Targeted derepression of the human immunodeficiency virus type 1 long terminal repeat by pyrrole-imidazole polyamides

Abstract

The host factor LSF represses the human immunodeficiency virus type 1 long terminal repeat (LTR) by mediating recruitment of histone deacetylase. We show that pyrrole-imidazole polyamides targeted to the LTR can specifically block LSF binding both in vitro and within cells via direct access to chromatin, resulting in increased LTR expression.

Figures

FIG. 1.

RCS-binding polyamides specifically inhibit LSF binding to the RCS of the HIV-1 LTR in vitro. (A) Sequences of the RCS (gray)- and LSF-binding motifs (underlined), sequences targeted by polyamides 1, 2, 3 and 4 (boxed), and oligonucleotide probes (thick lines below) used in the EMSA. (B) Chemical structure of each polyamide (1, 2, 3, and 4) and their mismatched controls (M1, M2, and M3/4). (C) Ball-and-stick model of polyamides juxtaposed to their target sites; the Ka value for each molecule is indicated. Polyamide 4 binds in a 3′ to 5′, N to C orientation as a single mismatch at a discrete site with high affinity and selectivity. (D) EMSA of RCS alone (lane 1) and that retarded by his-LSF (lane 2). Lanes 3 and 4 show competition by unlabeled RCS or Sp1 consensus binding sequence oligonucleotides (50:1 ratio of competitor to probe), and lanes 5 and 6 show supershift induced by the addition of antibody to LSF or nonspecific immunoglobulin G (IgG). Lanes 7 to 14 show competition by polyamides 1, 2, M1, and M2 (the concentration of the polyamide is shown). (E) EMSA of RCS-S (+4 to +35) alone (lane 1) and that retarded by his-LSF (lane 2), and competition by RCS or Sp1 (lanes 3 and 4), polyamide 3 (lanes 5 and 6), or M3/4 (lanes 7 and 8). (F) EMSA of RCS-L (−10 to +34) alone (lane 1) and that retarded by his-LSF (lane 2), and competition by RCS or Sp1 (lanes 3 and 4), polyamides 1 and 3 (lanes 5 and 6), M1 and M3/4 (lanes 7 and 8), 2 and 3 (lanes 9 and 10), or M2 and M3/4 (lanes 11 and 12).

FIG. 1.

RCS-binding polyamides specifically inhibit LSF binding to the RCS of the HIV-1 LTR in vitro. (A) Sequences of the RCS (gray)- and LSF-binding motifs (underlined), sequences targeted by polyamides 1, 2, 3 and 4 (boxed), and oligonucleotide probes (thick lines below) used in the EMSA. (B) Chemical structure of each polyamide (1, 2, 3, and 4) and their mismatched controls (M1, M2, and M3/4). (C) Ball-and-stick model of polyamides juxtaposed to their target sites; the Ka value for each molecule is indicated. Polyamide 4 binds in a 3′ to 5′, N to C orientation as a single mismatch at a discrete site with high affinity and selectivity. (D) EMSA of RCS alone (lane 1) and that retarded by his-LSF (lane 2). Lanes 3 and 4 show competition by unlabeled RCS or Sp1 consensus binding sequence oligonucleotides (50:1 ratio of competitor to probe), and lanes 5 and 6 show supershift induced by the addition of antibody to LSF or nonspecific immunoglobulin G (IgG). Lanes 7 to 14 show competition by polyamides 1, 2, M1, and M2 (the concentration of the polyamide is shown). (E) EMSA of RCS-S (+4 to +35) alone (lane 1) and that retarded by his-LSF (lane 2), and competition by RCS or Sp1 (lanes 3 and 4), polyamide 3 (lanes 5 and 6), or M3/4 (lanes 7 and 8). (F) EMSA of RCS-L (−10 to +34) alone (lane 1) and that retarded by his-LSF (lane 2), and competition by RCS or Sp1 (lanes 3 and 4), polyamides 1 and 3 (lanes 5 and 6), M1 and M3/4 (lanes 7 and 8), 2 and 3 (lanes 9 and 10), or M2 and M3/4 (lanes 11 and 12).

FIG. 1.

