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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

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          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.

          Author Summary

          γ herpesviruses are characterized by their life-long persistence in the infected host. This is due in part to their ability to establish latency in infected cells. Epstein-Barr virus (EBV) is almost ubiquitous within the human population. The virus establishes latency in B-lymphocytes and is thought to reactivate and undergo replication following physiological stimuli that lead to B-cell activation. During latency the DNA genome of EBV is methylated, resulting in epigenetic control of viral gene expression. A viral protein that is key to viral reactivation and replication, Zta, is unique amongst transcription factors in displaying enhanced binding to methylated DNA sites. Here, we define the molecular interactions that predispose Zta to interact with methylated binding sites and we identify a mutant of Zta that has lost the preference for methylated sites. This allows us to probe the relevance of the recognition of methylated DNA for the reactivation of EBV from latency and to suggest that EBV has evolved a mechanism to allow it to specifically recognize methylated DNA and overturn epigenetic silencing of its genome imposed by the host.

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          Most cited references23

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          Improved methods for building protein models in electron density maps and the location of errors in these models.

          Map interpretation remains a critical step in solving the structure of a macromolecule. Errors introduced at this early stage may persist throughout crystallographic refinement and result in an incorrect structure. The normally quoted crystallographic residual is often a poor description for the quality of the model. Strategies and tools are described that help to alleviate this problem. These simplify the model-building process, quantify the goodness of fit of the model on a per-residue basis and locate possible errors in peptide and side-chain conformations.
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            The p120 catenin partner Kaiso is a DNA methylation-dependent transcriptional repressor.

            We describe a novel mammalian DNA binding activity that requires at least two symmetrically methylated CpG dinucleotides in its recognition sequence, preferably within the sequence 5'CGCG. A key component of the activity is Kaiso, a protein with POZ and zinc-finger domains that is known to associate with p120 catenin. We find that Kaiso behaves as a methylation-dependent transcriptional repressor in transient transfection assays. Kaiso is a constituent of one of two methyl-CpG binding complexes originally designated as MeCP1. The data suggest that zinc-finger motifs are responsible for DNA binding, and may therefore target repression to specific methylated regions of the genome. As Kaiso associates with p120 catenin, Kaiso may link events at the cell surface with DNA methylation-dependent gene silencing.
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              The Epstein-Barr virus lytic program is controlled by the co-operative functions of two transactivators.

              The propagation of herpesviruses has long been viewed as a temporally regulated sequential process that results from the consecutive expression of specific viral transactivators. As a key step in this process, lytic viral DNA replication is considered as a checkpoint that controls the expression of the late structural viral genes. In a novel genetic approach, we show that both hypotheses do not hold true for the Epstein-Barr virus (EBV). The study of viral mutants of EBV in which the early genes BZLF1 and BRLF1 are deleted allowed a precise assignment of the function of these proteins. Both transactivators were absolutely essential for viral DNA replication. Both BZLF1 and BRLF1 were required for full expression of the EBV proteins expressed during the lytic program, although the respective influence of these molecules on the expression of various viral target genes varied greatly. In replication-defective viral mutants, neither early gene expression nor DNA replication was a prerequisite for late gene expression. This work shows that BRLF1 and BZLF1 harbor distinct but complementary functions that influence all stages of viral production.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plpa
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                March 2008
                March 2008
                7 March 2008
                : 4
                : 3
                : e1000005
                Affiliations
                [1 ]School of Life Sciences, University of Sussex, Brighton, United Kingdom
                [2 ]EMBL Grenoble, BP 181, Grenoble, France
                University of Wisconsin-Madison, United States of America
                Author notes

                Conceived and designed the experiments: QHK CS EV CP AJS. Performed the experiments: QHK CS EV CP. Analyzed the data: QHK CS EV CP AJS. Wrote the paper: AJS.

                Article
                07-PLPA-RA-0715R2
                10.1371/journal.ppat.1000005
                2267006
                18369464
                bf612608-572b-4664-9fe5-0c33ab70439d
                Karlsson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 10 October 2007
                : 22 January 2008
                Page count
                Pages: 10
                Categories
                Research Article
                Biochemistry/Protein Folding
                Biochemistry/Transcription and Translation
                Infectious Diseases/Viral Infections
                Virology
                Virology/Viral Replication and Gene Regulation
                Virology/Viruses and Cancer

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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