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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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ZtaC189S binding to meZREs in Rp is compromised in vitro.A. EMSA analysis was undertaken for meZRE2 with the indicated proteins or an unprogrammed lysate (IVT). B. Zta, ZtaC189A and ZtaC189S were generated in vitro and analyzed by SDS-PAGE. C. EMSA analysis of equivalent amounts of the indicated proteins was undertaken with meZRE3 as described above.
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ppat-1000005-g003: ZtaC189S binding to meZREs in Rp is compromised in vitro.A. EMSA analysis was undertaken for meZRE2 with the indicated proteins or an unprogrammed lysate (IVT). B. Zta, ZtaC189A and ZtaC189S were generated in vitro and analyzed by SDS-PAGE. C. EMSA analysis of equivalent amounts of the indicated proteins was undertaken with meZRE3 as described above.

Mentions: We next asked what effect methylation of ZRE2 and ZRE3 had on binding by Zta and ZtaC189S (ZRE1 lacks a CpG motif and hence is not subject to methylation). Note that methylation yields four methyl-cytosines in ZRE3 (two on each strand) but only two in ZRE2 (Figure 2). In line with previous reports, methylation of ZRE3 converted it from a marginal to a strong binding site for Zta and methylation of ZRE2 also enhanced binding (Figure 3 and [3],[12],[4]). Importantly, the ability of ZtaC189S to bind meZRE3 was markedly reduced compared to that of wild-type Zta (Figure 3). Binding of ZtaC189S to meZRE2 was only modestly reduced, to approximately 75% compared to wild type. An independent mutation of C189 to alanine (ZtaC189A) also resulted in decreased binding to meZRE3, suggesting that the decreased binding of the C189S mutant was due to loss of the cysteine and not, for example, merely due to phosphorylation of the newly introduced serine.


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)

ZtaC189S binding to meZREs in Rp is compromised in vitro.A. EMSA analysis was undertaken for meZRE2 with the indicated proteins or an unprogrammed lysate (IVT). B. Zta, ZtaC189A and ZtaC189S were generated in vitro and analyzed by SDS-PAGE. C. EMSA analysis of equivalent amounts of the indicated proteins was undertaken with meZRE3 as described above.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2267006&req=5

ppat-1000005-g003: ZtaC189S binding to meZREs in Rp is compromised in vitro.A. EMSA analysis was undertaken for meZRE2 with the indicated proteins or an unprogrammed lysate (IVT). B. Zta, ZtaC189A and ZtaC189S were generated in vitro and analyzed by SDS-PAGE. C. EMSA analysis of equivalent amounts of the indicated proteins was undertaken with meZRE3 as described above.
Mentions: We next asked what effect methylation of ZRE2 and ZRE3 had on binding by Zta and ZtaC189S (ZRE1 lacks a CpG motif and hence is not subject to methylation). Note that methylation yields four methyl-cytosines in ZRE3 (two on each strand) but only two in ZRE2 (Figure 2). In line with previous reports, methylation of ZRE3 converted it from a marginal to a strong binding site for Zta and methylation of ZRE2 also enhanced binding (Figure 3 and [3],[12],[4]). Importantly, the ability of ZtaC189S to bind meZRE3 was markedly reduced compared to that of wild-type Zta (Figure 3). Binding of ZtaC189S to meZRE2 was only modestly reduced, to approximately 75% compared to wild type. An independent mutation of C189 to alanine (ZtaC189A) also resulted in decreased binding to meZRE3, suggesting that the decreased binding of the C189S mutant was due to loss of the cysteine and not, for example, merely due to phosphorylation of the newly introduced serine.

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