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Journal of Virology, September 2003, p. 9590-9612, Vol. 77, No. 17
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.17.9590-9612.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

CCAAT/Enhancer-Binding Protein-{alpha} Is Induced during the Early Stages of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Lytic Cycle Reactivation and Together with the KSHV Replication and Transcription Activator (RTA) Cooperatively Stimulates the Viral RTA, MTA, and PAN Promoters

Shizhen Emily Wang,1 Frederick Y. Wu,2,{dagger} Yanxing Yu,1 and Gary S. Hayward1,2*

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

Received 30 January 2003/ Accepted 3 June 2003

During the immediate-early (IE) phase of reactivation from latency, the Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator protein (RTA) (or ORF50) is thought to be the most critical trigger that upregulates expression of many downstream viral lytic cycle genes, including the delayed-early (DE) gene encoding the replication-associated protein (RAP) (or K8). RAP physically interacts with and stabilizes the cellular transcription factor CCAAT/enhancer-binding protein-{alpha} (C/EBP{alpha}), leading to upregulated expression of the cellular C/EBP{alpha} and p21CIP-1 proteins followed by G0/G1 cell cycle arrest. Furthermore, RTA also interacts with C/EBP{alpha}, and both RAP and RTA cooperate with C/EBP{alpha} to activate the RAP promoter through binding to a strong proximal C/EBP binding site that also serves as an RTA-responsive element (RRE). Here we show that C/EBP{alpha} also activates the IE RTA promoter in transient-cotransfection reporter gene assays and that addition of either RTA or RAP enhances the effect. Electrophoretic mobility shift assay and deletion analysis revealed three C/EBP binding sites that mediate cooperative transactivation of the RTA promoter by C/EBP{alpha} and RTA. Furthermore, chromatin immunoprecipitation assay results showed that the endogenous C/EBP{alpha}, RTA, and RAP proteins all associate with RTA promoter sequences in tetradecanoyl phorbol acetate-induced primary effusion lymphoma (PEL) cells. Induction of endogenous KSHV RTA mRNA in PEL cells by exogenously introduced C/EBP{alpha} was confirmed by reverse transcription-PCR analysis and by double-label indirect immunofluorescence assays. Reciprocally, expression of exogenous RTA also led to an increase of endogenous C/EBP{alpha} expression that could be detected by Western immunoblot assays even in KSHV-negative DG75 cells. Cotransfected RTA also increased positive C/EBP{alpha} autoregulation of the C/EBP{alpha} promoter in transient-cotransfection reporter gene assays. Finally, C/EBP{alpha} proved to strongly activate the promoters of two other KSHV DE genes encoding PAN (polyadenylated nuclear) RNA and MTA (ORF57), which was again mediated by C/EBP binding sites that also contribute to RTA activation. Overall, these results support a model in which the cellular transcription factor C/EBP{alpha} and RTA:C/EBP{alpha} interactions play important roles both upstream and downstream of the two major KSHV regulatory proteins RTA and RAP during the early stages of lytic cycle reactivation.

* 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.

{dagger} Present address: Viral Oncology Program, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, Md.

Journal of Virology, September 2003, p. 9590-9612, Vol. 77, No. 17
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.17.9590-9612.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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