Prostratin induces HIV reactivation and downregulates HIV receptors and co-receptor expression in PBMCs.

Joaquín Rullasº, Mercedes Bermejoº, Javier García-Pérezº, Manuela Beltránº, Stephen J.Brown+ and José Alcamíº. º AIDS Immunopathogenesis Unit. Centro Nacional de Microbiología. Instituto de Salud Carlos III. 28220 Majadahonda (Spain); + AIDS Research Alliance, 621-A North San Vincente Blvd., West Hollywood, CA 90069 (USA).

Background: Induction of HIV reactivation, through lymphocyte activation has been proposed as a strategy to purge latent reservoirs. Prostratin (Pr) is a non-tumorogenic phorbol ester that delays HIV replication “in vitro”, but paradoxically, Pr can reactivate HIV in latently infected PBMCs. To get a better insight into the mechanisms of action of Pr, we have analyzed the effect of Pr on HIV reactivation and coreceptor expression in lymphocytes. Material and Methods: We have developed an efficient transfection system of resting lymphocytes. PBMCs were transfected with the following plasmids: luciferase expression constructs under the control of wild type HIV-LTR (pLTR-luc), LTR kB-deleted sequences (pLTR-DkB-luc) or a plasmid containing a full length provirus (pNL-luc). Cells were stimulated with Pr (0.1-10 µM) or PMA (25 ng/ml). The effect of Pr on HIV transcription was assessed by quantifying luciferase activity in cell extracts and p24 levels in culture supernatants. Induction of cRel/NF-kB family of transcription factors was analyzed by gel-shift assay. The expression of CD4, CCR5, CXCR4, CD69, and CD25 on PBMCs was assessed by flow cytometry 6 and 24 hours after stimulation. For “in vitro” infection experiments, PBMCs were infected with NL4.3 or BaL HIV strains. Results: Pr induced HIV transcription as detected by an increase in luciferase activity from LTR-driven plasmids. Treatment with Pr also produced an increase in p24 levels when PBMCs were transfected with a plasmid containing a full length provirus. Pr induced NF-kB translocation and inhibited the expression of HIV coreceptors CCR5 and CXCR4 on PBMCs. Downregulation of HIV receptors by Pr correlated with a decrease in propagation of both R5 and X4 HIV strains in PBMCs. Conclusion: Our data show two different effects for prostratin: induction of HIV transcription and down-regulation of HIV coreceptors. This latter effect could explain the delay in HIV propagation in prostratin-treated cultures. Altogether these results support the use of prostratin in combination with other antiretrovirals to reactivate HIV from latency and purge viral reservoirs.