RBP-J (CSL) is essential for activation of the K14/vGPCR promoter of Kaposi's sarcoma-associated herpesvirus by the lytic switch protein RTA.
The Kaposi's sarcoma-associated herpesvirus (KSHV) gene product virally encoded G protein-coupled receptor (vGPCR) is a homolog of cellular GPCRs and has been proposed to play important roles in KSHV-induced angiogenesis. The most abundant vGPCR-containing transcripts are K14/vGPCR bicistronic RNAs that are strongly induced during lytic reactivation. Here we show that the promoter governing this transcript is strongly responsive to activation by the viral lytic switch protein RTA. By deletion mapping and scanning mutation analyses, we have identified three putative RTA response elements (A, B, and C) in this promoter. However, none of these sites appear to directly bind RTA in electrophoretic mobility shift assays (EMSA). Site C corresponds to a canonical binding site for RBP-J, a sequence-specific transcriptional repressor that is normally the target of Notch signaling. RBP-J can bind RTA and recruit it to its cognate recognition site; when this happens, the activation function of RTA can relieve RBP-J-mediated repression and upregulate expression of the targeted gene. EMSA studies reveal that both sites A and C can bind to RBP-J; sequence inspection reveals that site A is a novel functional variant of known RBP-J recognition sites. (Site B corresponds to an as-yet-unknown host DNA-binding protein.) The importance of sites A and C in vivo is underscored by the observation that K14/vGPCR promoter function is dramatically inhibited in cells genetically deficient in RBP-J. The regulation of K14/vGPCR transcripts by RBP-J raises the possibility that other modulators of Notch signaling might be able to induce expression of this RNA outside the context of lytic KSHV replication.
Liang
Yuying
Y
Howard Hughes Medical Institute and Department of Microbiology, University of California Medical Center, San Francisco, 94143-0414, USA. yliang5@emory.edu
Ganem
Don
D
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism*; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D009687:Nuclear Proteins; C497570:RBPJ protein, human; D043562:Receptors, G-Protein-Coupled; C417604:Rta protein, Human herpesvirus 8; D015534:Trans-Activators; D014764:Viral Proteins