Retroviral delivery of promoter-targeted shRNA induces long-term silencing of HIV-1 transcription

We previously reported prolonged HIV-1 transcriptional gene silencing by an RNA duplex targeting a sequence located within the NF-kappa B binding motif of the HIV-1 promoter in a susceptible HeLa cell line. Here we report extremely prolonged suppression of productive HIV-1 infection in a T-cell line (Molt-4) by a retrovirally delivered short-hairpin RNA (shRNA) targeting the same region (sh kappa B). Following retroviral delivery of an shRNA we established shRNA-expressing CD4(+) T-cell lines. HIV-1 gene expression was profoundly Suppressed for I year. Results of nuclear run-on assays and HIV-1 LTR-luciferase reporter assays revealed that sh kappa B acted by inhibition of HIV-1 transcription. The effect was reversed by a histone deacetylase inhibitor, trichostatin-A (TSA), but not by a DNA methyltransferase inhibitor, 5-azacytidine (5-AzaC). Furthermore, chromatin immunoprecipitation assays (Chip) demonstrated rapid, sustained induction of heterochromatin structures within the HIV-1 promoter region, with enrichment of histone 3 lysine 27 tri-methylation (H3K27me3) and H3K9 methylation. H3K27me3 enrichment was the most pronounced. This prolonged suppression could not be recapitulated by either retrovirally delivered anti-sense or sense strands alone or in combination. Our data strongly suggest that sh kappa B induces high level, Sustained transcriptional gene silencing of HIV-1 and offers the possibility of new therapeutic strategies. (C) 2009 Elsevier Masson SAS. All rights reserved.