Drug 9AA reactivates p21/Waf1 and Inhibits HIV-1 progeny formation

Abstract

It has been demonstrated that the p53 pathway plays an important role in HIV-1 infection. Previous work from our lab has established a model demonstrating how p53 could become inactivated in HIV-1 infected cells through binding to Tat. Subsequently, p53 was inactivated and lost its ability to transactivate its downstream target gene p21/waf1. P21/waf1 is a well-known cdk inhibitor (CKI) that can lead to cell cycle arrest upon DNA damage. Most recently, the p21/waf1 function was further investigated as a molecular barrier for HIV-1 infection of stem cells. Therefore, we reason that the restoration of the p53 and p21/waf1 pathways could be a possible theraputical arsenal for combating HIV-1 infection. In this current study, we show that a small chemical molecule, 9-aminoacridine (9AA) at low concentrations, could efficiently reactivate p53 pathway and thereby restoring the p21/waf1 function. Further, we show that the 9AA could significantly inhibit virus replication in activated PBMCs, likely through a mechanism of inhibiting the viral replication machinery. A mechanism study reveals that the phosphorylated p53ser15 may be dissociated from binding to HIV-1 Tat protein, thereby activating the p21/waf1 gene. Finally, we also show that the 9AA-activated p21/waf1 is recruited to HIV-1 preintegration complex, through a mechanism yet to be elucidated.

Figure 1

9AA activates phosphorylation of p53 at Ser15. CEM and ACH2 cells were treated with drug 9AA, P44 peptide or DMSO as mock control, respectively. Cells were harvested 24 hrs after treatment. Cells were then lysed and subjected to western blot for p53, p53ser15 and p21/waf1 (anti-p53, anti-p53 ser15 from Cell Signaling, anti-p21/waf1 from Santa Cruz Biotechnology). (A) CEM cells, uninfected cells. (B) ACH2 cells, HIV-1 infected cells. (C) ACH2 cells were treated with 9AA at a concentration of 2.5 uM. Cells were collected at different time points and then subjected to western blot for detection of the phosphorylation of p53ser 15. Actin was used as a loading control.

Figure 2

9AA-induced inhibitory effects on cdk2/cyclin E activity in infected and uninfected cells. (A) ACH2 and CEM cells were treated with various concentrations of 9AA (0.1, 0.5, 1.0 uM) for 24 hrs. Cells were harvested and lysed for immunoprecipitation (IP) with α-Cyc E ab followed by kinase assays. Histone H1 was used as substrate and was added to each reaction tube along with (γ-³²P) ATP (3000 Ci/mmol). Reactions were incubated at 37°C for 30 minutes and stopped by the addition Laemmli buffer. The samples were then separated on a 4–20% Tris-Glycine gel. The gel was dried and exposed to a PhosphorImager cassette and analyzed utilizing Molecular Dynamic's ImageQuant Software. (B) The lysates from (A) were subjected to western blot to evaluate the levels of cdk2 in samples treated with 9AA at different concentrations (0, 0.1, 0.5, 1.0 uM).

Figure 3

9AA inhibits HIV-1 viral replication in PBMCs. Phytohemagglutinin-activated PBMCs were kept in culture for 2 days prior to infection. Isolation and treatment of PBMCs were performed by following the guidelines of the Centers for Disease Control. Approximately 5 × 10⁶ PBMCs were infected with pNL4 (MOI: 1.0). 9AA treatment (0, 0.1, 0.5 and 1.0 uM) was performed immediately after the addition of fresh medium. (A) Samples were collected every 6th day and stored at -20°C for RT assays. (B) Cells were also counted (~100/date) for viability using trypan blue staining.

Figure 4

Phosphorylated p53ser15 doesn't interact with HIV-1 Tat protein. (A) ACH2 cells were transfected with FLAG-Tat and treated with 9AA or DMSO as a mock control. Expression of FLAG-Tat was detected by anti-FLAG (Sigma). Reactivation of p53ser15 was evaluated by anti-p53ser15 (Cell Signaling). (B) The cell lysates were then used for immunoprecipitation with anti-p53 or anti-p53ser15 (Cell Signaling). The immunopreciptated complexes were separated on 4–20% SDS-PAGE gels and then submitted to western blot to detect the presence of FLAG-Tat.

Figure 5

P21/waf1 is recruited to HIV-1 preintegration complex (PIC). ACH2 cells were treated with 9AA or DMSO as mock control. Cells were then harvested, lysed and submitted to immunoprecipitation with anti-MA or anti-RT (ABI Inc., Columbia, MD). The immunopreciptated complexes were separated on 4–20% SDS-PAGE gels and then submitted to western blot to detect the presence p21/waf1. P21/waf1 is found to be present in the anti-MA immunoprecipitation complex.