: Modeling of Z binding to methylated ZRE sites versus AP1.(A) The predicted structure and hydrogen interactions between WT Z bound to Nap ZRE2 (upper panel, stereoview) is compared to Z(S186A) bound to the AP1 site (lower panel, stereoview). Z is shown as a green ribbon; hydrogen interactions are indicated by dotted black lines and the two DNA strands are shown as orange and cyan tubes. The left half-sites of Nap ZRE2 and AP1 are shown in cyan basepair stick as indicated. (B) Stereoview illustration of Z binding to the right half-site of methylated Nap ZRE1 is shown. A purple ball-and-stick model highlights the key difference of the Z/AP1 structure (at position −3' thymine and +3 adenine) compared to the Z/Nap ZRE1 structure, as well as the different sidechain conformations of Asn182'. (C) Z Cys189 forms van der Waals contacts with the −2' thymine unique to Rp ZRE3. Van der Waals contacts between the −2' thymine position unique to the Rp ZRE3 DNA molecule and Cys189 are highlighted (yellow contour). This interaction is greatly reduced with a guanine present at the −2' position (purple contour). Additionally, mutation of Cys189 to a serine residue greatly reduces the van der Waals interaction (red contour) with the −2' Rp ZRE3 thymine.

Mentions: To explore why methylation of some ZRE sites is required for Z binding, we used the Sybyl program to model the interaction of Z with the newly discovered methylated Nap ZREs versus the AP1 site. Consistent with previous observations modeling the binding of Z to methylated ZREs in the Rp [35],[46], our model of Z binding to the methylated Nap ZRE2 left-half site (Fig. 10A, upper panel) indicates that the cytosine methyl group at +1' occupies the same position as the thymine methyl group (+1') in the AP1 site (Fig. 10A, lower panel). Interestingly, this cytosine is present in the same location within the left half-sites of all four methylation-sensitive ZREs (Fig. 9).


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)

Modeling of Z binding to methylated ZRE sites versus AP1.(A) The predicted structure and hydrogen interactions between WT Z bound to Nap ZRE2 (upper panel, stereoview) is compared to Z(S186A) bound to the AP1 site (lower panel, stereoview). Z is shown as a green ribbon; hydrogen interactions are indicated by dotted black lines and the two DNA strands are shown as orange and cyan tubes. The left half-sites of Nap ZRE2 and AP1 are shown in cyan basepair stick as indicated. (B) Stereoview illustration of Z binding to the right half-site of methylated Nap ZRE1 is shown. A purple ball-and-stick model highlights the key difference of the Z/AP1 structure (at position −3' thymine and +3 adenine) compared to the Z/Nap ZRE1 structure, as well as the different sidechain conformations of Asn182'. (C) Z Cys189 forms van der Waals contacts with the −2' thymine unique to Rp ZRE3. Van der Waals contacts between the −2' thymine position unique to the Rp ZRE3 DNA molecule and Cys189 are highlighted (yellow contour). This interaction is greatly reduced with a guanine present at the −2' position (purple contour). Additionally, mutation of Cys189 to a serine residue greatly reduces the van der Waals interaction (red contour) with the −2' Rp ZRE3 thymine.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000356-g010: Modeling of Z binding to methylated ZRE sites versus AP1.(A) The predicted structure and hydrogen interactions between WT Z bound to Nap ZRE2 (upper panel, stereoview) is compared to Z(S186A) bound to the AP1 site (lower panel, stereoview). Z is shown as a green ribbon; hydrogen interactions are indicated by dotted black lines and the two DNA strands are shown as orange and cyan tubes. The left half-sites of Nap ZRE2 and AP1 are shown in cyan basepair stick as indicated. (B) Stereoview illustration of Z binding to the right half-site of methylated Nap ZRE1 is shown. A purple ball-and-stick model highlights the key difference of the Z/AP1 structure (at position −3' thymine and +3 adenine) compared to the Z/Nap ZRE1 structure, as well as the different sidechain conformations of Asn182'. (C) Z Cys189 forms van der Waals contacts with the −2' thymine unique to Rp ZRE3. Van der Waals contacts between the −2' thymine position unique to the Rp ZRE3 DNA molecule and Cys189 are highlighted (yellow contour). This interaction is greatly reduced with a guanine present at the −2' position (purple contour). Additionally, mutation of Cys189 to a serine residue greatly reduces the van der Waals interaction (red contour) with the −2' Rp ZRE3 thymine.
Mentions: To explore why methylation of some ZRE sites is required for Z binding, we used the Sybyl program to model the interaction of Z with the newly discovered methylated Nap ZREs versus the AP1 site. Consistent with previous observations modeling the binding of Z to methylated ZREs in the Rp [35],[46], our model of Z binding to the methylated Nap ZRE2 left-half site (Fig. 10A, upper panel) indicates that the cytosine methyl group at +1' occupies the same position as the thymine methyl group (+1') in the AP1 site (Fig. 10A, lower panel). Interestingly, this cytosine is present in the same location within the left half-sites of all four methylation-sensitive ZREs (Fig. 9).

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