Background:  Most HAART regimens eventually fail to provide complete and long-term suppression of virus replication due to the inability to fully clear virus replication in non-dividing cellular reservoirs. The HIV-1 viral protein R, Vpr, increases virus replication in T cells and is necessary for the infection of primary monocytes/macrophages and other non-dividing cells. It has been shown that Vpr interacts with the cellular glucocorticoid teceptor (GR) and transactivates the HIV-1 LTR through GRE and that this event can be blocked by the GR inhibitor, mifepristone. Based on these observations, we believe that targeting Vpr-mediated virus transcription with glucocorticoid antagonists can demonstrate a potent anti-retroviral therapy.

Methods:  Inhibition of HIV-1 Vpr-mediated GRE and LTR transactivation was demonstrated in 293T, HeLa, and COS-1 cells. HIV-1 LTR-luciferase or GRE-luciferase reporter plasmids were co-transfected with pVpr in 293T and HeLa cells and co-transfected with both pVpr and pGR in COS-1 cells in the presence of mifepristone and its analogs. Antiviral effects of mifepristone and analog compounds were determined through infectivity assays using dual-tropic and macrophage-tropic viruses. Normal donor PBMC and U1 cells were infected and compounds were added in a dose-dependent and time-dependent manner and p24 was analyzed by ELISA. Cytotoxicity assays were performed using PBMC following standard MTT protocol.

Results:  Our results show that Vpr-induced transactivation of both autologous and heterologous promoters was inhibited by mifepristone and analogues in a dose-dependent manner. Three analogues inhibited LTR or GRE transactivation by as much as >90% at a 1 mM concentration. Infectivity assays using dual-tropic and macrophage-tropic viruses, demonstrated antiviral effects on a dose-dependent regimen of mifepristone analogs. The effects of mifepristone analogs were also tested in HIV-1 latent cells that could be activated with extracellular Vpr protein. Cytotoxic effects of mifepristone and its analogs demonstrated that the CT50 of these compounds range from 10 to 40 mM in normal human primary cells.

Conclusions:  By utilizing the relationship between Vpr and the glucocortoicoid receptor, glucocorticoid antagonists such as mifepristone and mifepristone analogs hold promise for anti-retroviral therapy.  

Keywords: Antiviral; Viral Inhibition; LTR-Induced Transactivation