title: Inhibition of HIV-1 Tat-dependent trans activation by steric block chimeric 2'-O-methyl/LNA oligoribonucleotides.
abstract: The HIV-1 trans-activation responsive element (TAR) RNA 59-residue stem-loop interacts with the HIV trans-activator protein Tat and other cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR). Inhibition of these interactions blocks full-length HIV transcription and hence replication. We have found that three types of 12-residue oligonucleotide analogues, namely, a 2'-O-methyl oligoribonucleotide (OMe), a chimeric oligonucleotide containing 7xOMe and 5x5-methyl C locked nucleic acid (LNA) residues, and a peptide nucleic acid (PNA), inhibit Tat-dependent in vitro transcription in HeLa cell nuclear extract equally efficiently (50% inhibition at 100-200 nM) and sequence specifically. The results are correlated with surprisingly similar binding strengths to a model 39-residue TAR under transcription conditions. A 12-mer containing 11 contiguous LNA residues was less effective in both Tat-dependent transcription inhibition and TAR 39 binding. Anti-TAR 3'-carboxyfluorescein- (FAM-) labeled OMe and OMe/LNA chimeric 12-mers were also efficient Tat-dependent in vitro transcription inhibitors as were 3'-FAM-labeled OMe oligonucleotides containing some phosphorothioate (PS) linkages. By use of a HeLa cell line containing stably integrated plasmids expressing firefly luciferase under HIV-LTR/Tat dependence as well as a Renilla luciferase constitutive control, we showed submicromolar, selective, dose-dependent, and sequence-dependent intracellular inhibition of Tat-TAR trans activation by the anti-TAR 3'-FAM 12-residue 7xOMe/5xLNA oligonucleotide when delivered by cationic lipid. No intracellular activity was observed for the corresponding anti-TAR 3'-FAM OMe 12-mer. An alternating PS-containing 3'-FAM OMe 12-mer oligonucleotide exhibited partial inhibition of trans-activation activity, but this was correlated with a similar effect on control gene expression, suggesting nonspecific inhibition.
authors.affiliation: Laboratory of Molecular Biology, Medical Research Council, Hills Road, Cambridge CB2 2QH, UK.
authors.forename: A
authors.initials: A
authors.lastname: Arzumanov
authors.forename: A P
authors.initials: AP
authors.lastname: Walsh
authors.forename: V K
authors.initials: VK
authors.lastname: Rajwanshi
authors.forename: R
authors.initials: R
authors.lastname: Kumar
authors.forename: J
authors.initials: J
authors.lastname: Wengel
authors.forename: M J
authors.initials: MJ
authors.lastname: Gait
mesh_terms: D002412:Cations / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D005452:Fluoresceins; D015696:Gene Products, tat / Q000037:antagonists & inhibitors* / Q000235:genetics; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D050356:Lipid Metabolism; D008156:Luciferases / Q000378:metabolism; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013752:Tetracycline / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000187:drug effects*; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D002412:Cations; D017931:DNA Primers; D005452:Fluoresceins; D015696:Gene Products, tat; D016376:Oligonucleotides, Antisense; D010446:Peptide Fragments; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus; C024098:6-carboxyfluorescein; D008156:Luciferases; D013752:Tetracycline