9-Aminoacridine inhibition of HIV-1 Tat dependent transcription

Abstract

As part of a continued search for more efficient anti-HIV-1 drugs, we are focusing on the possibility that small molecules could efficiently inhibit HIV-1 replication through the restoration of p53 and p21WAF1 functions, which are inactivated by HIV-1 infection. Here we describe the molecular mechanism of 9-aminoacridine (9AA) mediated HIV-1 inhibition. 9AA treatment resulted in inhibition of HIV LTR transcription in a specific manner that was highly dependent on the presence and location of the amino moiety. Importantly, virus replication was found to be inhibited in HIV-1 infected cell lines by 9AA in a dose-dependent manner without inhibiting cellular proliferation or inducing cell death. 9AA inhibited viral replication in both p53 wildtype and p53 mutant cells, indicating that there is another p53 independent factor that was critical for HIV inhibition. p21WAF1 is an ideal candidate as p21WAF1 levels were increased in both p53 wildtype and p53 mutant cells, and p21WAF1 was found to be phosphorylated at S146, an event previously shown to increase its stability. Furthermore, we observed p21WAF1 in complex with cyclin T1 and cdk9 in vitro, suggesting a direct role of p21WAF1 in HIV transcription inhibition. Finally, 9AA treatment resulted in loss of cdk9 from the viral promoter, providing one possible mechanism of transcriptional inhibition. Thus, 9AA treatment was highly efficient at reactivating the p53 - p21WAF1 pathway and consequently inhibiting HIV replication and transcription.

Figure 1

9AA inhibits HIV-LTR transcription. A) CEM cells were transfected with 2.5 μg HIV-LTR CAT and 0.5 μg of pc-Tat by electroporation. Twenty-four hours post-transfection, cells were treated with DMSO, 9AA (0.1, 0.5, 1, or 2.5 μM), or 100 nM Flavo (flavopiridol). Cells were harvested 48 hours post transfection and processed for CAT assays. CAT assays were performed with 4 mM acetyl CoA, 5 μl of 14C-chloramphenicol (40 mCi/mmole), 10 μl of protein extracts, and 18 μl of water. Reactions were carried out at 37°C for 30 minutes. Samples were extracted with ethyl acetate, dried, and separated by TLC. B) TZM-bl cells were transfected with 1.0 μg of Tat and treated the next day with DMSO, 9AA (0.1, 0.5, 1, or 2.5 μM), or 100 nM flavopiridol. Cells were processed 48 hours post drug treatment for luciferase assays. Assays were performed in triplicate and an average value is shown plus standard deviation. C) Structures of 9AA and derivatives. D) TZM-bl cells were transfected with 1.0 μg of Tat and treated the next day with DMSO, 1 or 10 μM of 9AA, 2AA, 4AA, AH, 4AQ, or 100 nM flavopiridol. Cells were processed 48 hours post drug treatment for luciferase assays. Assays were performed in duplicate and an average value is shown.

Figure 2

9AA inhibits viral replication in cells with mutant p53. HIV-1 infected (J1.1 and ACH2) T-cells were treated with 0.5, 1, and 5 μM of 9AA. A) RT activity was determined by at seven day post treatment. B) Cell viability was determined by trypan blue staining (~100/sample) seven days post treatment. C) J1.1 cells were treated with DMSO, 1, 2.5, and 5 μM 9AA and collected after 48 hours. Western blots were performed with anti-p21WAF1, anti-p53, anti-p53-phospho-S15, and actin antibodies.

Figure 3

9AA does not inhibit proliferation of uninfected cells. Uninfected (CEM and Jurkat) and HIV-1 infected (ACH2 and J1.1) T-cells were treated with DMSO, 1, 10, or 50 μM of A) 9AA, B) 2AA, C) 4AA, D) AH. Cell proliferation/viability was determined by MTT assays. Treatments were performed in triplicate and samples analyzed at 48 hours.

Figure 4

9AA induces AKT activity and stabilization of p21WAF1. Uninfected (CEM and Jurkat) and HIV-1 infected (ACH2 and J1.1) T-cells were treated with DMSO, 1, 2.5, or 5 μM of 9AA and collected 48 hours later. Western blot analysis was performed for Akt (pan), phospho-Akt (S473), phospho-Akt (T308), GSK3-β (pan), phospho-GSK3-β (S9), phospho-PDK1 (S241), phospho-p21WAF1 (S146), and actin.

Figure 5

p21WAF1 binds to cyclin T and cdk9 in vitro. One μg of GST, GST-p21, GST-p21 (C), or GST-p21 (N) were added to 1 mg of CEM cell lysates and allowed to bind overnight. The next day, complexes were bound to glutathione sepharose beads, washed, and analyzed by SDS-PAGE, followed by western blotting for cyclin T and cdk9 (Panel A) or cdk2 (Panel B). ext = Extract.

Figure 6

9AA treatment results in loss of cdk9 from the HIV-1 LTR. ACH2 cells were treated with either DMSO or 2.5 μM 9AA for 48 hours, cross-linked and collected for ChIP analysis. Antibodies against cdk9, histone H3-phospho-Ser10 (H3-pS10), RNA Polymerase II (Pol II), and rabbit IgG were utilized.

Figure 7

Model of 9AA induced activation of p21WAF1 in HIV-1 infected cells. In HIV-1 infected cells, p53 is inactivated through the binding to Tat. Upon 9AA treatment, p53 becomes phosphorylated at Ser15, resulting in loss of Tat binding and transcription of p53 dependent promoters, specifically p21WAF1. p21WAF1 protein levels are increased and this newly formed p21WAF1 is able to bind to both cycT/cdk9 and cycE/cdk2 complexes. Binding of p21WAF1 to cyclin/cdk complexes results in inhibition of HIV-1 LTR transcription possibly through decreased RNA Pol II and/or histone H1 phosphorylation. p21WAF1 can also be activated by 9AA treatment in p53 mutant cells by a yet undefined mechanism.