PF74 Inhibits HIV-1 Integration by Altering the Composition of the Preintegration Complex

Abstract

The HIV-1 capsid protein (CA) facilitates reverse transcription and nuclear entry of the virus. However, CA's role in post-nuclear entry steps remains speculative. We describe a direct link between CA and integration by employing the capsid inhibitor PF74 as a probe coupled with the biochemical analysis of HIV-1 preintegration complexes (PICs) isolated from acutely infected cells. At a low micromolar concentration, PF74 potently inhibited HIV-1 infection without affecting reverse transcription. Surprisingly, PF74 markedly reduced proviral integration owing to inhibition of nuclear entry and/or integration. However, a 2-fold reduction in nuclear entry by PF74 did not quantitatively correlate with the level of antiviral activity. Titration of PF74 against the integrase inhibitor raltegravir showed an additive antiviral effect that is dependent on a block at the post-nuclear entry step. PF74's inhibitory effect was not due to the formation of defective viral DNA ends or a delay in integration, suggesting that the compound inhibits PIC-associated integration activity. Unexpectedly, PICs recovered from cells infected in the presence of PF74 exhibited elevated integration activity. PF74's effect on PIC activity is CA specific since the compound did not increase the integration activity of PICs of a PF74-resistant HIV-1 CA mutant. Sucrose gradient-based fractionation studies revealed that PICs assembled in the presence of PF74 contained lower levels of CA, suggesting a negative association between CA and PIC-associated integration activity. Finally, the addition of a CA-specific antibody or PF74 inhibited PIC-associated integration activity. Collectively, our results demonstrate that PF74's targeting of PIC-associated CA results in impaired HIV-1 integration.IMPORTANCE Antiretroviral therapy (ART) that uses various combinations of small molecule inhibitors has been highly effective in controlling HIV. However, the drugs used in the ART regimen are expensive, cause side effects, and face viral resistance. The HIV-1 CA plays critical roles in the virus life cycle and is an attractive therapeutic target. While currently there is no CA-based therapy, highly potent CA-specific inhibitors are being developed as a new class of antivirals. Efforts to develop a CA-targeted therapy can be aided through a clear understanding of the role of CA in HIV-1 infection. CA is well established to coordinate reverse transcription and nuclear entry of the virus. However, the role of CA in post-nuclear entry steps of HIV-1 infection is poorly understood. We show that a CA-specific drug PF74 inhibits HIV-1 integration revealing a novel role of this multifunctional viral protein in a post-nuclear entry step of HIV-1 infection.

Keywords: HIV-1; PF74; capsid; integration; preintegration complex.

FIG 1

PF74 inhibits HIV-1 infection without reducing reverse transcription and partially blocking nuclear entry. (A) Anti-viral activity of PF74. SupT1 cells (5 × 10⁴ cells) were infected with HIV-1 GFP reporter virions (∼15 ng of p24) and cultured in the presence of increasing concentrations of PF74 (0 to 20 μM). At 24 h postinfection, the cells were collected, and the intracellular GFP fluorescence was measured by flow cytometry. The inhibitory effect of PF74 was determined from the percent reduction in GFP fluorescence of infected cells cultured in the presence of PF74 relative to infected cells cultured without the inhibitor. (B to D) Effect of PF74 on HIV-1 reverse transcription. SupT1 cells were inoculated with three different amounts (1,500 ng/ml [B], 150 ng/ml [C], or 15 ng/ml [D] of p24) of HIV-1 virions and cultured in the absence or presence of 2 and 10 μM PF74. At 16 h postinfection, the cells were harvested, and the total DNA isolated from these cells was analyzed by qPCR using primers specific for amplification of late RT products. The levels of viral DNA were quantified by calculating the copies of viral DNA from a standard curve generated in parallel and under same conditions of qPCR using 10-fold serial dilutions of known copy numbers (10⁰ to 10⁸) of an HIV-1 molecular clone. (E and F) Effects of PF74 on HIV-1 nuclear entry. SupT1 cells were inoculated with an equivalent amount of HIV-1 virions (150 ng/ml) and cultured in the absence or presence of 2 and 10 μM PF74. At 16 h postinfection, the cells were harvested, and the total DNA was isolated from these cells. Then, 100 ng of the DNA was subjected to qPCR to amplify the junctions of the HIV-1 2-LTR (E) and 1-LTR circles (F) using specific primer sets. The copy numbers of the 2-LTR and 1-LTR circles were calculated by extrapolating the qPCR data to a standard curve generated using 10-fold serial dilutions of the p2LTR and p1LTR plasmids, respectively. Data shown in panels B to F are mean values from at least three independent experiments, with error bars representing the standard errors of the mean.

