394 Prostratin Induces Internalization and Degradation of HIV-specific Receptor and Co-receptor through Activation of Novel PKCs M. Hezareh*1, P. Dudognon2, M. Pondarzewski2, S. J. Brown1, J.-L. Carpentier2, M. Foti2 1AIDS Res Alliance, West Hollywood, CA and 2Ctr Med Univ, Geneva, Switzerland
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. Prostratinís lack of tumor and its ability to up-regulate latent HIV-1 provirus expression could be exploited therapeutically to target latent reservoirs in patients (pts) on HAART. The antiviral mechanism of prostratin is not well defined. Based on previous studies it is likely that the anti-viral activity is related to the down-regulation of HIV specific receptor (CD4) and co-receptor (CCR5, CXCR4) expression. In this study, we investigate mechanisms by which prostratin down-regulates HIV receptor and co-receptors surface expression in lymphocytic and monocytic cell lines.
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.
Results: Prostratin induces down-regulation of surface expression of CD4 and CXCR4, but not CCR5, in lymphocytic and monocytic cell lineages. The extent of CD4 and CXCR4 down-regulation by prostratin is cell-type dependent and can reach down-regulation levels induced by PMA. Down-regulation 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 down-regulation in monocytic cell lines.
We examined the potential contribution of various PKC isoforms to down-regulate CD4 and CXCR4 in response to prostratin and/or PMA stimulation. Whereas a short exposure of cells to prostratin or PMA induces the translocation of various isoforms to membranes, the use of specific PKC inhibitors revealed that conventional and novel PKCs are the main mediators of CD4 and CXCR4 down-regulation by prostratin.
Conclusion: Prostratin, similarly to PMA, rapidly reduces cell surface expression of CD4 and CXCR4, but not CCR5, by inducing their endocytosis. Internalization of CD4 and CXCR4 is mediated by the activation of conventional and novel PKCs in response to prostratin/PMA.