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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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Zta and ZtaC189S interact with non-methylated Rp ZREs equivalently.A. Schematic diagram showing the location of ZREs 1–3 in Rp. Transcription of this gene occurs in a leftwards direction with respect to the viral genome. The numbering relates to the type I EBV genome accession number NC_007605. Asterixes mark the methylated Cytosine residues. B. Zta and Zta C189s were generated in an in vitro translation system and fractionated on SDS-PAGE, together with a non-programmed translation reaction (IVT). C.–E. Equivalent amounts of the indicated proteins were subject to EMSA analysis with the probes indicated above. A reaction with no added protein was also included, indicated probe. F. The three ZREs associated with Rp are aligned and their areas of conservation indicated by boxes. The interactions of Zta and ZtaC189S with each site are summarized.
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ppat-1000005-g002: Zta and ZtaC189S interact with non-methylated Rp ZREs equivalently.A. Schematic diagram showing the location of ZREs 1–3 in Rp. Transcription of this gene occurs in a leftwards direction with respect to the viral genome. The numbering relates to the type I EBV genome accession number NC_007605. Asterixes mark the methylated Cytosine residues. B. Zta and Zta C189s were generated in an in vitro translation system and fractionated on SDS-PAGE, together with a non-programmed translation reaction (IVT). C.–E. Equivalent amounts of the indicated proteins were subject to EMSA analysis with the probes indicated above. A reaction with no added protein was also included, indicated probe. F. The three ZREs associated with Rp are aligned and their areas of conservation indicated by boxes. The interactions of Zta and ZtaC189S with each site are summarized.

Mentions: To investigate the molecular basis for this defect, we compared the ability of Zta and ZtaC189S to bind to the three ZREs present in Rp (Figure 2). All three of the ZREs contribute to Rp activity and are important for activation of the endogenous viral gene by chemical stimuli [14], although ZRE1 appears to be dispensable on a fully-methylated template [3]. Zta and ZtaC189S were produced by coupled transcription and translation in vitro and their ability to interact with each ZRE was assessed by EMSA (Figure 2). As summarized in Figure 2F, the proteins were equally capable of binding to non-methylated ZRE1 and ZRE2 but, in agreement with previous reports for Zta [3],[12], neither Zta not ZtaC189S showed a detectable interaction with non-methylated ZRE3. Thus, the reduced ability of ZtaC189S to transactivate BRLF1 is not due to an inherent defect in binding to the ZREs.


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)

Zta and ZtaC189S interact with non-methylated Rp ZREs equivalently.A. Schematic diagram showing the location of ZREs 1–3 in Rp. Transcription of this gene occurs in a leftwards direction with respect to the viral genome. The numbering relates to the type I EBV genome accession number NC_007605. Asterixes mark the methylated Cytosine residues. B. Zta and Zta C189s were generated in an in vitro translation system and fractionated on SDS-PAGE, together with a non-programmed translation reaction (IVT). C.–E. Equivalent amounts of the indicated proteins were subject to EMSA analysis with the probes indicated above. A reaction with no added protein was also included, indicated probe. F. The three ZREs associated with Rp are aligned and their areas of conservation indicated by boxes. The interactions of Zta and ZtaC189S with each site are summarized.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2267006&req=5

ppat-1000005-g002: Zta and ZtaC189S interact with non-methylated Rp ZREs equivalently.A. Schematic diagram showing the location of ZREs 1–3 in Rp. Transcription of this gene occurs in a leftwards direction with respect to the viral genome. The numbering relates to the type I EBV genome accession number NC_007605. Asterixes mark the methylated Cytosine residues. B. Zta and Zta C189s were generated in an in vitro translation system and fractionated on SDS-PAGE, together with a non-programmed translation reaction (IVT). C.–E. Equivalent amounts of the indicated proteins were subject to EMSA analysis with the probes indicated above. A reaction with no added protein was also included, indicated probe. F. The three ZREs associated with Rp are aligned and their areas of conservation indicated by boxes. The interactions of Zta and ZtaC189S with each site are summarized.
Mentions: To investigate the molecular basis for this defect, we compared the ability of Zta and ZtaC189S to bind to the three ZREs present in Rp (Figure 2). All three of the ZREs contribute to Rp activity and are important for activation of the endogenous viral gene by chemical stimuli [14], although ZRE1 appears to be dispensable on a fully-methylated template [3]. Zta and ZtaC189S were produced by coupled transcription and translation in vitro and their ability to interact with each ZRE was assessed by EMSA (Figure 2). As summarized in Figure 2F, the proteins were equally capable of binding to non-methylated ZRE1 and ZRE2 but, in agreement with previous reports for Zta [3],[12], neither Zta not ZtaC189S showed a detectable interaction with non-methylated ZRE3. Thus, the reduced ability of ZtaC189S to transactivate BRLF1 is not due to an inherent defect in binding to the ZREs.

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