Mechanisms of HIV receptor and co-Receptor Downregulation by Prostratin; a Potential Candidate for Inductive Adjuvant Therapy for HAART.

Marjan Hezareh², Moulay Moukil¹, Malgorzata Pondarzewski¹, Stephen J. Brown², Jean-Louis Carpentier¹ and Michelangelo Foti^{1¶ 1}Dept of Morphology, Faculty of Medicine, Geneva (Switzerland) and ²AIDS Research Alliance (ARA), West Hollywood, California (USA).

Background: Prostratin, a non-tumor promoting phorbol ester exhibits potent antiviral activity against acute infection by multiple strains of HIV. It also activates viral gene expression from latently infected cells. Prostratin’s lack of tumor promotion combined with its ability to up-regulate latent HIV-1 provirus expression could be exploited therapeutically to target latent reservoirs in patients on HAART. We therefore investigated the mechanism of prostratin’s activity, by characterizing intracellular events leading to the post transcriptional downregulation of surface HIV receptor and coreceptors expression. Material and Methods: Cell surface expression and trafficking of CD4, CCR5, and CXCR4 was investigated by FACS-based analyses. Protein kinase C translocations were assessed by subcellular fractionation followed by western analysis or immunofluorescence microscopy. Results: Prostratin induces downregulation of surface expression of CD4 and CXCR4, but not CCR5, in lymphocytic and monocytic cell lineages. The extent of CD4 and CXCR4 downregulation by prostratin is cell type dependent and can reach downregulation levels induced by PMA. Downregulation of CD4 and CXCR4 by prostratin and PMA is achieved by receptor mediated internalization in lymphocytic cell lines, whereas both receptor-mediated endocytosis and macropinocytosis contribute to CD4 downregulation in monocytic cell lines. We examined the potential contribution of various PKC isoforms to downregulate CD4 and CXCR4 in response to prostratin and/or PMA stimulation. Although a short exposure of cells to prostratin or PMA induces the translocation of various PKC isoforms to plasma membranes, the use of specific PKC inhibitors revealed that conventional and novel PKC are the main mediators of CD4 and CXCR4 downregulation by prostratin. Conclusion: Altogether these results showed that prostratin, similarly to PMA, through the activation of novel PKC isoforms, rapidly reduces cell surface expression of CD4 and CXCR4, but not CCR5, by inducing their internalization and degradation. Future potential adjunctive use of prostratin, would probably envision a pulse or cycle administration of this compound to successfully deplete latent viral reservoirs. This rules out permanent internalization of CD4 and CXCR4 receptors and also downregulation of PKC due to the long term presence of an activator.