Abstract
BackgroundThe 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.ResultsWe 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.ConclusionInitial 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.
Highlights
The positive transcription elongation factor, positive transcription elongation factor b (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
Inhibition of Cdk9 kinase activity by P-TEFb inhibitors To determine the effectiveness of preparations of the cyclin dependent kinase inhibitors, flavopiridol and seliciclib, we performed in vitro kinase assays with recombinant P-TEFb
This is because, except for flavopiridol, all P-TEFb inhibitors are competitive with ATP and the absolute infection by 50% (IC50)'s are dependent on the ATP concentration [22]
Summary
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. During HIV-1 replication, the host polymerase (Pol II) is recruited to the viral promoter within the long terminal repeat (LTR) and initiates transcription [1]. The HIV-1 transcription transactivator Tat binds to the bulge of the HIV-1 RNA stem loop termed TAR that is found in all nascent HIV-1 messages and recruits positive transcription elongation factor b (P-TEFb) to the LTR [reviewed in [4,5]]. It is hypothesized that the large form of P-TEFb serves a reservoir for the free form
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