Role of CCAAT/enhancer-binding protein alpha (C/EBPα) 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, Frederick Y. Wu, Masahiro Fujimuro, Jianchao Zong, S. Diane Hayward, Gary S. Hayward

Research output: Contribution to journalArticlepeer-review

Abstract

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/EBPαs) and p21CIP-1 expression, resulting in G0/G1 cell cycle arrest during the lytic cycle. RAP and C/EBPα 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/EBPα; this analysis showed that one of these sites has a very high affinity for C/EBPα. Luciferase (LUC) assays performed with a target RAP promoter-LUC reporter gene confirmed that C/EBPα 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/EBPα together resulted in a threefold increase in activity over that obtained with C/EBPα alone. Importantly, the introduction of exogenous Flag-tagged C/EBPα 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/EBPα 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/EBPα and RTA together cooperated to elevate RAP promoter activity four- to sixfold more than either alone. Furthermore, the addition of RAP, C/EBPα, and RTA in LUC reporter cotransfection assays resulted in 7- to 15-fold more activation than that seen with either C/EBPα or RTA alone. Site-specific mutational analysis of the RAP promoter showed that the strong C/EBP binding site is crucial for C/EBPα-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/EBPα 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/EBPα 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/EBPα 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/EBPα and RAP, as assayed both in vitro and by immunoprecipitation. Binding to C/EBPα 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/EBPα and DNA binding activities as well as all RTA transactivation and the cooperativity with C/EBPα. Therefore, we suggest that RTA transactivation of the RAP RRE is mediated by an interaction with DNA-bound C/EBPα but that full activity requires more than just the core C/EBP binding site.

Original languageEnglish (US)
Pages (from-to)600-623
Number of pages24
JournalJournal of virology
Volume77
Issue number1
DOIs
StatePublished - Jan 2003

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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