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CCAAT/Enhancer Binding Protein α Interacts with ZTA and Mediates ZTA-Induced p21(CIP-1) Accumulation and G(1) Cell Cycle Arrest during the Epstein-Barr Virus Lytic Cycle

By Frederick Y. Wu, Honglin Chen, Shizhen Emily Wang, Collette M. J. apRhys, Gangling Liao, Masahiro Fujimuro, Christopher J. Farrell, Jian Huang, S. Diane Hayward and Gary S. Hayward

Abstract

Cellular CCAAT/enhancer binding protein α (C/EBPα) promotes cellular differentiation and has antimitotic activities involving cell cycle arrest at G(1)/S through stabilization of p21(CIP-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 G(1)/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 G(1)/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 G(1) 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 G(1) 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

Topics: Virus-Cell Interactions
Publisher: American Society for Microbiology
Year: 2003
DOI identifier: 10.1128/JVI.77.2.1481-1500.2003
OAI identifier: oai:pubmedcentral.nih.gov:140856
Provided by: PubMed Central
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