HIV-1 replication is induced by an excess of iron and iron chelation by desferrioxamine (DFO) inhibits viral replication by reducing proliferation of infected cells. Treatment of cells with DFO and 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) inhibit expression of proteins that regulate cell-cycle progression, including cycle-dependent kinase 2 (CDK2). Our recent studies showed that CDK2 participates in HIV-1 transcription and viral replication suggesting that inhibition of CDK2 by iron chelators might also affect HIV-1 transcription. Here we evaluated the effect of a clinically approved orally effective iron chelator, 4-[3,5-bis-(hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid (ICL670) and 311 on HIV-1 transcription. Both ICL670 and 311 inhibited Tat-induced HIV-1 transcription in CEM-T cells, 293T and HeLa cells. Neither ICL670 nor 311 induced cytotoxicity at concentrations that inhibited HIV-1 transcription. The chelators decreased cellular activity of CDK2 and reduced HIV-1 Tat phosphorylation by CDK2. Neither ICL670A or 311 decreased 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 CDK2 and CDK9. Further consideration should be given to the development of iron chelators for future anti-retroviral therapeutics.

Background: Transcription of HIV-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of RNA polymerase II (RNAPII) C-terminal domain (CTD) by CDK9/cyclin T1. Earlier we showed that CDK2/cyclin E phosphorylates HIV-1 Tat in vitro. We also showed that CDK2 induces HIV-1 transcription in vitro and that inhibition of CDK2 expression by RNA interference inhibits HIV-1 transcription and viral replication in cultured cells. In the present study, we analyzed whether Tat is phosphorylated in cultured cells by CDK2 and whether Tat phosphorylation has a regulatory effect on HIV-1 transcription.

HIV transcription is induced by the HIV-1 Tat protein, in concert with cellular co-factors including CDK9, CDK2, NF-κB, and others. The cells of most of the body’s organs are exposed to ~3–6% oxygen, but most in vitro studies of HIV replication are conducted at 21% oxygen. We hypothesized that activities of host cell factors involved in HIV-1 replication may differ at 3% versus 21% O2, and that such differences may affect HIV-1 replication. Here we show that Tat-induced HIV-1 transcription was reduced at 3% O2 compared to 21% O2. HIV-1 replication was also reduced in acutely or chronically infected cells cultured at 3% O2 compared to 21% O2. This reduction was not due the decreased cell growth or increased cellular toxicity and also not due to the induction of hypoxic response. At 3% O2, the activity of CDK9/cyclin T1 was inhibited and Sp1 activity was reduced, whereas the activity of other host cell factors such as CDK2 or NF-κB was not affected. CDK9-specific inhibitor ARC was much less efficient at 3% compared to 21% O2 and also expression of CDK9/cyclin T1-dependent IκB inhibitor α was repressed. Our results suggest that lower HIV-1 transcription at 3% O2 compared to 21% O2 may be mediated by lower activity of CDK9/cyclin T1 and Sp1 at 3% O2 and that additional host cell factors such as CDK2 and NF-κB might be major regulators of HIV-1 transcription at low O2 concentrations.

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1509 Poster Board I-532 Background The hypoxic response is an important component of the body 's reaction to impaired tissue oxygenation associated with the anemia and vasoocclusive episodes of sickle cell disease (SCD). It has been reported that HIV infection progresses relatively slowly in patients with SCD (Am J Hematol 1998; 59:199-207). We recently showed that HIV-1 transcription and replication is significantly reduced in cells cultured at 3% versus 21% oxygen (J Cell Physiol 2009; in press). Our previous studies indicated that protein phosphatase-1 (PP1) interacts with HIV-1 transcriptional activator, Tat, and thereby participates in the regulation of HIV-1 transcription. Sickle cell patients are in chronically hypoxic state and we hypothesized that HIV-1 replication in their peripheral blood mononuclear cells (PBMCs) would be slower then in controls. Methods We isolated PBMCs from patients with SCD and from normal subjects, activated the cells with phytohemagglutinin and IL-2 for 24 h, and infected with pseudotyped HIV-1 virus expressing Luciferase. The infected cells were cultured at 3% of oxygen for 72 h. Results We show here that PP1 association with cellular regulatory subunits is modified and that PP1 activity is significantly reduced by 20-40% in different cell lines at 3% versus 21% oxygen. One round of replication of pseudotyped HIV-1 Luciferase virus normalized to the number of the cells in culture was significantly reduced in SCD PBMCs comparing to normal controls. Conclusions Our results provide a direct evidence of that HIV-1 replication may be slower in SCD-derived PBMCs. In future, we will analyze PP1 activity and the association of PP1 with regulatory subunits in SCD PBMCs. Understanding of how oxygen status influences HIV-1 replication might open new possibilities for treatment of hidden HIV-1 reservoirs that harbor non-replicating HIV-1 virus. Acknowledgments This work was supported by NHLBI Research Grant 2 R25 HL003679-08 from the National Institutes of Health and The Office of Research on Minority Health. Disclosures No relevant conflicts of interest to declare.