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Article: Cell cycle arrest by Kaposi's sarcoma-associated herpesvirus replication-associated protein is mediated at both the transcriptional and posttranslational levels by binding to CCAAT/enhancer-binding protein α and p21CIP-1

TitleCell cycle arrest by Kaposi's sarcoma-associated herpesvirus replication-associated protein is mediated at both the transcriptional and posttranslational levels by binding to CCAAT/enhancer-binding protein α and p21CIP-1
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. 16, p. 8893-8914 How to Cite?
AbstractLytic-cycle replication of Kaposi's sarcoma-associated herpesvirus (KSHV) in PEL cells causes G1 cell cycle arrest mediated by the virus-encoded replication-associated protein (RAP) (or K8 protein), which induces high-level expression of the cellular C/EBPα and p21 proteins. Here we have examined the mechanism of this induction at both the transcriptional and posttranslational levels. RAP proved to bind very efficiently to both C/EBPα and p21 and stabilized them by up to 10-fold from proteasome-mediated degradation in vitro. Cross-linking revealed that RAP itself forms stable dimers and tetramers in solution and forms higher-order complexes but not heterodimers with CC/EBPα. Cotransfection of RAP with C/EBPα cooperatively stimulated both the C/EBPα and p21 promoters in luciferase reporter gene assays. Only the basic/leucine zipper region of RAP was needed for interaction with and stabilization of C/EBPα, but both the N-terminal and C-terminal domains were required for transcriptional augmentation. In vitro-translated RAP interfered with DNA binding by C/EBPα in electrophonetic mobility shift assay (EMSA) experiments but did not itself bind to the target C/EBPα sites or form supershifted bands. However, in endogenous chromatin immunoprecipitation (ChIP) assays with tetradecanoyl phorbol acetate-induced PEL cells, RAP proved to specifically associate with the C/EBPα promoter in vivo, but only in a C/EBPα-dependent manner, implying an in vivo piggyback interaction with DNA-bound C/EBPα. Expression of exogenous RAP (Ad-RAP) caused G1/S cell cycle arrest in human dermal microvascular endothelial cells and also induced both the C/EBPα and p21 proteins, which formed punctate nuclear patterns that colocalized with RAP in PML nuclear bodies. In the presence of RAP, C/EBPα was also efficiently recruited into viral DNA replication compartments in both infected and cotransfected cells. In support of a direct role for this interaction in viral DNA replication, three C/EBPα binding sites were identified by in vitro EMSA experiments within a 220-bp core segment of the duplicated KSHV Ori-Lyt region, and although RAP did not bind to Ori-Lyt DNA directly in vitro, both endogenous RAP and C/EBPα were found to be associated with the Ori-Lyt region by ChIP assays in lytically induced PEL cells. Finally, we found that the KSHV lytic cycle could not be triggered by either synchronizing KSHV latently infected PEL cells in G1 phase or inducing p21 in a C/EBPα-independent process.
Persistent Identifierhttp://hdl.handle.net/10722/157365
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.authorWang, SEen_US
dc.contributor.authorTang, QQen_US
dc.contributor.authorFujimuro, Men_US
dc.contributor.authorChiou, CJen_US
dc.contributor.authorZheng, Qen_US
dc.contributor.authorChen, Hen_US
dc.contributor.authorHayward, SDen_US
dc.contributor.authorLane, MDen_US
dc.contributor.authorHayward, GSen_US
dc.date.accessioned2012-08-08T08:49:21Z-
dc.date.available2012-08-08T08:49:21Z-
dc.date.issued2003en_US
dc.identifier.citationJournal Of Virology, 2003, v. 77 n. 16, p. 8893-8914en_US
dc.identifier.issn0022-538Xen_US
dc.identifier.urihttp://hdl.handle.net/10722/157365-
