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      Essential role of RBP-Jkappa in activation of the K8 delayed-early promoter of Kaposi's sarcoma-associated herpesvirus by ORF50/RTA.

      Biology
      Animals, Basic-Leucine Zipper Transcription Factors, genetics, Binding Sites, Cell Line, DNA-Binding Proteins, metabolism, physiology, Gene Expression Regulation, Viral, Herpesvirus 8, Human, Humans, Immediate-Early Proteins, Immunoglobulin J Recombination Signal Sequence-Binding Protein, Mice, Open Reading Frames, Point Mutation, Promoter Regions, Genetic, Repressor Proteins, Sequence Deletion, Trans-Activators, Viral Proteins, Virus Activation

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          Abstract

          KSHV K8 gene is activated by virally encoded transactivator RTA in delayed-early stage of viral reactivation. Three RTA-responsive elements (RREs) were identified in the promoter. Among them, RRE-II was found to be the most critical cis-acting element for RTA transactivation. In this report, the mechanism underlying RTA-mediated activation of the K8 delayed-early promoter was investigated. A DNA affinity purification study demonstrated that RRE-II was bound by cellular protein RBP-Jkappa, a sequence-specific DNA binding protein and a primary target of the Notch signaling pathway. Inspection of the RRE-II sequence revealed a potential recognition sequence for RBP-Jkappa (GTGAGAA) between the nucleotides -102 and -108 relative to the transcription initial site. Removal or mutation of the motif abolished RBP-Jkappa binding to the K8 promoter and as a consequence, RTA failed to bind to and activate the promoter. An essential role of RBP-Jkappa in the transcription of the K8 promoter was demonstrated by diminishment of the promoter activity in RBP-Jkappa-null murine embryonic fibroblasts. Taken together, RTA activates the K8 promoter through an indirect binding mechanism, i.e. being recruited to the K8 promoter through interaction with RBP-Jkappa bound to an RBP-Jkappa motif in the promoter.

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