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...

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/EBPalpha), leading to upregulated expression of the cellular C/EBPalpha and p21(CIP-1) proteins followed by G(0)/G(1) cell cycle arrest. Furthermore, RTA also interacts with C/EBPalpha, and both RAP and RTA cooperate with C/EBPalpha 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/EBPalpha 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/EBPalpha and RTA. Furthermore, chromatin immunoprecipitation assay results showed that the endogenous C/EBPalpha, 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/EBPalpha 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/EBPalpha expression that could be detected by Western immunoblot assays even in KSHV-negative DG75 cells. Cotransfected RTA also increased positive C/EBPalpha autoregulation of the C/EBPalpha promoter in transient-cotransfection reporter gene assays. Finally, C/EBPalpha 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/EBPalpha and RTA:C/EBPalpha 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.