Transcription factor NF-B binding sites in the CMV promoter as an inhibitor of HIV replication
Nejat Düzgüneş: 0000-0001-6159-1391
32nd Annual Meeting of the American Association for Dental Research
San Antonio, TX
March 12-15, 2003
Date of Presentation
Journal of Dental Research
Journal Volume Number
82 (special issue A)
Objectives: The cellular transcription factor NF-kB is a major regulator of transcriptional activity at the human immunodeficiency virus (HIV) 5’- long terminal repeat promoter (LTR). This suggests NF-kB as a potential target for anti-HIV therapy. We are exploring the use of the Cytomegalovirus (CMV) promoter element as a DNA decoy for cellular transcription factors including NF-kB. Structural analysis of the CMV promoter element using the transfac database demonstrates the presence of three NF-kB-binding consensus sequences, suggesting that the CMV promoter may sequester sufficient NF-kB to interfere with transcriptional activation at the HIV LTR promoter. Methods: To evaluate the possibility of NF-kB-specific competition between the CMV and LTR promoter elements, we co-transfected HeLa cells with the HIV pro-viral clone HXBDBgl, and effector plasmids containing: 1) CMV promoter, 2) RSV promoter, 3) SV-40 promoter, SV-40 enhancer, and SV-40 promoter/enhancer complex, 4) anti-NF-kB ribozyme under the transcriptional regulation of the CMV promoter. p24 viral protein concentration in the supernatant was assayed using ELISA to determine expression levels of HXBDBgl. We also evaluated these effectors using an LTR-luciferase reporter plasmid, to validate that their effect was mediated at the LTR promoter, rather than by post-transcriptional regulation of p24. Results: CMV-promoter containing plasmids reduced LTR-mediated expression of p24 and luciferase by 10-fold and 5-fold respectively, when compared to an RSV promoter-containing plasmid. Whether the anti-NF-kB ribozyme generated an additive effect beyond the reduction in HIV replication mediated by the CMV promoter was not evident. Conclusions: Inhibition of HIV replication by the CMV promoter is mediated at the HIV LTR. Studies on the generation of a mutant plasmid lacking the NF-kB consensus sequence in the CMV promoter, and an anti-NF-kB ribozyme expressing plasmid under the transcriptional regulation of the RSV promoter, are in progress. This work was supported by an AADR Student Research Fellowship.
Transcription factor NF-B binding sites in the CMV promoter as an inhibitor of HIV replication.
Paper presented at 32nd Annual Meeting of the American Association for Dental Research in San Antonio, TX.