title: Retroviral delivery of promoter-targeted shRNA induces long-term silencing of HIV-1 transcription.
abstract: We previously reported prolonged HIV-1 transcriptional gene silencing by an RNA duplex targeting a sequence located within the NF-kappaB 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 (shkappaB). Following retroviral delivery of an shRNA we established shRNA-expressing CD4(+) T-cell lines. HIV-1 gene expression was profoundly suppressed for 1 year. Results of nuclear run-on assays and HIV-1 LTR-luciferase reporter assays revealed that shkappaB 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 shkappaB induces high level, sustained transcriptional gene silencing of HIV-1 and offers the possibility of new therapeutic strategies.
authors.affiliation: Laboratory of Tumor Cell Biology, Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, Japan. kk620004@ims.u-tokyo.ac.jp
authors.forename: Makoto
authors.initials: M
authors.lastname: Yamagishi
authors.forename: Takaomi
authors.initials: T
authors.lastname: Ishida
authors.forename: Ariko
authors.initials: A
authors.lastname: Miyake
authors.forename: David A
authors.initials: DA
authors.lastname: Cooper
authors.forename: Anthony D
authors.initials: AD
authors.lastname: Kelleher
authors.forename: Kazuo
authors.initials: K
authors.lastname: Suzuki
authors.forename: Toshiki
authors.initials: T
authors.lastname: Watanabe
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology; D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000821:virology; D020868:Gene Silencing; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics*; D014158:Transcription, Genetic; D014161:Transduction, Genetic; D014779:Virus Replication
chemical_list: D019380:Anti-HIV Agents; D034741:RNA, Small Interfering; D012367:RNA, Viral