title: Role of CCAAT/enhancer-binding protein alpha (C/EBPalpha) 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.
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/EBPalpha) and p21(CIP-1) expression, resulting in G(0)/G(1) cell cycle arrest during the lytic cycle. RAP and C/EBPalpha 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/EBPalpha; this analysis showed that one of these sites has a very high affinity for C/EBPalpha. Luciferase (LUC) assays performed with a target RAP promoter-LUC reporter gene confirmed that C/EBPalpha 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/EBPalpha together resulted in a threefold increase in activity over that obtained with C/EBPalpha alone. Importantly, the introduction of exogenous Flag-tagged C/EBPalpha 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/EBPalpha 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/EBPalpha and RTA together cooperated to elevate RAP promoter activity four- to sixfold more than either alone. Furthermore, the addition of RAP, C/EBPalpha, and RTA in LUC reporter cotransfection assays resulted in 7- to 15-fold more activation than that seen with either C/EBPalpha or RTA alone. Site-specific mutational analysis of the RAP promoter showed that the strong C/EBP binding site is crucial for C/EBPalpha-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/EBPalpha 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/EBPalpha 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/EBPalpha 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/EBPalpha and RAP, as assayed both in vitro and by immunoprecipitation. Binding to C/EBPalpha 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/EBPalpha and DNA binding activities as well as all RTA transactivation and the cooperativity with C/EBPalpha. Therefore, we suggest that RTA transactivation of the RAP RRE is mediated by an interaction with DNA-bound C/EBPalpha but that full activity requires more than just the core C/EBP binding site.
authors.affiliation: Viral Oncology Program, Sidney Kimmel Comprehensive Cancer Center, School of Medicine, The Johns Hopkins University, Baltimore, Maryland 21231-1000, USA.
authors.forename: Shizhen Emily
authors.initials: SE
authors.lastname: Wang
authors.forename: Frederick Y
authors.initials: FY
authors.lastname: Wu
authors.forename: Masahiro
authors.initials: M
authors.lastname: Fujimuro
authors.forename: Jianchao
authors.initials: J
authors.lastname: Zong
authors.forename: S Diane
authors.initials: SD
authors.lastname: Hayward
authors.forename: Gary S
authors.initials: GS
authors.lastname: Hayward
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D001665:Binding Sites; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000502:physiology*; D002352:Carrier Proteins / Q000235:genetics* / Q000502:physiology; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D019288:Herpesvirus 8, Human / Q000235:genetics* / Q000502:physiology; D006801:Humans; D017874:Immediate-Early Proteins / Q000502:physiology*; D008223:Lymphoma / Q000821:virology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D012097:Repressor Proteins; D020218:Response Elements / Q000502:physiology; D015534:Trans-Activators / Q000502:physiology*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000235:genetics* / Q000502:physiology*; D014779:Virus Replication
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D022763:CCAAT-Enhancer-Binding Protein-alpha; D002352:Carrier Proteins; D017874:Immediate-Early Proteins; C118265:K8 protein, Human herpesvirus 8; D012333:RNA, Messenger; D012097:Repressor Proteins; C417604:Rta protein, Human herpesvirus 8; D015534:Trans-Activators; D014764:Viral Proteins; D004247:DNA