dc.description.abstractLytic-cycle replication of Kaposi's sarcoma-associated herpesvirus (KSHV) in PEL cells causes G1 cell cycle arrest mediated by the virus-encoded replication-associated protein (RAP) (or K8 protein), which induces high-level expression of the cellular C/EBPα and p21 proteins. Here we have examined the mechanism of this induction at both the transcriptional and posttranslational levels. RAP proved to bind very efficiently to both C/EBPα and p21 and stabilized them by up to 10-fold from proteasome-mediated degradation in vitro. Cross-linking revealed that RAP itself forms stable dimers and tetramers in solution and forms higher-order complexes but not heterodimers with CC/EBPα. Cotransfection of RAP with C/EBPα cooperatively stimulated both the C/EBPα and p21 promoters in luciferase reporter gene assays. Only the basic/leucine zipper region of RAP was needed for interaction with and stabilization of C/EBPα, but both the N-terminal and C-terminal domains were required for transcriptional augmentation. In vitro-translated RAP interfered with DNA binding by C/EBPα in electrophonetic mobility shift assay (EMSA) experiments but did not itself bind to the target C/EBPα sites or form supershifted bands. However, in endogenous chromatin immunoprecipitation (ChIP) assays with tetradecanoyl phorbol acetate-induced PEL cells, RAP proved to specifically associate with the C/EBPα promoter in vivo, but only in a C/EBPα-dependent manner, implying an in vivo piggyback interaction with DNA-bound C/EBPα. Expression of exogenous RAP (Ad-RAP) caused G1/S cell cycle arrest in human dermal microvascular endothelial cells and also induced both the C/EBPα and p21 proteins, which formed punctate nuclear patterns that colocalized with RAP in PML nuclear bodies. In the presence of RAP, C/EBPα was also efficiently recruited into viral DNA replication compartments in both infected and cotransfected cells. In support of a direct role for this interaction in viral DNA replication, three C/EBPα binding sites were identified by in vitro EMSA experiments within a 220-bp core segment of the duplicated KSHV Ori-Lyt region, and although RAP did not bind to Ori-Lyt DNA directly in vitro, both endogenous RAP and C/EBPα were found to be associated with the Ori-Lyt region by ChIP assays in lytically induced PEL cells. Finally, we found that the KSHV lytic cycle could not be triggered by either synchronizing KSHV latently infected PEL cells in G1 phase or inducing p21 in a C/EBPα-independent process.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.meshBase Sequenceen_US
dc.subject.meshCcaat-Enhancer-Binding Protein-Alpha - Genetics - Metabolismen_US
dc.subject.meshCell Cycle - Physiologyen_US
dc.subject.meshCell Lineen_US
dc.subject.meshCyclin-Dependent Kinase Inhibitor P21en_US
dc.subject.meshCyclins - Metabolismen_US
dc.subject.meshDna Primersen_US
dc.subject.meshFluorescent Antibody Techniqueen_US
dc.subject.meshHerpesvirus 8, Human - Physiologyen_US
dc.subject.meshPromoter Regions, Geneticen_US
dc.subject.meshProtein Bindingen_US
dc.subject.meshProtein Processing, Post-Translational - Physiologyen_US
dc.subject.meshTranscription, Genetic - Physiologyen_US
dc.subject.meshViral Proteins - Physiologyen_US
dc.titleCell cycle arrest by Kaposi's sarcoma-associated herpesvirus replication-associated protein is mediated at both the transcriptional and posttranslational levels by binding to CCAAT/enhancer-binding protein α and p21CIP-1en_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.77.16.8893-8914.2003en_US
dc.identifier.pmid12885907-
dc.identifier.scopuseid_2-s2.0-0041488861en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0041488861&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume77en_US
dc.identifier.issue16en_US
dc.identifier.spage8893en_US
dc.identifier.epage8914en_US
dc.identifier.isiWOS:000184462800030-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridWu, FY=36991458700en_US
dc.identifier.scopusauthoridWang, SE=8765314100en_US
dc.identifier.scopusauthoridTang, QQ=7201631934en_US
dc.identifier.scopusauthoridFujimuro, M=6603781101en_US
dc.identifier.scopusauthoridChiou, CJ=7202682681en_US
dc.identifier.scopusauthoridZheng, Q=8679121500en_US
dc.identifier.scopusauthoridChen, H=26643315400en_US
dc.identifier.scopusauthoridHayward, SD=7102776214en_US
dc.identifier.scopusauthoridLane, MD=7401977437en_US
dc.identifier.scopusauthoridHayward, GS=7101602499en_US

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