Prostratin Antagonizes HIV Latency by Activating NF-κB

Samuel A Williams,Lin-Feng Chen,Hakju Kwon,David Fenard,Dwayne Bisgrove,Eric Verdin,Warner C Greene

doi:10.1074/jbc.m402124200

Abstract

A subset of quiescent memory CD4 T cells harboring integrated but transcriptionally silent proviruses poses a currently insurmountable barrier to the eradication of the human immunodeficiency virus (HIV) in infected patients. Induction of HIV gene expression in these latently infected cells by immune activating agents has been proposed as one approach to confer sensitivity to antiretroviral therapy. Interest has recently focused on the non-tumor-promoting phorbol ester, prostratin, as a potential agent to activate latent HIV proviruses. Using multiple Jurkat T cell lines containing integrated but transcriptionally latent HIV proviruses (J-Lat cells), we now demonstrate that prostratin effectively activates HIV gene expression in these latently infected cells. We further show that prostratin acts by stimulating IKK-dependent phosphorylation and degradation of IkappaBalpha, leading to the rapid nuclear translocation of NF-kappaB and activation of the HIV-1 long terminal repeat in a kappaB enhancer-dependent manner. In contrast, NFAT and AP-1 are not induced by prostratin. Using chromatin immunoprecipitation assays to identify host transcription factors recruited to the latent HIV-1 promoter in living cells, we find that prostratin induces RelA binding. Analysis of potential upstream signal transducers demonstrates that prostratin stimulates membrane translocation of classical, novel, and atypical protein kinase C (PKC) isoforms. Studies with isoform-specific PKC inhibitors suggest that the novel PKCs play a particularly prominent role in the prostratin response. These findings provide new insights into the molecular pathway through which prostratin antagonizes HIV latency highlighting a central role for the action of NF-kappaB.

Highlights

A small but clinically important fraction of CD4ϩ memory T cells in human immunodeficiency virus (HIV)-1 1-infected patients contain integrated but transcriptionally inactive proviruses [1]
Prostratin Activation of the HIV long terminal repeat (LTR) Is Mediated through NF-␬B—We explored the signaling pathway activated by prostratin in J-Lat cells that mediates activation of the latent HIV proviruses
Prostratin, TNF-␣, and phorbol 12-myristate 13-acetate (PMA) each induced strong green fluorescent protein (GFP) responses, and these responses were not impaired by the addition of cyclosporin A (Fig. 2C). Combined stimulation of these cells with PMA and ionomycin induced higher levels of GFP expression than observed with PMA alone, and this response was partially inhibited by cyclosporin A. These findings suggest that NFAT induction plays a role in the activation of latent HIV provirus in J-Lat cells stimulated with PMA and ionomycin

Summary

Introduction

A small but clinically important fraction of CD4ϩ memory T cells in HIV-1 1-infected patients contain integrated but transcriptionally inactive proviruses [1]. TNF-␣ activated GFP expression in each of the latently infected cell lines to levels comparable with that induced by prostratin. These studies demonstrate that prostratin induces dose-dependent transcriptional activation of the latent HIV provirus present in four independently derived J-Lat cell lines.

Results

Conclusion