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Methylated DNA recognition during the reversal of epigenetic silencing is regulated by cysteine and serine residues in the Epstein-Barr virus lytic switch protein.

Karlsson QH, Schelcher C, Verrall E, Petosa C, Sinclair AJ - PLoS Pathog. (2008)

Bottom Line: Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with various malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma.The mutant is compromised both in vitro and in vivo for binding two methylated ZREs in Rp (ZRE2 and ZRE3), although the effect is striking only for ZRE3.Our results suggest that C189 regulates the enhanced interaction of Zta with methylated DNA in overturning the epigenetic control of viral latency.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Sussex, Brighton, United Kingdom.

ABSTRACT
Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with various malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma. Like all herpesviruses, the EBV life cycle alternates between latency and lytic replication. During latency, the viral genome is largely silenced by host-driven methylation of CpG motifs and, in the switch to the lytic cycle, this epigenetic silencing is overturned. A key event is the activation of the viral BRLF1 gene by the immediate-early protein Zta. Zta is a bZIP transcription factor that preferentially binds to specific response elements (ZREs) in the BRLF1 promoter (Rp) when these elements are methylated. Zta's ability to trigger lytic cycle activation is severely compromised when a cysteine residue in its bZIP domain is mutated to serine (C189S), but the molecular basis for this effect is unknown. Here we show that the C189S mutant is defective for activating Rp in a Burkitt's lymphoma cell line. The mutant is compromised both in vitro and in vivo for binding two methylated ZREs in Rp (ZRE2 and ZRE3), although the effect is striking only for ZRE3. Molecular modeling of Zta bound to methylated ZRE3, together with biochemical data, indicate that C189 directly contacts one of the two methyl cytosines within a specific CpG motif. The motif's second methyl cytosine (on the complementary DNA strand) is predicted to contact S186, a residue known to regulate methyl-ZRE recognition. Our results suggest that C189 regulates the enhanced interaction of Zta with methylated DNA in overturning the epigenetic control of viral latency. As C189 is conserved in many bZIP proteins, the selectivity of Zta for methylated DNA may be a paradigm for a more general phenomenon.

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Structural model of Zta bound to meZRE3.A. Alignment of Zta recognition sequences. The numbering convention is shown for base pair positions (bold italics) and individual nucleotides (plain font). Cytosines modified by methylation are indicated by a dot. B. Model of the Zta-meZRE3 complex viewed along the pseudodyad. The methylation sensitive C189 residue (red asterisk) and bidentate hydrogen bond interactions between R190Left and Guanine0′ (dotted black lines) are indicated. Cytosine methyl groups are semi-transparent. C. Orthogonal view. The hydrophobic contact between Cytosine1′ and S186Left (broken blue line) and hydrogen bond network involving S186Left, N182Left and Guanine2′ (dotted black lines) are shown. D. Schematic summary of contacts. van der Waals contacts involving the CpG methyl groups and Zta residues are shown as broken lines.
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ppat-1000005-g006: Structural model of Zta bound to meZRE3.A. Alignment of Zta recognition sequences. The numbering convention is shown for base pair positions (bold italics) and individual nucleotides (plain font). Cytosines modified by methylation are indicated by a dot. B. Model of the Zta-meZRE3 complex viewed along the pseudodyad. The methylation sensitive C189 residue (red asterisk) and bidentate hydrogen bond interactions between R190Left and Guanine0′ (dotted black lines) are indicated. Cytosine methyl groups are semi-transparent. C. Orthogonal view. The hydrophobic contact between Cytosine1′ and S186Left (broken blue line) and hydrogen bond network involving S186Left, N182Left and Guanine2′ (dotted black lines) are shown. D. Schematic summary of contacts. van der Waals contacts involving the CpG methyl groups and Zta residues are shown as broken lines.

Mentions: To better understand the above observations, we modeled the structure of Zta's DNA-binding domain bound to methylated ZRE3 and compared it to the previously reported model of Zta-bound meZRE2 [15]. In the following description, we designate residues within the two Zta monomers and the corresponding DNA half-sites as “Left” or “Right”, and the two CpG motifs as motifs 1 and 2, as summarized in Figures 6A and D. The modeled ZRE2 and ZRE3 structures, both based on the crystal structure of Zta bound to an AP-1 site, differ exclusively in the right half-site, as the two ZREs are identical except at base pairs 1 and 2 (Figure 6A).


