HIV-1 replication is certainly induced by the excess of iron and
HIV-1 replication is certainly induced by the excess of iron and iron chelation by desferrioxamine (DFO) inhibits viral replication by reducing proliferation of infected cells. at concentrations that inhibited HIV-1 transcription. The chelators decreased cellular activity of CDK2 and reduced HIV-1 Tat phosphorylation by CDK2. The ICL670 and 311 did not decrease CDK9 protein level but significantly reduced association of CDK9 with cyclin T1 and reduced phosphorylation of Ser-2 residues of RNA polymerase II C-terminal domain. In conclusion our findings add to the evidence that iron chelators can inhibit HIV-1 transcription by deregulating inhibiting CDK2 and CDK9. Further consideration should be given to the development of iron chelators for future anti-retroviral therapeutics. (Ammosova et al. 2006 Ammosova et al. 2005 Deng et al. 2002 Nekhai et al. 2002 and that inhibition of CDK2 by CYC202 (R-roscovitine) (Agbottah et al. 2005 or by siRNA (Ammosova et al. 2005 efficiently blocks replication of HIV-1. Moreover Tat is phosphorylated by CDK2 in cultured cells and inhibition of this phosphorylation by mutation of Ser16 and Ser46 residues of Tat blocked HIV-1 transcription and viral replication (Ammosova et al. 2006 Richardson and colleagues showed that the iron chelator 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) inhibited the expression of CDK2 (Gao and Richardson 2001 Thus the effect of iron chelators could conceivably affect the activity of CDK2 and thus inhibit HIV-1 transcription and viral replication. In the present study we analyzed the effect of iron chelators ICL670 and 311 on HIV-1 transcription and on the expression and activities of CDK2 and CDK9 in cultured cells. RESULTS Iron chelators 311 and ICL670 inhibit Tat-induced HIV-1 transcription in TAK-441 CEM T-cells and 293T cells We examined the effect of iron chelators 311 and ICL670 (see their structure in supplemental Fig. 1) on HIV-1 transcription in CEM cells containing an integrated HIV-1 LTR – GFP (CEM-GFP). We infected CEM-GFP cells with adenovirus expressing Tat (Ad-Tat) (Ammosova et al. 2003 Nekhai et TAK-441 al. 2006 In CEM-GFP cells HIV-1 transcription was detectable after infection with Ad-Tat but not with non-relevant Ad-virus (Fig. 1a). Treatment of the Ad-Tat infected CEM-GFP cells with either ICL670 (100 μM) or 311 (10 μM) resulted in the inhibition of Tat-mediated HIV-1 transactivation as visualized by fluorescence (Fig. 1b). Titration of the iron chelators showed that ICL670 inhibited HIV-1 transcription in CEM-GFP cells with IC50=23 μM (Fig 1c) and that 311 inhibited HIV-1 transcription with IC50=2 μM (Fig 1c). To determine whether the effect of the iron chelators might be due to reduced TAK-441 expression of Myod1 Tat we induced Tat-transactivation with purified recombinant Tat protein added to the media in the presence of chloroquine (Frankel and Pabo 1988 Recombinant Tat potently induced HIV-1 transcription in CEM-GFP cells (Fig. TAK-441 2a lane 2). Treatment with 100 μM ICL670 or 10 μM 311 inhibited HIV-1 transcription induced by the extracellular Tat (Fig. 2a lanes 3 and 4). Thus inhibition of HIV-1 transcription by iron chelators was not the result of decreased expression of Tat. We next analyzed the effect of ICL670 and 311 on HIV-1 transcription from HIV-1 genomic construct pNL4-3 Luc in 293T cells. The 293T cells were transfected with pNL4-3 Luc construct and simultaneously treated with 100 μM ICL670 or 10 μM 311. Treatment with chelators inhibited HIV-1 transcription as evidenced by the decrease of luciferase activity (Fig. 2b lanes 2 and 3). We next analyzed the effect of chelators on HIV-1 basal transcription by transiently transfecting 293T cells with HIV-1 LTR-reporter combined with cytomegalovirus (CMV)-EGFP vector to normalize transfection. We also used HIV-1 LTR-in which the TAR-coding sequence was deleted (HIV-1 LTR ΔTAR (Ammosova et al. 2003 Treatment with 10 μM 311 or 100 μM ICL670 inhibited basal HIV-1 transcription from WT or TAR-deleted HIV-1 LTR (Fig. 3a). To determine whether the chelators TAK-441 exclusively affect the HIV-1 promoter we analyzed their effect on viral CMV or cellular phosphoglycerate kinase (PGK) promoters. Neither chelator had a significant effect on transcription from the CMV promoter (Fig. 3b lane 2). In the case of the PGK promoter 311 did not inhibit transcription whereas ICL670 had an inhibitory effect although not as prominent as for the HIV-1 (Fig. 3b lane 1). Fig. 1 Iron chelators 311 TAK-441 and ICL670 inhibit Tat-induced HIV-1 transcription in CEM T-cells Fig. 2 (a) Iron chelators inhibit HIV-1 transcription induced by extracellular Tat Fig. 3 Iron chelators inhibit basal HIV-1.