title: Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent trans-activation in cells.
abstract: The trans-activation response (TAR) RNA stem-loop that occurs at the 5' end of HIV RNA transcripts is an important antiviral target and is the site of interaction of the HIV-1 Tat protein together with host cellular factors. Oligonucleotides and their analogues targeted to TAR are potential antiviral candidates. We have investigated a range of cell penetrating peptide (CPP) conjugates of a 16mer peptide nucleic acid (PNA) analogue targeted to the apical stem-loop of TAR and show that disulfide-linked PNA conjugates of two types of CPP (Transportan or a novel chimeric peptide R6-Penetratin) exhibit dose-dependent inhibition of Tat-dependent trans-activation in a HeLa cell assay when incubated for 24 h. Activity is reached within 6 h if the lysosomotropic reagent chloroquine is co-administered. Fluorescein-labelled stably-linked conjugates of Tat, Transportan or Transportan TP10 with PNA were inactive when delivered alone, but attained trans-activation inhibition in the presence of chloroquine. Confocal microscopy showed that such fluorescently labelled CPP-PNA conjugates were sequestered in endosomal or membrane-bound compartments of HeLa cells, which varied in appearance depending on the CPP type. Co-administration of chloroquine was seen in some cases to release fluorescence from such compartments into the nucleus, but with different patterns depending on the CPP. The results show that CPP-PNA conjugates of different types can inhibit Tat-dependent trans-activation in HeLa cells and have potential for development as antiviral agents. Endosomal or membrane release is a major factor limiting nuclear delivery and trans-activation inhibition.
authors.affiliation: Laboratory of Molecular Biology, Medical Research Council, Hills Road, Cambridge CB2 2QH, UK.
authors.forename: John J
authors.initials: JJ
authors.lastname: Turner
authors.forename: Gabriela D
authors.initials: GD
authors.lastname: Ivanova
authors.forename: Birgit
authors.initials: B
authors.lastname: Verbeure
authors.forename: Donna
authors.initials: D
authors.lastname: Williams
authors.forename: Andrey A
authors.initials: AA
authors.lastname: Arzumanov
authors.forename: Saïd
authors.initials: S
authors.lastname: Abes
authors.forename: Bernard
authors.initials: B
authors.lastname: Lebleu
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Gait
mesh_terms: D000998:Antiviral Agents / Q000032:analysis / Q000737:chemistry* / Q000494:pharmacology*; D002463:Cell Membrane Permeability; D002738:Chloroquine / Q000494:pharmacology; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D015696:Gene Products, tat / Q000037:antagonists & inhibitors*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry; D018613:Microscopy, Confocal; D020135:Peptide Nucleic Acids / Q000032:analysis / Q000737:chemistry* / Q000494:pharmacology*; D015533:Transcriptional Activation / Q000187:drug effects*; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D000998:Antiviral Agents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015696:Gene Products, tat; D020135:Peptide Nucleic Acids; D054322:tat Gene Products, Human Immunodeficiency Virus; C024098:6-carboxyfluorescein; D002738:Chloroquine