During HIV-1 replication, the host polymerase (Pol II) is recruited to the viral promoter within the long terminal repeat (LTR) and initiates transcription . Pol II initiates transcription, but elongation of most of the transcripts is blocked by negative elongation factors [2, 3]. 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]]. P-TEFb phosphorylates both the carboxyl-terminal domain (CTD) of Pol II  and the negative elongation factors [2, 7] allowing Pol II to transition from abortive to productive elongation .
P-TEFb is found within a cell in two forms referred to as large and free forms [9, 10]. The kinase active, free form contains Cdk9 and one of several cyclin regulatory subunits, cyclin T1, cyclin T2a, cyclin T2b or cyclin K, with cyclin T1 being the predominantly associated cyclin in many cell types [11, 12]. The kinase inactive, large form of P-TEFb additionally contains 7SK RNA [9, 10] and hexamethylene bisacetamide-induced protein 1 (HEXIM1) [13, 14] or HEXIM2 . In HeLa cells, between 50% and 90% of P-TEFb is present in the large form of the complex while the remainder of P-TEFb is in the kinase active, free form [9, 10, 14, 15]. It is hypothesized that the large form of P-TEFb serves a reservoir for the free form.
All currently approved anti-HIV therapies target viral proteins that have been shown to rapidly evolve under the selective pressure of highly active anti-retroviral therapy (HAART) [16–18]. Mutations in the viral genome that decrease the effectiveness of HAART arise as a result of the selection of random mutations generated by the lack of proofreading activity in HIV reverse transcriptase [17, 19] and by G to A hypermutation that is believed to result from APOBEC3G restriction . Thus, identification and characterization of additional anti-virals is a necessity. Anti-virals against cellular targets that are required for virus replication may prove to be highly effective. Furthermore, evolution of HIV resistance to this group of compounds might be less likely. Consistent with this possibility, an extensive 6 month study aimed at generating a HIV-1 strain resistant to the cyclin-dependent kinase inhibitor, roscovitine, proved unsuccessful .
Targeting P-TEFb kinase activity as an anti-HIV therapy is potentially attractive, but has not been extensively evaluated. The P-TEFb inhibitors DRB and flavopiridol have been demonstrated to effectively inhibit HIV Tat-dependent transcription in cell lines [22, 23]. Limited studies of the effect of these inhibitors on HIV replication demonstrate a significant reduction of replication at concentrations with limited cytotoxicity [22, 23]. The anti-retroviral activity of roscovitine or the R-enantiomer of roscovitine (seliciclib or Cyc202) has also been explored. This inhibitor has a spectrum of inhibitory activities against a number of cyclin dependent kinases including Cdk 1, 2, 7 and 9 . A previous examination of the effect of seliciclib on HIV replication had focused on its inhibition of Cdk2 activity .
The use of P-TEFb inhibitors as chemotherapeutic agents against cancers has also been proposed . Flavopiridol and seliciclib showed modest cytotoxicity when tested in clinical trials against different kinds of cancers [reviewed on ]. In phase II cancer clinical trials, fatigue, venous thromboses and diarrhea were the primarily side effects of flavopiridol infusions that achieved plasma flavopiridol levels of approximately 400 nM during a 72 hour treatment period [28–31]. Phase II monotherapy trials with flavopiridol have proved disappointing  and newer studies have combined flavopiridol with other chemotherapeutic agents [32, 33]. Seliciclib has recently been tested as a chemotherapeutic agent in Phase I trials and was shown to cause fatigue and elevated creatinine at the highest tested doses that achieved maximal plasma levels of 2 to 4 μg/ml [24, 34].
In this study, we sought to characterize the anti-HIV activity of the cyclin-dependent kinase inhibitors DRB, flavopiridol and seliciclib. In HeLa cells, we found that the anti-HIV activity of these compounds correlated with concentrations that released free P-TEFb from the large form of the complex. These concentrations were not cytotoxic to cells despite the known requirement of P-TEFb activity for Pol II-dependent transcript elongation. However, the concentration of these compounds that was needed to inhibit HIV replication in PBLs and MDMs was higher. Compound cytotoxicity was also greater in these primary cells decreasing the likely utility of these compounds in controlling HIV replication in infected individuals.