Methylated DNA recognition during the reversal of epigenetic silencing is regulated by cysteine and serine residues in the Epstein-Barr virus lytic switch protein.

Karlsson QH, Schelcher C, Verrall E, Petosa C, Sinclair AJ - PLoS Pathog. (2008)

Structural model of Zta bound to meZRE3.A. Alignment of Zta recognition sequences. The numbering convention is shown for base pair positions (bold italics) and individual nucleotides (plain font). Cytosines modified by methylation are indicated by a dot. B. Model of the Zta-meZRE3 complex viewed along the pseudodyad. The methylation sensitive C189 residue (red asterisk) and bidentate hydrogen bond interactions between R190Left and Guanine0′ (dotted black lines) are indicated. Cytosine methyl groups are semi-transparent. C. Orthogonal view. The hydrophobic contact between Cytosine1′ and S186Left (broken blue line) and hydrogen bond network involving S186Left, N182Left and Guanine2′ (dotted black lines) are shown. D. Schematic summary of contacts. van der Waals contacts involving the CpG methyl groups and Zta residues are shown as broken lines.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2267006&req=5

ppat-1000005-g006: Structural model of Zta bound to meZRE3.A. Alignment of Zta recognition sequences. The numbering convention is shown for base pair positions (bold italics) and individual nucleotides (plain font). Cytosines modified by methylation are indicated by a dot. B. Model of the Zta-meZRE3 complex viewed along the pseudodyad. The methylation sensitive C189 residue (red asterisk) and bidentate hydrogen bond interactions between R190Left and Guanine0′ (dotted black lines) are indicated. Cytosine methyl groups are semi-transparent. C. Orthogonal view. The hydrophobic contact between Cytosine1′ and S186Left (broken blue line) and hydrogen bond network involving S186Left, N182Left and Guanine2′ (dotted black lines) are shown. D. Schematic summary of contacts. van der Waals contacts involving the CpG methyl groups and Zta residues are shown as broken lines.
Mentions: To better understand the above observations, we modeled the structure of Zta's DNA-binding domain bound to methylated ZRE3 and compared it to the previously reported model of Zta-bound meZRE2 [15]. In the following description, we designate residues within the two Zta monomers and the corresponding DNA half-sites as “Left” or “Right”, and the two CpG motifs as motifs 1 and 2, as summarized in Figures 6A and D. The modeled ZRE2 and ZRE3 structures, both based on the crystal structure of Zta bound to an AP-1 site, differ exclusively in the right half-site, as the two ZREs are identical except at base pairs 1 and 2 (Figure 6A).

Bottom Line: Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with various malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma.The mutant is compromised both in vitro and in vivo for binding two methylated ZREs in Rp (ZRE2 and ZRE3), although the effect is striking only for ZRE3.Our results suggest that C189 regulates the enhanced interaction of Zta with methylated DNA in overturning the epigenetic control of viral latency.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Sussex, Brighton, United Kingdom.

ABSTRACT
Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with various malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma. Like all herpesviruses, the EBV life cycle alternates between latency and lytic replication. During latency, the viral genome is largely silenced by host-driven methylation of CpG motifs and, in the switch to the lytic cycle, this epigenetic silencing is overturned. A key event is the activation of the viral BRLF1 gene by the immediate-early protein Zta. Zta is a bZIP transcription factor that preferentially binds to specific response elements (ZREs) in the BRLF1 promoter (Rp) when these elements are methylated. Zta's ability to trigger lytic cycle activation is severely compromised when a cysteine residue in its bZIP domain is mutated to serine (C189S), but the molecular basis for this effect is unknown. Here we show that the C189S mutant is defective for activating Rp in a Burkitt's lymphoma cell line. The mutant is compromised both in vitro and in vivo for binding two methylated ZREs in Rp (ZRE2 and ZRE3), although the effect is striking only for ZRE3. Molecular modeling of Zta bound to methylated ZRE3, together with biochemical data, indicate that C189 directly contacts one of the two methyl cytosines within a specific CpG motif. The motif's second methyl cytosine (on the complementary DNA strand) is predicted to contact S186, a residue known to regulate methyl-ZRE recognition. Our results suggest that C189 regulates the enhanced interaction of Zta with methylated DNA in overturning the epigenetic control of viral latency. As C189 is conserved in many bZIP proteins, the selectivity of Zta for methylated DNA may be a paradigm for a more general phenomenon.

Show MeSH
Related in: MedlinePlus