FIG 2

PF74 treatment markedly reduces HIV-1 integration. (A to C) Effects of PF74 on proviral DNA integration. SupT1 cells were inoculated with three different amounts (1,500 ng/ml [A], 150 ng/ml [B], or 15 ng/ml [C] of p24) of HIV-1 particles and cultured in the absence or presence of 2 and 10 μM PF74. At 16 h postinfection, the cells were harvested, and the total DNA from these cells was subjected to Alu-based qPCR to measure proviral DNA integration into the host genome. The copies of viral DNA integration were calculated by extrapolating the integration data into a standard curve generated using known copy numbers (10⁰ to 10⁸) of an HIV-1 molecular clone. Data in panels A to C represent mean values from three independent experiments, with standard errors of the mean. (D) Cumulative effect of PF74 on HIV-1 reverse transcription, LTR circles, and proviral DNA integration. To analyze PF74’s antiviral effect, the observed data for PF74’s effect on infection (Fig. 1A), reverse transcription (Fig. 1C), 2-LTR circles (Fig. 1E), 1-LTR circles (Fig. 1F), and proviral integration (Fig. 2B) were plotted as the percent versus controls (untreated cells). (E and F) Effects of RAL treatment on PF74-induced LTR circle formation. SupT1 cells were inoculated with an equivalent amount of HIV-1 virions (150 ng/ml) and cultured in the absence or presence of 2 μM PF74, 1 μM RAL, or a combined treatment of 2 μM PF74 and 1 μM RAL. At 16 h postinfection, the total DNA from the infected cells was isolated and analyzed by qPCR to amplify the junctions of the HIV-1 2-LTR and 1-LTR circles. Copies of the 2-LTR circles (E) and 1-LTR circles (F) were quantified using a standard curve, as described for Fig. 1E and F. Data shown in panels A, B, C, E, and F are mean values from three independent experiments, with error bars representing the standard errors of the mean.

FIG 3

Effects of RAL treatment on the antiviral activity of PF74. TZM-bl cells were inoculated with 5 ng/ml of pseudotyped HIV-1 particles and cultured in the absence or presence of RAL (5 to 100 nM) (A) or PF74 (0.1 to 2 μM) (B). At 48 h postinfection, cellular lysates were prepared and subjected to luciferase activity measurements as an indicator of infection. Data shown are mean values from three independent experiments, with error bars representing the standard errors of the mean. (C and D) Titration of PF74 against RAL. TZM-bl cells inoculated with pseudotyped HIV-1 were treated with either 25 nM RAL (C) or 100 nM RAL (D) in the absence or presence of a range of concentrations of PF74 (0.2 to 2 μM). At 48 h postinfection, the luciferase activity was measured in the cellular lysates, and the data are plotted as the percent infection versus the controls. (E) Titration of RAL against PF74. TZM-bl cells were inoculated with pseudotyped HIV-1 and treated with PF74 (2 μM) in the absence or presence of RAL (5 to 100 nM). At 48 h postinfection, the luciferase activity was measured and plotted as the percent infection versus the controls. Data presented in panels C to E are representative of three independent experiments.

FIG 4

Time-of-addition assay of PF74 and/or RAL and measurement of HIV-1 infectivity. TZM-bl cells inoculated with pseudotyped HIV-1 particles (5 ng/ml) were subjected to a time-of-addition assay in the absence or presence of RAL, PF74, or RAL plus PF74. At 48 h postinfection, cellular lysates were prepared and subjected to luciferase activity measurements as an indicator of infection. (A and B) Time of RAL addition assay. Cells were cultured in the absence (A) or presence (B) of PF74 (2 μM). To these cells, RAL (25 nM) was added at 0, 1, 2, 5, 8, 12, and 16 h relative to infection. At 48 h postinfection, the luciferase activity was measured in the cellular lysates and plotted as the percent infection versus the controls. (C and D) Time-of-PF74-addition assay. Cells were cultured in the absence (C) or presence (D) of RAL (25 nM). PF74 (2 μM) was added at different time points (0, 1, 2, 5, 8, 12, and 16 h) relative to the infection to these cells. At 48 h postinfection, cellular lysates were prepared, and the luciferase activity was measured. The data are plotted as the percent infection versus the controls. Data presented are representative from three independent experiments, with error bars representing the standard errors of the mean.

