CCAAT/Enhancer Binding Protein α Binds to the Epstein-Barr Virus (EBV) ZTA Protein through Oligomeric Interactions and Contributes to Cooperative Transcriptional Activation of the ZTA Promoter through Direct Binding to the ZII and ZIIIB Motifs during Induction of the EBV Lytic Cycle

Frederick Y. Wu, Shizhen Emily Wang, Honglin Chen, Ling Wang, S. Diane Hayward, Gary Selwyn Hayward

Research output: Contribution to journalArticle

Abstract

The Epstein-Barr virus (EBV)-encoded ZTA protein interacts strongly with and stabilizes the cellular CCAAT/enhancer binding protein α (C/EBPα), leading to the induction of p21-mediated G1 cell cycle arrest. Despite the strong interaction between these two basic leucine zipper (bZIP) family proteins, the ZTA and C/EBPα subunits do not heterodimerize, as indicated by an in vitro cross-linking assay with in vitro-cotranslated 35S-labeled C/EBPα and 35S-labeled ZTA protein. Instead, they evidently form a higher-order oligomeric complex that competes with C/EBPα 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/EBPα. 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/EBPα 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/EBPα activated the ZTA promoter even more effectively than cotransfected ZTA. However, synergistic activation of the ZTA promoter was not observed when ZTA and C/EBPα were cotransfected together in either HeLa or DG75 cells. Mutagenesis of either the ZII or the ZIlIB sites in the ZTA promoter strongly reduced C/EBPα transactivation, suggesting that these sites act cooperatively. Furthermore, the introduction of exogenous C/EBPα 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/EBPα-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.

Original languageEnglish (US)
Pages (from-to)4847-4865
Number of pages19
JournalJournal of Virology
Volume78
Issue number9
DOIs
StatePublished - May 2004

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CCAAT-Enhancer-Binding Proteins
Human herpesvirus 4
transcriptional activation
Human Herpesvirus 4
Transcriptional Activation
cooperatives
binding proteins
promoter regions
Proteins
assays
proteins
Leucine Zippers
leucine zipper
protein binding
Electrophoretic Mobility Shift Assay
Protein Binding
chromatin
protein synthesis
cells
Virus Latency

ASJC Scopus subject areas

  • Immunology

Cite this

@article{7c1dcb71bfbd4fde878fa16a8a6af862,
title = "CCAAT/Enhancer Binding Protein α Binds to the Epstein-Barr Virus (EBV) ZTA Protein through Oligomeric Interactions and Contributes to Cooperative Transcriptional Activation of the ZTA Promoter through Direct Binding to the ZII and ZIIIB Motifs during Induction of the EBV Lytic Cycle",
abstract = "The Epstein-Barr virus (EBV)-encoded ZTA protein interacts strongly with and stabilizes the cellular CCAAT/enhancer binding protein α (C/EBPα), leading to the induction of p21-mediated G1 cell cycle arrest. Despite the strong interaction between these two basic leucine zipper (bZIP) family proteins, the ZTA and C/EBPα subunits do not heterodimerize, as indicated by an in vitro cross-linking assay with in vitro-cotranslated 35S-labeled C/EBPα and 35S-labeled ZTA protein. Instead, they evidently form a higher-order oligomeric complex that competes with C/EBPα 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/EBPα. 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/EBPα 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/EBPα activated the ZTA promoter even more effectively than cotransfected ZTA. However, synergistic activation of the ZTA promoter was not observed when ZTA and C/EBPα were cotransfected together in either HeLa or DG75 cells. Mutagenesis of either the ZII or the ZIlIB sites in the ZTA promoter strongly reduced C/EBPα transactivation, suggesting that these sites act cooperatively. Furthermore, the introduction of exogenous C/EBPα 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/EBPα-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.",
author = "Wu, {Frederick Y.} and Wang, {Shizhen Emily} and Honglin Chen and Ling Wang and Hayward, {S. Diane} and Hayward, {Gary Selwyn}",
year = "2004",
month = "5",
doi = "10.1128/JVI.78.9.4847-4865.2004",
language = "English (US)",
volume = "78",
pages = "4847--4865",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "9",

}

TY - JOUR

T1 - CCAAT/Enhancer Binding Protein α Binds to the Epstein-Barr Virus (EBV) ZTA Protein through Oligomeric Interactions and Contributes to Cooperative Transcriptional Activation of the ZTA Promoter through Direct Binding to the ZII and ZIIIB Motifs during Induction of the EBV Lytic Cycle

AU - Wu, Frederick Y.

AU - Wang, Shizhen Emily

AU - Chen, Honglin

AU - Wang, Ling

AU - Hayward, S. Diane

AU - Hayward, Gary Selwyn

PY - 2004/5

Y1 - 2004/5

N2 - The Epstein-Barr virus (EBV)-encoded ZTA protein interacts strongly with and stabilizes the cellular CCAAT/enhancer binding protein α (C/EBPα), leading to the induction of p21-mediated G1 cell cycle arrest. Despite the strong interaction between these two basic leucine zipper (bZIP) family proteins, the ZTA and C/EBPα subunits do not heterodimerize, as indicated by an in vitro cross-linking assay with in vitro-cotranslated 35S-labeled C/EBPα and 35S-labeled ZTA protein. Instead, they evidently form a higher-order oligomeric complex that competes with C/EBPα 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/EBPα. 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/EBPα 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/EBPα activated the ZTA promoter even more effectively than cotransfected ZTA. However, synergistic activation of the ZTA promoter was not observed when ZTA and C/EBPα were cotransfected together in either HeLa or DG75 cells. Mutagenesis of either the ZII or the ZIlIB sites in the ZTA promoter strongly reduced C/EBPα transactivation, suggesting that these sites act cooperatively. Furthermore, the introduction of exogenous C/EBPα 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/EBPα-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.

AB - The Epstein-Barr virus (EBV)-encoded ZTA protein interacts strongly with and stabilizes the cellular CCAAT/enhancer binding protein α (C/EBPα), leading to the induction of p21-mediated G1 cell cycle arrest. Despite the strong interaction between these two basic leucine zipper (bZIP) family proteins, the ZTA and C/EBPα subunits do not heterodimerize, as indicated by an in vitro cross-linking assay with in vitro-cotranslated 35S-labeled C/EBPα and 35S-labeled ZTA protein. Instead, they evidently form a higher-order oligomeric complex that competes with C/EBPα 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/EBPα. 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/EBPα 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/EBPα activated the ZTA promoter even more effectively than cotransfected ZTA. However, synergistic activation of the ZTA promoter was not observed when ZTA and C/EBPα were cotransfected together in either HeLa or DG75 cells. Mutagenesis of either the ZII or the ZIlIB sites in the ZTA promoter strongly reduced C/EBPα transactivation, suggesting that these sites act cooperatively. Furthermore, the introduction of exogenous C/EBPα 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/EBPα-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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U2 - 10.1128/JVI.78.9.4847-4865.2004

DO - 10.1128/JVI.78.9.4847-4865.2004

M3 - Article

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JO - Journal of Virology

JF - Journal of Virology

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