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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Manipulation of P-TEFb control machinery by HIV: recruitment of P-TEFb from the large form by Tat and binding of HEXIM1 to TAR

Basal transcription of the HIV LTR is highly repressed and requires Tat to recruit the positive transcription elongation factor, P-TEFb, which functions to promote the transition of RNA polymerase II from abortive to productive elongation. P-TEFb is found in two forms in cells, a free, active form and a large, inactive complex that also contains 7SK RNA and HEXIM1 or HEXIM2. Here we show that HIV infection of cells led to the release of P-TEFb from the large form. Consistent with Tat being the cause of this effect, transfection of a FLAG-tagged Tat in 293T cells caused a dramatic shift of P-TEFb out of the large form to a smaller form containing Tat. In vitro, Tat competed with HEXIM1 for binding to 7SK, blocked the formation of the P-TEFb–HEXIM1–7SK complex, and caused the release P-TEFb from a pre-formed P-TEFb–HEXIM1–7SK complex. These findings indicate that Tat can acquire P-TEFb from the large form. In addition, we found that HEXIM1 binds tightly to the HIV 5′ UTR containing TAR and recruits and inhibits P-TEFb activity. This suggests that in the absence of Tat, HEXIM1 may bind to TAR and repress transcription elongation of the HIV LTR

Characterization of P-TEFb retention by HeLa cell nuclei using differential salt extraction

<p><b>Copyright information:</b></p><p>Taken from "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"</p><p>http://www.retrovirology.com/content/4/1/47</p><p>Retrovirology 2007;4():47-47.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1948018.</p><p></p> 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

The P-TEFb inhibitors DRB, seliciclib and flavopiridol release P-TEFb from the large form

<p><b>Copyright information:</b></p><p>Taken from "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"</p><p>http://www.retrovirology.com/content/4/1/47</p><p>Retrovirology 2007;4():47-47.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1948018.</p><p></p> 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

Inhibition of HIV-1 infectivity by non-cytotoxic concentrations of the P-TEFb inhibitors DRB, seliciclib and flavopiridol

<p><b>Copyright information:</b></p><p>Taken from "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"</p><p>http://www.retrovirology.com/content/4/1/47</p><p>Retrovirology 2007;4():47-47.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1948018.</p><p></p> HeLa37 cells were infected with HIV-1and 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