RCS-binding polyamides specifically inhibit LSF binding to the RCS of the HIV-1 LTR in vitro. (A) Sequences of the RCS (gray)- and LSF-binding motifs (underlined), sequences targeted by polyamides 1, 2, 3 and 4 (boxed), and oligonucleotide probes (thick lines below) used in the EMSA. (B) Chemical structure of each polyamide (1, 2, 3, and 4) and their mismatched controls (M1, M2, and M3/4). (C) Ball-and-stick model of polyamides juxtaposed to their target sites; the Ka value for each molecule is indicated. Polyamide 4 binds in a 3′ to 5′, N to C orientation as a single mismatch at a discrete site with high affinity and selectivity. (D) EMSA of RCS alone (lane 1) and that retarded by his-LSF (lane 2). Lanes 3 and 4 show competition by unlabeled RCS or Sp1 consensus binding sequence oligonucleotides (50:1 ratio of competitor to probe), and lanes 5 and 6 show supershift induced by the addition of antibody to LSF or nonspecific immunoglobulin G (IgG). Lanes 7 to 14 show competition by polyamides 1, 2, M1, and M2 (the concentration of the polyamide is shown). (E) EMSA of RCS-S (+4 to +35) alone (lane 1) and that retarded by his-LSF (lane 2), and competition by RCS or Sp1 (lanes 3 and 4), polyamide 3 (lanes 5 and 6), or M3/4 (lanes 7 and 8). (F) EMSA of RCS-L (−10 to +34) alone (lane 1) and that retarded by his-LSF (lane 2), and competition by RCS or Sp1 (lanes 3 and 4), polyamides 1 and 3 (lanes 5 and 6), M1 and M3/4 (lanes 7 and 8), 2 and 3 (lanes 9 and 10), or M2 and M3/4 (lanes 11 and 12).

FIG. 2.

Polyamides block binding of LSF to the RCS in vivo. ChIP assays show an increase of acetylated histone H4 near Nuc-1 of the LTR after treatment with TSA and a decrease in LSF at this region of the LTR after exposure to RCS-binding polyamides. (A) PCR amplification of the 5′ LTR or β-actin promoter performed by using cell extracts (−) and cells exposed to TSA (+). Shown are products of ChIP and PCR amplification of the 5′ LTR or β-actin promoter using anti-acetylated histone H4 or mock immunoprecipitation (IP) with rabbit immunoglobulin G (IgG) and control PCR amplification of the 5′ LTR or β-actin promoter using extract prior to immunoprecipitation. Results are representative of three independent experiments. (B) ChIP with anti-LSF. PCR products of extracts of untreated cells, cells exposed to TSA, and polyamides 1 and 4, 2 and 4, M1 and M3/4, or M2 and M3/4. Shown are products of ChIP and PCR amplification of the 5′ LTR using anti-LSF or mock immunoprecipitation with rabbit immunoglobulin G and control PCR amplification of the 5′ LTR or β-actin promoter using extract prior to immunoprecipitation. PCR amplification of the β-actin promoter with extract following immunoprecipitation with anti-LSF yielded no product (data not shown). These two experiments are representative of four independent experiments.

FIG.3.

RCS-binding polyamides modulate expression of an integrated HIV-1 LTR in tissue culture model systems. (A) RCS-binding polyamides (5 μM) induce an increase in expression of an integrated LTR-GFP reporter gene in HUT78 cells in the absence of Tat, as shown by an increased GFP fluorescence. Significant changes in mean fluorescence are indicated by an asterisk (P < 0.01); the mean of five independent samples was compared by using the unpaired t test. (B) HIV-1 p24 gag production was measured in triplicate cultures of ACH2 cells after 24 h of exposure to polyamide 2 or M2, 100 nM TSA, or medium control. An asterisk indicates statistically significant difference from the results of the control culture (P < 0.05 by unpaired t test). (C) Expression of the integrated reporter gene in HeLa-LTR-CAT can be activated by transfection of Tat, and this activation is blunted by cotransfection of YY1 (6, 11, 14). Inhibition of pAR-Tat activation (50 ng) by CMV-YY1 (1.5 μg) is expressed as 100%. The effect of exposure of cells to binding polyamide 1 or mismatch polyamide M1 is displayed. An asterisk indicates statistically significant difference from 100% repression (P < 0.05). Results of four independent transfections are shown.