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Journal of Virology, August 2003, p. 8893-8914, Vol. 77, No. 16
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.16.8893-8914.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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 {alpha} and p21CIP-1

Frederick Y. Wu,1,2 Shizhen Emily Wang,2 Qi-Qun Tang,3 Masahiro Fujimuro,2 Chuang-Jiun Chiou,1 Qizhi Zheng,2 Honglin Chen,2 S. Diane Hayward,1,2 M. Daniel Lane,3 and Gary S. Hayward1,2*

Molecular Virology Laboratories, Department of Pharmacology and Molecular Sciences,1 Viral Oncology Program, Sidney Kimmel Comprehensive Cancer Center,2 Department of Biological Chemistry The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231-10003

Received 30 January 2003/ Accepted 12 May 2003

Lytic-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/EBP{alpha} 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/EBP{alpha} 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 C/EBP{alpha}. Cotransfection of RAP with C/EBP{alpha} cooperatively stimulated both the C/EBP{alpha} 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/EBP{alpha}, but both the N-terminal and C-terminal domains were required for transcriptional augmentation. In vitro-translated RAP interfered with DNA binding by C/EBP{alpha} in electrophonetic mobility shift assay (EMSA) experiments but did not itself bind to the target C/EBP{alpha} 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/EBP{alpha} promoter in vivo, but only in a C/EBP{alpha}-dependent manner, implying an in vivo piggyback interaction with DNA-bound C/EBP{alpha}. Expression of exogenous RAP (Ad-RAP) caused G1/S cell cycle arrest in human dermal microvascular endothelial cells and also induced both the C/EBP{alpha} and p21 proteins, which formed punctate nuclear patterns that colocalized with RAP in PML nuclear bodies. In the presence of RAP, C/EBP{alpha} 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/EBP{alpha} 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/EBP{alpha} 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/EBP{alpha}-independent process.

* 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, August 2003, p. 8893-8914, Vol. 77, No. 16
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.16.8893-8914.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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