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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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Single amino acid in basic region of Zta blocks the ability to transactivate Rp in BL cells.A. Schematic diagram showing the relationship between Zta expression and activation of the BRLF1 promoter. B. Expression vectors for Zta, ZtaC189S and the relevant “empty” vector (pBABE) were introduced into Raji cells and their ability to activate the endogenous BRLF1 gene determined. 24 hours after transfection, RNA was prepared, cDNA was synthesized then amplified using quantitative PCR with specific primers for the BRLF1 transcript and a housekeeping gene, L32. Expression of BRLF1 mRNA, following normalization for expression of L32 is shown, relative to that seen following Zta transfection (100%). C. Expression of Zta and ZtaC89S were determined by quantitative PCR and expressed relative to expression of Zta (100%).
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ppat-1000005-g001: Single amino acid in basic region of Zta blocks the ability to transactivate Rp in BL cells.A. Schematic diagram showing the relationship between Zta expression and activation of the BRLF1 promoter. B. Expression vectors for Zta, ZtaC189S and the relevant “empty” vector (pBABE) were introduced into Raji cells and their ability to activate the endogenous BRLF1 gene determined. 24 hours after transfection, RNA was prepared, cDNA was synthesized then amplified using quantitative PCR with specific primers for the BRLF1 transcript and a housekeeping gene, L32. Expression of BRLF1 mRNA, following normalization for expression of L32 is shown, relative to that seen following Zta transfection (100%). C. Expression of Zta and ZtaC89S were determined by quantitative PCR and expressed relative to expression of Zta (100%).

Mentions: We and others have previously shown that altering a single cysteine residue within the DNA contact region of Zta (ZtaC189S) impairs its ability to disrupt EBV latency [13],[12]. Here we asked whether ZtaC189S is competent to initiate one of the earliest events in latency disruption, the transcriptional activation of BRLF1 (Figure 1A). Expression vectors encoding Zta and ZtaC189S were introduced into a Burkitt's lymphoma derived cell line, Raji, and subsequent transactivation of the endogenous viral Rp was assessed (Figure 1B). Basal expression of BRLF1 is low in the absence of Zta, but is enhanced 33-fold following Zta expression. In contrast, expression of an equivalent amount of ZtaC189S resulted in only 4-fold enhancement of BRLF1 expression. Therefore, ZtaC189S is severely compromised for its ability to transactivate the BRLF1 gene in Raji cells.


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)

Single amino acid in basic region of Zta blocks the ability to transactivate Rp in BL cells.A. Schematic diagram showing the relationship between Zta expression and activation of the BRLF1 promoter. B. Expression vectors for Zta, ZtaC189S and the relevant “empty” vector (pBABE) were introduced into Raji cells and their ability to activate the endogenous BRLF1 gene determined. 24 hours after transfection, RNA was prepared, cDNA was synthesized then amplified using quantitative PCR with specific primers for the BRLF1 transcript and a housekeeping gene, L32. Expression of BRLF1 mRNA, following normalization for expression of L32 is shown, relative to that seen following Zta transfection (100%). C. Expression of Zta and ZtaC89S were determined by quantitative PCR and expressed relative to expression of Zta (100%).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000005-g001: Single amino acid in basic region of Zta blocks the ability to transactivate Rp in BL cells.A. Schematic diagram showing the relationship between Zta expression and activation of the BRLF1 promoter. B. Expression vectors for Zta, ZtaC189S and the relevant “empty” vector (pBABE) were introduced into Raji cells and their ability to activate the endogenous BRLF1 gene determined. 24 hours after transfection, RNA was prepared, cDNA was synthesized then amplified using quantitative PCR with specific primers for the BRLF1 transcript and a housekeeping gene, L32. Expression of BRLF1 mRNA, following normalization for expression of L32 is shown, relative to that seen following Zta transfection (100%). C. Expression of Zta and ZtaC89S were determined by quantitative PCR and expressed relative to expression of Zta (100%).
Mentions: We and others have previously shown that altering a single cysteine residue within the DNA contact region of Zta (ZtaC189S) impairs its ability to disrupt EBV latency [13],[12]. Here we asked whether ZtaC189S is competent to initiate one of the earliest events in latency disruption, the transcriptional activation of BRLF1 (Figure 1A). Expression vectors encoding Zta and ZtaC189S were introduced into a Burkitt's lymphoma derived cell line, Raji, and subsequent transactivation of the endogenous viral Rp was assessed (Figure 1B). Basal expression of BRLF1 is low in the absence of Zta, but is enhanced 33-fold following Zta expression. In contrast, expression of an equivalent amount of ZtaC189S resulted in only 4-fold enhancement of BRLF1 expression. Therefore, ZtaC189S is severely compromised for its ability to transactivate the BRLF1 gene in Raji cells.

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