Inhibition of HIV-1 replication by P-TEFb inhibitors DRB, seliciclib and flavopiridol correlates with release of free P-TEFb from the large, inactive form of the complex

Abstract

Background: The positive transcription elongation factor, P-TEFb, comprised of cyclin dependent kinase 9 (Cdk9) and cyclin T1, T2 or K regulates the productive elongation phase of RNA polymerase II (Pol II) dependent transcription of cellular and integrated viral genes. P-TEFb containing cyclin T1 is recruited to the HIV long terminal repeat (LTR) by binding to HIV Tat which in turn binds to the nascent HIV transcript. Within the cell, P-TEFb exists as a kinase-active, free form and a larger, kinase-inactive form that is believed to serve as a reservoir for the smaller form.

Results: We developed a method to rapidly quantitate the relative amounts of the two forms based on differential nuclear extraction. Using this technique, we found that titration of the P-TEFb inhibitors flavopiridol, DRB and seliciclib onto HeLa cells that support HIV replication led to a dose dependent loss of the large form of P-TEFb. Importantly, the reduction in the large form correlated with a reduction in HIV-1 replication such that when 50% of the large form was gone, HIV-1 replication was reduced by 50%. Some of the compounds were able to effectively block HIV replication without having a significant impact on cell viability. The most effective P-TEFb inhibitor flavopiridol was evaluated against HIV-1 in the physiologically relevant cell types, peripheral blood lymphocytes (PBLs) and monocyte derived macrophages (MDMs). Flavopiridol was found to have a smaller therapeutic index (LD50/IC50) in long term HIV-1 infectivity studies in primary cells due to greater cytotoxicity and reduced efficacy at blocking HIV-1 replication.

Conclusion: Initial short term studies with P-TEFb inhibitors demonstrated a dose dependent loss of the large form of P-TEFb within the cell and a concomitant reduction in HIV-1 infectivity without significant cytotoxicity. These findings suggested that inhibitors of P-TEFb may serve as effective anti-HIV-1 therapies. However, longer term HIV-1 replication studies indicated that these inhibitors were more cytotoxic and less efficacious against HIV-1 in the primary cell cultures.

Figure 1

Effects of P-TEFb inhibitors on the kinase activity of P-TEFb in vitro and on the large form of P-TEFb in cells. In vitro P-TEFb kinase assays were performed using recombinant P-TEFb, Pol II CTD or DSIF in the presence of increasing concentrations of seliciclib (A) or flavopiridol (B). The kinase reactions were resolved by SDS-PAGE and the amount of incorporated γ-³²P-ATP was quantitated with a Packard InstantImager. (C and D) Glycerol gradient analysis of HeLa37 cells treated with DRB. (C) HeLa37 cells were treated with increasing amounts of DRB for 1 hour and lysed to extract both forms of P-TEFb from the nucleus. The lysates were subjected to glycerol gradient sedimentation and the fractions were examined by immunoblotting for Cdk9 and cyclin T1. (D) The Cdk9 and cyclin T1 signals in the free (fractions 3–6) and large (fractions 8–11) forms of P-TEFb were calculated and plotted as a function of the concentration of DRB used in the cell treatment.

Figure 2

Characterization of P-TEFb retention by HeLa cell nuclei using differential salt extraction. Untreated HeLa cells and HeLa cells treated for 1 hour with 100 μM DRB were lysed with a buffer containing the indicated amounts of NaCl to generate cytosolic extracts (CE). The CE and the nuclear pellet (NP) were examined by immunoblotting with the indicated antibodies for the presence of P-TEFb or the TFIIH components p62, Cdk7 and cyclin H.

Figure 3

The P-TEFb inhibitors DRB, seliciclib and flavopiridol release P-TEFb from the large form. Low-salt cytosolic extract (CE) containing the large form of P-TEFb and high-salt nuclear extracts (NE) containing the free form of P-TEFb were generated from (A) DRB-treated HeLa cells, (B) DRB treated Jurkat cells, (C) seliciclib-treated HeLa37 cells or (D) flavopiridol-treated Jurkat cells. Quantitative western blotting was performed on low salt cytosolic extracts (CE) and high-salt nuclear extracts (NE) to detect the percentage of Cdk9 and cyclin T1 present in the free and large form of the P-TEFb complex. The percent of P-TEFb in the large form of the complex (low-salt or CE) was calculated as a fraction of the total amount of P-TEFb (low-salt + high-salt P-TEFb) and plotted as a function of the concentration of P-TEFb inhibitor.

Figure 4

Inhibition of HIV-1 infectivity by non-cytotoxic concentrations of the P-TEFb inhibitors DRB, seliciclib and flavopiridol. HeLa37 cells were infected with HIV-1_p256 and treated with the indicated amounts of (A) DRB, (B) seliciclib and (C) flavopiridol. After 40 hours the cells were fixed, immunostained for HIV antigens and the number of HIV positive cells counted. The number of infected cells (solid circles) was normalized to the control infection and plotted. Cell viability studies were performed in parallel. Values form cytotoxicity studies (open circles) were normalized to the mock treated cells and plotted.

Figure 5

Inhibition of HIV replication in PBLs by flavopiridol. Isolated PBLs from three independent donors (A, B and C) were infected with HIV-1_p256 and treated with increasing concentrations of flavopiridol. Supernatants were collected at 4, 8, 12 and 16 days post-infection. The amount of HIV-1 infection was measured by quantifying the amount of HIV-1 reverse transcriptase enzyme (RT) in the supernatants on the indicated days (BOTTOM graph for of each panel). Cytotoxicity studies were performed on uninfected PBLs by treating cells with increasing concentrations of flavopiridol for 4, 8, 12 and 16 days. Cell viability was estimated by performing ATPLite assay. The light readings were normalized to the mock treated cells and plotted (TOP graph for each panel).

Figure 6

Inhibition of HIV-1 replication in MDMs by flavopiridol. MDMs were isolated from healthy donors and infected with HIV-1_p256 along with increasing concentrations of flavopiridol. Supernatants were collected at 4, 8, 12 and 16 days post infection. The amount of HIV-1 infection was measured by quantifying the amount of HIV reverse transcriptase enzyme (RT) in the supernatants on the indicated days (BOTTOM graph). Cytotoxicity studies were performed on uninfected MDMs by treating cells with different concentrations of flavopiridol for 4, 8, 12 and 16 days and measuring cell viability by the ATPLite assay. The light readings were normalized to the mock treated cells and plotted (TOP graph). The experiment was performed in MDMs twice and the data from both experiments was pooled, averaged and graphed.