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Journal of Virology, January 2003, p. 1481-1500, Vol. 77, No. 2
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.2.1481-1500.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

CCAAT/Enhancer Binding Protein {alpha} Interacts with ZTA and Mediates ZTA-Induced p21CIP-1 Accumulation and G1 Cell Cycle Arrest during the Epstein-Barr Virus Lytic Cycle

Frederick Y. Wu,1,2 Honglin Chen,2 Shizhen Emily Wang,2 Collette M. J. apRhys,2 Gangling Liao,2 Masahiro Fujimuro,2 Christopher J. Farrell,1 Jian Huang,1 S. Diane Hayward,1,2 and Gary S. Hayward1,2*

Molecular Virology Laboratories, Department of Pharmacology and Molecular Sciences,1 Viral Oncology Program, The Sidney Kimmel Comprehensive Cancer Center, School of Medicine, The Johns Hopkins University, Baltimore, Maryland 21231-10002

Received 23 September 2002/ Accepted 27 September 2002

Cellular CCAAT/enhancer binding protein {alpha} (C/EBP{alpha}) promotes cellular differentiation and has antimitotic activities involving cell cycle arrest at G1/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/EBP{alpha} 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/EBP{alpha} 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/EBP{alpha} by coimmunoprecipitation in EBV-Akata cells and with DNA-bound C/EBP{alpha} 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/EBP{alpha} from degradation in a protein stability assay with a non-EBV-induced Akata cell proteasome extract. Furthermore, both C/EBP{alpha} and ZTA were found to specifically associate with the C/EBP{alpha} promoter in chromatin immunoprecipitation assays, but the interaction with ZTA appeared to be mediated by C/EBP{alpha} because it was abolished by clearing with anti-C/EBP{alpha} antibody. ZTA did not bind to or activate the C/EBP{alpha} promoter directly but cooperatively enhanced the positive autoregulation of the C/EBP{alpha} promoter by cotransfected C/EBP{alpha} 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/EBP{alpha}, ZTA enhanced the level of C/EBP{alpha} activation. This effect proved to require a previously unrecognized region in the proximal p21 promoter that contains three high-affinity C/EBP{alpha} binding sites. Finally, in C/EBP{alpha}-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/EBP{alpha} 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/EBP{alpha} leading to greatly enhanced C/EBP{alpha} and p21 levels through both transcriptional and posttranslational mechanisms.

* Corresponding author. Mailing address: CRB-3M08, 1650 Orleans St., Baltimore, MD 21231-1000. Phone: (410) 955-8684. Fax: (410) 955-8685. E-mail: ghayward{at}jhmi.edu.

Journal of Virology, January 2003, p. 1481-1500, Vol. 77, No. 2
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.2.1481-1500.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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