FIG 5

Effect of PF74 on the integration activity of HIV-1 PICs. (A) Measurement of PIC-associated integration activity in vitro. SupT1 cells were inoculated with high-titer HIV-1 particles (1,500 ng/ml of p24), and PIC-containing cytoplasmic extracts (Cy-PICs) were isolated from these cells. In vitro integration assays using the Cy-PICs as the source of integration activity and quantification of viral DNA integration by nested qPCR were carried out as described previously (76, 77). Several controls were included in parallel to ascertain the specificity of the PIC-associated integration activity. (B) Integration activity with different amounts of PICs. Various amounts of Cy-PICs were used as the source of integration activity in the assay, and viral DNA integration was calculated as described for panel A. (C) Relative integration activity. Copies of integrated viral DNA in diluted Cy-PICs were plotted as the percentage versus the copies of viral DNA integration relative to undiluted (200 μl) Cy-PICs. (D to G) Integration activity of wild-type HIV-1 PICs assembled in the presence of PF74. (D) Integration activity of Cy-PICs. Acutely infected SupT1 cells were cultured in the absence or presence of PF74. The cells were harvested, and cytoplasmic extracts containing PICs were isolated from these cells. The integration activities of these PICs were measured in vitro, and the viral DNA copy numbers were determined. Mean values from three independent experiments are shown, with error bars representing the standard errors of the mean. (E) Viral DNA copy numbers in the Cy-PICs. Copies of viral DNA in Cy-PICs assembled in the absence or presence of PF74 were quantified by qPCR using a standard curve generated according to the method described for Fig. 1B. (F) Integration activity of Nu-PICs. To generate Nu-PICs, nuclear pellets of acutely infected SupT1 cells were homogenized, and the PIC-containing nuclear extracts (Nu-PICs) were isolated as described in Materials and Methods. In vitro integration assays of Nu-PICs and quantification of integrated viral DNA were performed using a standard curve as described for Fig. 5A. (G) Viral DNA copy numbers in the Nu-PICs. Copies of Nu-PIC-associated viral DNA were quantified by qPCR. The data shown are mean values from three independent experiments, with error bars representing the standard errors of the mean. (H to K) Integration activities of PF74-resistant HIV-1 5Mut PICs assembled in the presence of PF74. SupT1 cells were inoculated with a high titer (∼1,500 ng/ml of p24) of HIV-1 5Mut particles, and Cy-PICs and Nu-PICs were prepared from these cells. The integration activities of Cy-PICs (H) and Nu-PICs (I) were measured, and the copy numbers of viral DNA Cy-PICs (J) and Nu-PICs (K) were determined by qPCR as described in the text. Mean values from three independent experiments are shown, with error bars representing the standard errors of the mean.

FIG 6

Analysis of PIC composition. (A) Fractionation of Cy-PICs and integration activity measurements. Portions (1 ml) of Cy-PICs were overlaid on a freshly prepared, 10 to 50% linear gradient of sucrose and subjected to ultracentrifugation for 2 h at 4°C. Fractions of 1 ml were collected starting from the top and analyzed for integration activity using 200-μl aliquots of each fraction. (B) PIC activity. Integration assays were performed in the presence of the integrase inhibitor RAL using fraction 14 as the source of PIC activity. (C) Viral DNA content in the fractionated PICs. Aliquots (200 μl) of each fraction were subjected to qPCR to quantitate copies of vDNA using a standard curve. (D and E) Quantification of CA in the fractionated PICs. Aliquots (100 μl) of each fraction were subjected to ELISA to quantitate the amount of CA by extrapolating the data to a standard curve generated using purified recombinant CA. The data show the CA levels in the fractioned PICs isolated from cells inoculated with enveloped (D) and VSV-G-pseudotyped (E) HIV-1. The data shown are averages of three independent experiments. (F and G) Detection of PIC-associated CA by immunoprecipitation. To detect PIC-associated CA by pulldown assays, we used magnetic bead-coated GST-CypA (A) and anti-CA antibodies (B). The GST-CypA or antibody-coated beads or equivalent amounts of protein A/G-magnetic beads (negative control) were incubated with cPICs or CA-A14C/E45C tubes (positive control) or recombinant monomeric CA. The bound complexes were subjected to centrifugation and washed, and the pelleted CA was subjected to immunoblot analysis. Lanes: S, supernatant after centrifugation; W, wash; E, eluate. The data are representative of three independent experiments.

FIG 7

Effects of PF74 and CA antibody treatment on PIC-associated integration activity in vitro. (A and B) Integration activity measurement in the presence of PF74. Various concentrations of PF74 (0 to 10 μM) were added to an in vitro integration assay mixture containing wild type Cy-PICs (A) and 5Mut Cy-PICs (B). The integration activity was measured by qPCR, and copies of viral DNA integration were plotted relative to the respective control PICs. (C) Integration activity measurements in the presence of anti-CA antibody. Various amounts of an HIV-1 CA monoclonal antibody or the nonspecific control antibody (anti-gp120 antibody) were added to the in vitro integration assay mixture containing wild-type Cy-PICs, and the integration activities were quantified by qPCR. Viral DNA integration was plotted relative to the respective control PICs. Mean values from three independent experiments are shown, with error bars representing the standard errors of the mean.

FIG 8

PF74 treatment reduces CA levels in the PICs. Cy-PICs isolated from acutely infected SupT1 cells cultured in the presence of 2 μM PF74 were fractionated through a linear gradient of sucrose as described for Fig. 6. Fractions of 1 ml were collected and analyzed for PIC-associated CA by p24 ELISA. (B) CA levels in fractions 10 through 14. (C) Relative levels of CA in PICs of untreated cells versus PF74-treated cells. Representative data from three independent experiments are presented. The data in panels B and C are plotted as mean values from three independent experiments, with error bars representing the standard errors of the mean. *, P < 0.05 (comparison of untreated PICs to PF74-treated PICs).