Schematic representation of the gag and pol regions of HIV-1 (top) and the amino acid sequences of portions of p6^Gag protein and viral protease encoded by two different reading frames (bottom). The amino acid residues conserved in >98% of the majority HIV-1 substrains (group M), which include subtypes A, B, C, D, F, G, H, and J and circulating recombinant forms (AE, AG, AGI, and AB) (HIV Sequence Database [http://hiv-web.lanl.gov]), are shown in large capital letters. The B subtype consensus sequences are shown in small capital letters. The nucleotide sequence of our interest is shown in open letters. PR, protease; RH, RNase H; INT, integrase.

Effects of PNA oligomers on HIV-1 production in chronically HIV-1-infected cells. (A) Levels of p24 antigen in the supernatants of H9_LAI cells cultured in the absence or presence of various PNA oligomers used at a 100 μM concentration. All compounds were tested in triplicate, and the values shown are means with SDs. The p24 antigen level in a supernatant of H9_LAI treated with an HIV-1 protease inhibitor, indinavir, is shown as a reference. (B) Levels of p24 antigen in the supernatants of H9_LAI and H9_RF cells cultured in the absence or presence of various concentrations of PNA_PR2. Each dose was tested in triplicate. The values shown are means with SDs. (C) The degrees of inhibition of p24 antigen production in H9_LAI cells cultured with 100 μM PNA_PR2 or other PNA oligomers targeting the sequences proximate to the PNA_PR2-annealing site were compared. Each PNA oligomer was tested in triplicate. Each bar represents the target sequence shifted upstream (−) or downstream (+) by one to six nucleotides away from the PNA_PR2-annealing site. The values shown are relative antiviral effects (means ± SDs). (D) Amounts of p24 antigen in the supernatant (solid bars) and cell lysate (crosshatched bars) of H9_LAI cells cultured in the absence or presence of 30 and 60 μM PNA_PR2. The cell numbers are indicated by circles. The experimental results shown are representative of three separate experiments.

Effects of PNA_PR2 on Gag-Pol expression and virion production in HIV-1-infected cells (A and B) and on Gag protein expression in HLtat cells transfected with the Gag expression plasmid p55M1-10 (C). (A) The amounts of viral RNA in the culture supernatant (pelleted virion derived) (lanes 1 to 3) and in the cells (lanes 4 to 6) were evaluated by RT-PCR (top). Arrows indicate the pertinent PCR products for each primer pair. The virion-derived Gag protein in the culture supernatant (lanes 1 to 3) and intracellular Gag protein processing (lanes 4 to 6) were evaluated by Western blot analysis using anti-p24^Gag antibody (bottom). Shown are the positions of Pr160^Gag-Pol, Pr55^Gag, p41, and p24 Gag proteins. Lanes of each gel represent H9_LAI cells cultured alone (lanes 1 and 4) or incubated with 100 μM PNA_PR2 (lanes 2 and 5) and 2 μM indinavir (lanes 3 and 6). (B) Western blot analyses with anti-p24^Gag antibody and anti-p17^Gag antibody (top) and with anti-p6^Gag antibody (bottom) of cell lysates processed from H9_LAI cells cultured alone (lane 1) or in the presence of 30 μM PNA_PR2 (lane 2), 60 μM PNA_PR2 (lane 3), 2 μM indinavir alone (lane 4), 30 μM PNA_PR2 plus 2 μM indinavir (lane 5), or 60 μM PNA_PR2 plus 2 μM indinavir (lane 6). Cell lysate of uninfected H9 cells is shown in lane 7. PI − or + represents the absence or presence of a protease inhibitor, indinavir, respectively. The level of p24 antigen in each culture supernatant is indicated at the bottom. (C) Western blot analyses with anti-p24^Gag antibody (left) and anti-p6^Gag antibody (right) of cell lysates processed from HLtat cells transfected with pHXB2-based plasmid pSUM9 (136) (lanes 1) or with p55M1-10 and cultured alone (lanes 2) or in the presence of 100 μM PNA_RR2 (lanes 3). The cell lysate of mock-transfected HLtat cells is included as a reference (lanes 4). Sup, supernatant; Ag, antigen; Ab, antibody.

Cellular uptake of PNA. (A to C) Chronically HIV-1-infected H9_LAI cells were incubated with fluorescein-tagged PNA at various concentrations overnight, fixed with 3.7% formaldehyde for 20 min, washed and resuspended in PBS, and examined by fluorescence microscopy. Shown are the cells incubated with 0 (A), 30 (B), and 60 (C) μM fluorescein-tagged PNA_PR2. Note that the cells treated with fluorescein-tagged PNA_PR2 were resuspended in a slightly larger volume of PBS than was the untreated control so that each cell could be clearly visualized. The panel on the right-hand side is the fluorescence image, and that on the left is the phase-contrast image of the same field. The discernible fluorescent signal was seen in the majority of cells at 30 μM PNA or greater. (D) Shown at a higher magnification are the cells incubated with 30 μM PNA_PR2. Note that the fluorescent signal is virtually confined to the cytoplasm. (E) H9_LAI cells incubated with fluorescein-tagged PNA were also evaluated by fluorescence-activated cell sorter analysis. Curves a, b, and c represent H9_LAI cells incubated with 0, 30, and 60 μM fluorescein-tagged PNA_PR2, respectively. Similar results were obtained with fluorescein-tagged PNA_PR4 and PNA_RT2.

Electron microscopy of H9_LAI cells cultured in the absence (A) or presence (B) of 60 μM PNA_PR2. Shown are representatives of 100 cells surveyed in each sample (magnification, ×13,500). Virions are shown at a higher magnification (×54,000) in each inset on the right-hand side at the bottom.

Effects of PNA_PR2 on gag, pol, and env gene expression and virion production in COS-7 cells transfected with an infectious HIV-1 molecular clone, pNL4-3. Shown are results of Western blot analyses with anti-p24^Gag antibody and anti-p17^Gag antibody (A), antiprotease antibody and anti-p66^RT antibody (B), and anti-gp120 antibody (C) of pNL4-3-transfected COS-7 cells cultured with no drug (lanes 1), 100 μM PNA_PR2 (lanes 2), 1 μM saquinavir (also a protease inhibitor) (lanes 3), or 3 μM indinavir (lanes 4). The cell lysate of mock-transfected COS-7 cells is shown in lanes 5. Viral lysate from pelleted virion particles (vp) is included in the Western blot analyses for the pol gene products as a reference. The levels of p24 antigen in supernatant and the infectious titers determined by MAGI assay are shown at the bottom. Ab, antibody; Ag, antigen.