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Article: CCAAT/enhancer binding protein α interacts with ZTA and mediates ZTA-induced p21CIP-1 accumulation and G1 cell cycle arrest during the Epstein-Barr virus lytic cycle

TitleCCAAT/enhancer binding protein α interacts with ZTA and mediates ZTA-induced p21CIP-1 accumulation and G1 cell cycle arrest during the Epstein-Barr virus lytic cycle
Authors
Issue Date2003
PublisherAmerican Society for Microbiology. The Journal's web site is located at http://jvi.asm.org/
Citation
Journal Of Virology, 2003, v. 77 n. 2, p. 1481-1500 How to Cite?
AbstractCellular CCAAT/enhancer binding protein α (C/EBPα) promotes cellular differentiation and has antimitotic activities involving cell cycle arrest at GI/S through stabilization of p21CIP-1/WAF1 and through transcriptional activation of the p21 promoter. The Epstein-Barr virus lytic-cycle transactivator protein ZTA is known to arrest the host cell cycle at G1/S via a p53-independent p21 pathway, but the detailed molecular mechanisms involved have not been defined. To further evaluate the role of ZTA in cell cycle arrest, we constructed a recombinant adenovirus vector expressing ZTA (Ad-ZTA), whose level of expression at a low multiplicity of infection in normal human diploid fibroblast (HF) cells was lower than or equal to the physiological level seen in Akata cells lytically induced by EBV (EBV-Akata cells). Fluorescence-activated cell sorting analysis of HF cells infected with Ad-ZTA confirmed that G1/S cell cycle arrest occurred in the majority of ZTA-positive cells, but not with an adenovirus vector expressing green fluorescent protein. Double-label immunofluorescence assays (IFA) performed with Ad-ZTA-infected HF cells revealed that only ZTA-positive cells induced the expression of both endogenous C/EBPα and p21 and blocked the progression into S phase, as detected by a lack of incorporation of bromodeoxyuridine. The stimulation of endogenous ZTA protein expression either through treatment with tetradecanoyl phorbol acetate in D98/HR1 cells or through B-cell receptor cross-linking with anti-immunoglobulin G antibody in EBV-Akata cells also coincided with the induction of both C/EBPα and p21 and their mRNAs, as assayed by Northern blot, Western blot, and IFA experiments. Mechanistically, the ZTA protein proved to directly interact with C/EBPα by coimmunoprecipitation in EBV-Akata cells and with DNA-bound C/EBPα in electrophoretic mobility shift assay experiments, and the in vitro interaction domain encompassed the basic leucine zipper domain of ZTA. ZTA also specifically protected C/EBPα from degradation in a protein stability assay with a non-EBV-induced Akata cell proteasome extract. Furthermore, both C/EBPα and ZTA were found to specifically associate with the C/EBPα promoter in chromatin immunoprecipitation assays, but the interaction with ZTA appeared to be mediated by C/EBPα because it was abolished by clearing with anti-C/EBPα antibody. ZTA did not bind to or activate the C/EBPα promoter directly but cooperatively enhanced the positive autoregulation of the C/EBPα promoter by cotransfected C/EBPα in transient luciferase reporter gene assays with Vero and HeLa cells as well as with DG75 B lymphocytes. Similarly, ZTA alone had little effect on the p21 promoter in transient reporter gene assays, but in the presence of cotransfected C/EBPα, ZTA enhanced the level of C/EBPα activation. This effect proved to require a previously unrecognized region in the proximal p21 promoter that contains three high-affinity C/EBPα binding sites. Finally, in C/EBPα-deficient mouse embryonic fibroblasts (MEF), Ad-ZTA was unable to induce either p21 or G1 arrest, whereas it was able to induce both in wild-type MEF. Overall, we conclude that C/EBPα is essential for at least one pathway of ZTA-induced G1 arrest during EBV lytic-cycle DNA replication and that this process involves a physical piggyback interaction between ZTA and C/EBPα leading to greatly enhanced C/EBPα and p21 levels through both transcriptional and posttranslational mechanisms.
