This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, S. E.
Right arrow Articles by Hayward, G. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, S. E.
Right arrow Articles by Hayward, G. S.

 Previous Article  |  Next Article 

Journal of Virology, January 2003, p. 600-623, Vol. 77, No. 1
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.1.600-623.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Role of CCAAT/Enhancer-Binding Protein Alpha (C/EBP{alpha}) in Activation of the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Lytic-Cycle Replication-Associated Protein (RAP) Promoter in Cooperation with the KSHV Replication and Transcription Activator (RTA) and RAP

Shizhen Emily Wang,1 Frederick Y. Wu,1,2 Masahiro Fujimuro,1 Jianchao Zong,1 S. Diane Hayward,1,2 and Gary S. Hayward1,2*

Molecular Virology Laboratories, Department of Pharmacology and Molecular Sciences,2 Viral Oncology Program, Sidney Kimmel Comprehensive Cancer Center, School of Medicine, The Johns Hopkins University, Baltimore, Maryland 21231-10001

Received 28 June 2002/ Accepted 20 September 2002

The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded replication-associated protein (RAP, or K8) has been shown to induce both CCAAT/enhancer binding protein alpha (C/EBP{alpha}) and p21CIP-1 expression, resulting in G0/G1 cell cycle arrest during the lytic cycle. RAP and C/EBP{alpha} are also known to interact strongly both in vitro and in lytically infected cells. We recognized two potential consensus C/EBP binding sites in the RAP promoter and performed electrophoretic mobility shift assay (EMSA) analysis with in vitro-translated C/EBP{alpha}; this analysis showed that one of these sites has a very high affinity for C/EBP{alpha}. Luciferase (LUC) assays performed with a target RAP promoter-LUC reporter gene confirmed that C/EBP{alpha} can transcriptionally activate the RAP promoter up to 50-fold. Although RAP had no effect on its own promoter by itself, the addition of RAP and C/EBP{alpha} together resulted in a threefold increase in activity over that obtained with C/EBP{alpha} alone. Importantly, the introduction of exogenous Flag-tagged C/EBP{alpha} triggered RAP expression in BCBL-1 cells latently infected with KSHV, as detected by both reverse transcription-PCR and double-label immunofluorescence assay analyses, suggesting the presence of a self-reinforcing loop with C/EBP{alpha} and RAP activating each other. The RAP promoter can also be activated 50- to 120-fold by the KSHV lytic-cycle-triggering protein known as replication and transcription activator (RTA). C/EBP{alpha} and RTA together cooperated to elevate RAP promoter activity four- to sixfold more than either alone. Furthermore, the addition of RAP, C/EBP{alpha}, and RTA in LUC reporter cotransfection assays resulted in 7- to 15-fold more activation than that seen with either C/EBP{alpha} or RTA alone. Site-specific mutational analysis of the RAP promoter showed that the strong C/EBP binding site is crucial for C/EBP{alpha}-mediated transactivation of the RAP promoter. However, the C/EBP binding site also overlaps the previously reported 16-bp RTA-responsive element (RRE), and the same mutation also both reduced RTA-mediated transactivation and abolished the cooperativity between C/EBP{alpha} and RTA. Furthermore, in vitro-translated RTA, although capable of binding directly to the polyadenylated nuclear RNA (PAN) RRE motif, failed to bind to the RAP RRE and interfered with RRE-bound C/EBP{alpha} in EMSA experiments. Partial RTA responsiveness but no cooperativity could be transferred to a heterologous promoter containing added consensus C/EBP binding sites. A chromatin immunoprecipitation assay showed that all three proteins associated specifically with RAP promoter DNA in vivo and that, when C/EBP{alpha} was removed from a tetradecanoyl phorbol acetate-treated JSC-1 primary effusion lymphoma cell lysate, the levels of association of RTA and RAP with the RAP promoter were reduced 3- and 13-fold, respectively. Finally, RTA also proved to physically interact with both C/EBP{alpha} and RAP, as assayed both in vitro and by immunoprecipitation. Binding to C/EBP{alpha} occurred within the N-terminal DNA binding domain of RTA, and deletion of a 17-amino-acid basic motif of RTA abolished both the C/EBP{alpha} and DNA binding activities as well as all RTA transactivation and the cooperativity with C/EBP{alpha}. Therefore, we suggest that RTA transactivation of the RAP RRE is mediated by an interaction with DNA-bound C/EBP{alpha} but that full activity requires more than just the core C/EBP binding site.

* 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. 600-623, Vol. 77, No. 1
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.1.600-623.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Jeong, J. H., Orvis, J., Kim, J. W., McMurtrey, C. P., Renne, R., Dittmer, D. P. (2004). Regulation and Autoregulation of the Promoter for the Latency-associated Nuclear Antigen of Kaposi's Sarcoma-associated Herpesvirus. J. Biol. Chem. 279: 16822-16831 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»