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.

Figure 1

(A) Secondary structure of the TAR RNA apical stem–loop, the binding site on TAR and sequence of the PNA 16mer. (B) Stably integrated plasmids within the HeLa Tet-Off/Tat/luc-f/luc-R cell line used for trans-activation inhibition studies.

Figure 2

(A) Structures of various FAM-labelled stably-linked CPP–PNA and PNA–CPP conjugates 1–9 and their calculated and observed mass values. (B) Structure of various disulfide-linked CPP–PNA conjugates 10–15 and their calculated and observed mass values. The linkage is in all cases between an N-terminal Cys residue on the PNA and a Cys residue within the peptide either on the C-terminus (10–13) N-terminus (14) or an internal residue (15). These conjugates do not carry a FAM label.

Figure 3

Trans-activation inhibitory effects of disulfide-linked CPP–PNA conjugates 13–15 in the HeLa cell reporter assay with 6 h delivery (A) or 24 h (B). Firefly luciferase activity represents Tat-TAR dependent expression whilst Renilla luciferase activity represents control constitutive expression. Bars (left to right) in each case represent 2.5, 1.25, 0.625, 0.312 and 0 µM CPP–PNA concentrations.

Figure 4

Trans-activation inhibitory effects (firefly luciferase activity) of CPP–PNA conjugates in the HeLa cell reporter assay with delivery for 6 h in the presence of 100 µM chloroquine. (A) Stably-linked conjugates Tat–PNA (1), TP–PNA (2), TP10–PNA (3) and NLS–PNA–Tat (7). (B) Disulfide-linked conjugates R₆–Pen-S-S–PNA (13), mismatched conjugate 13 (PNA sequence, CTCCGCAGCTCAGATC), scrambled conjugate 13 (PNA sequence, ATCGCTCGCACCATGC), TP–S-S–PNA (14) and TP(int)–S-S–PNA (15). Bars (left to right) in each case represent 2.5, 1.25, 0.625, 0.312 and 0 µM (light shaded bar) CPP–PNA concentrations. Control (black bar), absence of CPP–PNA and absence of chloroquine.

Figure 5

Trans-activation inhibitory effects (firefly luciferase activity) of 2.5 µM Tat-PNA (1) incubated for 2, 4, 6 or 8 h, respectively, with HeLa reporter cells, cells washed and grown for 18 h before assay.

Figure 6

Confocal microscopy images of the uptake of fluorescein (FAM)-labelled CPP–PNA conjugates when incubated for 5.5 h with unfixed HeLa cells. Nuclei are stained red with hydroethidine. (A and C) Orange filter to view both the red colour of hydroethidine and the fluorescein fluorescence. (B and D) Green filter to view only the fluorescein fluorescence. (A and B) Show incubations in the absence of chloroquine, (C and D) Show incubations in the presence of 100 µM chloroquine. First line Tat–PNA (1); second line Transportan–PNA (2); third line NLS–PNA–Tat (7); fourth line K₈–PNA (9). In (C), second line, yellow dots are marked with arrows showing co-localization of hydroethidine dye and fluorescein fluorescence on the inner wall of the nucleus in several nuclei.