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The Epstein-Barr virus lytic cycle activator Zta interacts with methylated ZRE in the promoter of host target gene egr1.

ABSTRACT: Activation of the host gene egr1 is essential for the lytic replication of Epstein-Barr virus (EBV). egr1 is activated by Zta (BZLF1, ZEBRA). Zta interacts directly with DNA through a series of closely related Zta-response elements (ZREs). Here we dissect the mechanism used by Zta to interact with the egr1 promoter and identify a weak interaction with egr1ZRE that is dependent on the distal part of egr1ZRE. Furthermore, we demonstrate that the ability of Zta to interact with egr1ZRE is enhanced at least tenfold by methylation. The ability of Zta to transactivate a reporter construct driven by the egr1 promoter can be enhanced by methylation. As the ability of Zta to interact with a methylated ZRE in the EBV genome correlates with its ability to activate the expression of the endogenous viral gene BRLF1, this suggests that Zta may also have the capability to overturn epigenetic control of egr1.

SUBMITTER: Heather J

PROVIDER: S-EPMC2885059 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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The Epstein-Barr virus lytic cycle activator Zta interacts with methylated ZRE in the promoter of host target gene egr1.

Heather James J Flower Kirsty K Isaac Samine S Sinclair Alison J AJ

The Journal of general virology 20090304 Pt 6

Activation of the host gene egr1 is essential for the lytic replication of Epstein-Barr virus (EBV). egr1 is activated by Zta (BZLF1, ZEBRA). Zta interacts directly with DNA through a series of closely related Zta-response elements (ZREs). Here we dissect the mechanism used by Zta to interact with the egr1 promoter and identify a weak interaction with egr1ZRE that is dependent on the distal part of egr1ZRE. Furthermore, we demonstrate that the ability of Zta to interact with egr1ZRE is enhanced ...[more]

PMID: 19264650

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Project description:The human gammaherpesvirus Epstein-Barr virus (EBV) (human herpesvirus 4 [HHV4]) infects most adults and is an important contributor to the development of many types of lymphoid and epithelial cancers. Essential contributions of viral genes to viral replication are known, but the potential contributions of cell genes are less well delineated. A key player is the viral protein Zta (BZLF1, ZEBRA, or Z). This sequence-specific DNA-binding protein can disrupt EBV latency by driving the transcription of target genes and by interacting with the EBV lytic origin of replication. Here, we used an unbiased proteomics approach to identify the Zta-interactome in cells derived from Burkitt's lymphoma. Isolating Zta and associated proteins from Burkitt's lymphoma cells undergoing EBV replication, followed by tandem mass tag (TMT) mass spectrometry, resulted in the identification of 39 viral and cellular proteins within the Zta interactome. An association of Zta with the cellular protein NFATc2 was validated in independent experiments. Furthermore, the ability of Zta to attenuate the activity of an NFAT-dependent promoter was shown, which suggests a functional consequence for the association. The expression of Zta is itself regulated through NFAT activity, suggesting that Zta may contribute to a feedback loop that would limit its own expression, thus aiding viral replication by preventing the known toxic effects of Zta overexpression.IMPORTANCE Epstein-Barr virus infects most people across the world and causes several kinds of cancer. Zta is an important viral protein that makes the virus replicate by binding to its DNA and turning on the expression of some genes. We used a sensitive, unbiased approach to isolate and identify viral and cellular proteins that physically interact with Zta. This revealed 39 viral and cellular proteins. We found that one protein, termed NFATc2, was already known to be important for a very early step in viral replication. We identify that once this step has occurred, Zta reduces the effectiveness of NFATc2, and we suggest that this is important to prevent cells from dying before viral replication is complete and the mature virus is released from the cells.

Project description:The Epstein-Barr virus (EBV)-encoded ZTA protein interacts strongly with and stabilizes the cellular CCAAT/enhancer binding protein alpha (C/EBPalpha), leading to the induction of p21-mediated G(1) cell cycle arrest. Despite the strong interaction between these two basic leucine zipper (bZIP) family proteins, the ZTA and C/EBPalpha subunits do not heterodimerize, as indicated by an in vitro cross-linking assay with in vitro-cotranslated (35)S-labeled C/EBPalpha and (35)S-labeled ZTA protein. Instead, they evidently form a higher-order oligomeric complex that competes with C/EBPalpha binding but not with ZTA binding in electrophoretic mobility shift assays (EMSAs). Glutathione S-transferase affinity assays with mutant ZTA proteins revealed that the basic DNA binding domain and the key leucine zipper residues required for homodimerization are all required for the interaction with C/EBPalpha. ZTA is known to bind to two ZRE sites within the ZTA promoter and to positively autoregulate its own expression in transient cotransfection assays, but there is conflicting evidence about whether it does so in vivo. Examination of the proximal ZTA upstream promoter region by in vitro EMSA analysis revealed two high-affinity C/EBP binding sites (C-2 and C-3), which overlap the ZII and ZIIIB motifs, implicated as playing a key role in lytic cycle induction. A chromatin immunoprecipitation assay confirmed the in vivo binding of both endogenous C/EBPalpha and ZTA protein to the ZTA promoter after lytic cycle induction but not during the latent state in EBV-infected Akata cells. Reporter assays revealed that cotransfected C/EBPalpha activated the ZTA promoter even more effectively than cotransfected ZTA. However, synergistic activation of the ZTA promoter was not observed when ZTA and C/EBPalpha were cotransfected together in either HeLa or DG75 cells. Mutagenesis of either the ZII or the ZIIIB sites in the ZTA promoter strongly reduced C/EBPalpha transactivation, suggesting that these sites act cooperatively. Furthermore, the introduction of exogenous C/EBPalpha into EBV-infected HeLa-BX1 cells induced endogenous ZTA mRNA and protein expression, as demonstrated by both reverse transcription-PCR and immunoblotting assays. Finally, double-label immunofluorescence assays suggested that EAD protein expression was activated even better than ZTA expression in latently infected C/EBPalpha-transfected Akata cells, perhaps because of the presence of a strong B-cell-specific repressed chromatin conformation on the ZTA promoter itself during EBV latency.

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The Epstein-Barr virus lytic cycle activator Zta interacts with methylated ZRE in the promoter of host target gene egr1.

Publications

The Epstein-Barr virus lytic cycle activator Zta interacts with methylated ZRE in the promoter of host target gene egr1.

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