Persistent Identifierhttp://hdl.handle.net/10722/157351
ISSN
2015 Impact Factor: 4.606
2015 SCImago Journal Rankings: 3.347
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorWu, FYen_US
dc.contributor.authorChen, Hen_US
dc.contributor.authorWang, SEen_US
dc.contributor.authorAprhys, CMJen_US
dc.contributor.authorLiao, Gen_US
dc.contributor.authorFujimuro, Men_US
dc.contributor.authorFarrell, CJen_US
dc.contributor.authorHuang, Jen_US
dc.contributor.authorHayward, SDen_US
dc.contributor.authorHayward, GSen_US
dc.date.accessioned2012-08-08T08:49:09Z-
dc.date.available2012-08-08T08:49:09Z-
dc.date.issued2003en_US
dc.identifier.citationJournal Of Virology, 2003, v. 77 n. 2, p. 1481-1500en_US
dc.identifier.issn0022-538Xen_US
dc.identifier.urihttp://hdl.handle.net/10722/157351-
dc.description.abstractCellular CCAAT/enhancer binding protein α (C/EBPα) promotes cellular differentiation and has antimitotic activities involving cell cycle arrest at GI/S through stabilization of p21CIP-1/WAF1 and through transcriptional activation of the p21 promoter. The Epstein-Barr virus lytic-cycle transactivator protein ZTA is known to arrest the host cell cycle at G1/S via a p53-independent p21 pathway, but the detailed molecular mechanisms involved have not been defined. To further evaluate the role of ZTA in cell cycle arrest, we constructed a recombinant adenovirus vector expressing ZTA (Ad-ZTA), whose level of expression at a low multiplicity of infection in normal human diploid fibroblast (HF) cells was lower than or equal to the physiological level seen in Akata cells lytically induced by EBV (EBV-Akata cells). Fluorescence-activated cell sorting analysis of HF cells infected with Ad-ZTA confirmed that G1/S cell cycle arrest occurred in the majority of ZTA-positive cells, but not with an adenovirus vector expressing green fluorescent protein. Double-label immunofluorescence assays (IFA) performed with Ad-ZTA-infected HF cells revealed that only ZTA-positive cells induced the expression of both endogenous C/EBPα and p21 and blocked the progression into S phase, as detected by a lack of incorporation of bromodeoxyuridine. The stimulation of endogenous ZTA protein expression either through treatment with tetradecanoyl phorbol acetate in D98/HR1 cells or through B-cell receptor cross-linking with anti-immunoglobulin G antibody in EBV-Akata cells also coincided with the induction of both C/EBPα and p21 and their mRNAs, as assayed by Northern blot, Western blot, and IFA experiments. Mechanistically, the ZTA protein proved to directly interact with C/EBPα by coimmunoprecipitation in EBV-Akata cells and with DNA-bound C/EBPα in electrophoretic mobility shift assay experiments, and the in vitro interaction domain encompassed the basic leucine zipper domain of ZTA. ZTA also specifically protected C/EBPα from degradation in a protein stability assay with a non-EBV-induced Akata cell proteasome extract. Furthermore, both C/EBPα and ZTA were found to specifically associate with the C/EBPα promoter in chromatin immunoprecipitation assays, but the interaction with ZTA appeared to be mediated by C/EBPα because it was abolished by clearing with anti-C/EBPα antibody. ZTA did not bind to or activate the C/EBPα promoter directly but cooperatively enhanced the positive autoregulation of the C/EBPα promoter by cotransfected C/EBPα in transient luciferase reporter gene assays with Vero and HeLa cells as well as with DG75 B lymphocytes. Similarly, ZTA alone had little effect on the p21 promoter in transient reporter gene assays, but in the presence of cotransfected C/EBPα, ZTA enhanced the level of C/EBPα activation. This effect proved to require a previously unrecognized region in the proximal p21 promoter that contains three high-affinity C/EBPα binding sites. Finally, in C/EBPα-deficient mouse embryonic fibroblasts (MEF), Ad-ZTA was unable to induce either p21 or G1 arrest, whereas it was able to induce both in wild-type MEF. Overall, we conclude that C/EBPα is essential for at least one pathway of ZTA-induced G1 arrest during EBV lytic-cycle DNA replication and that this process involves a physical piggyback interaction between ZTA and C/EBPα leading to greatly enhanced C/EBPα and p21 levels through both transcriptional and posttranslational mechanisms.en_US
dc.languageengen_US
dc.publisherAmerican Society for Microbiology. The Journal's web site is located at http://jvi.asm.org/en_US
dc.relation.ispartofJournal of Virologyen_US
dc.subject.meshAdenoviridae - Geneticsen_US
dc.subject.meshCcaat-Enhancer-Binding Protein-Alpha - Metabolismen_US
dc.subject.meshCyclin-Dependent Kinase Inhibitor P21en_US
dc.subject.meshCyclins - Genetics - Metabolismen_US
dc.subject.meshDna-Binding Proteins - Metabolismen_US
dc.subject.meshElectrophoretic Mobility Shift Assayen_US
dc.subject.meshFluorescent Antibody Technique, Indirecten_US
dc.subject.meshG1 Phaseen_US
dc.subject.meshGenes, Reporteren_US
dc.subject.meshGenetic Vectorsen_US
dc.subject.meshHerpesvirus 4, Human - Physiologyen_US
dc.subject.meshPromoter Regions, Geneticen_US
dc.subject.meshProtein Bindingen_US
dc.subject.meshS Phaseen_US
dc.subject.meshTrans-Activators - Metabolismen_US
dc.subject.meshViral Proteinsen_US
dc.titleCCAAT/enhancer binding protein α interacts with ZTA and mediates ZTA-induced p21CIP-1 accumulation and G1 cell cycle arrest during the Epstein-Barr virus lytic cycleen_US
dc.typeArticleen_US
dc.identifier.emailChen, H:hlchen@hkucc.hku.hken_US
dc.identifier.authorityChen, H=rp00383en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1128/JVI.77.2.1481-1500.2003en_US
dc.identifier.pmid12502863-
dc.identifier.scopuseid_2-s2.0-0037223721en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0037223721&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume77en_US
dc.identifier.issue2en_US
dc.identifier.spage1481en_US
dc.identifier.epage1500en_US
dc.identifier.isiWOS:000180166600067-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridWu, FY=36991458700en_US
dc.identifier.scopusauthoridChen, H=26643315400en_US
dc.identifier.scopusauthoridWang, SE=8765314100en_US
dc.identifier.scopusauthoridApRhys, CMJ=6506181422en_US
dc.identifier.scopusauthoridLiao, G=7102949054en_US
dc.identifier.scopusauthoridFujimuro, M=6603781101en_US
dc.identifier.scopusauthoridFarrell, CJ=7102327845en_US
dc.identifier.scopusauthoridHuang, J=34770766800en_US
dc.identifier.scopusauthoridHayward, SD=7102776214en_US
dc.identifier.scopusauthoridHayward, GS=7101602499en_US

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