Small-molecule screening using a human primary cell model of HIV latency identifies compounds that reverse latency without cellular activation.

The development of highly active antiretroviral therapy (HAART) to treat individuals infected with HIV-1 has dramatically improved patient outcomes, but HAART still fails to cure the infection. The latent viral reservoir in resting CD4+ T cells is a major barrier to virus eradication. Elimination of this reservoir requires reactivation of the latent virus. However, strategies for reactivating HIV-1 through nonspecific T cell activation have clinically unacceptable toxicities. We describe here the development of what we believe to be a novel in vitro model of HIV-1 latency that we used to search for compounds that can reverse latency. Human primary CD4+ T cells were transduced with the prosurvival molecule Bcl-2, and the resulting cells were shown to recapitulate the quiescent state of resting CD4+ T cells in vivo. Using this model system, we screened small-molecule libraries and identified a compound that reactivated latent HIV-1 without inducing global T cell activation, 5-hydroxynaphthalene-1,4-dione (5HN). Unlike previously described latency-reversing agents, 5HN activated latent HIV-1 through ROS and NF-kappaB without affecting nuclear factor of activated T cells (NFAT) and PKC, demonstrating that TCR pathways can be dissected and utilized to purge latent virus. Our study expands the number of classes of latency-reversing therapeutics and demonstrates the utility of this in vitro model for finding strategies to eradicate HIV-1 infection.

Stimulated nuclear translocation of NF-kappaB and shuttling differentially depend on dynein and the dynactin complex.

Translocation from the cytoplasm to the nucleus is required for the regulation of gene expression by transcription factors of the nuclear factor kappa B (NF-kappaB) family. The p65:p50 NF-kappaB heterodimer that predominates in many cell types can undergo stimulated movement, following degradation of the IkappaB inhibitor, as well as shuttling in the absence of stimulation with IkappaB bound. Disruption of the dynactin complex and knockdown of endogenous dynein were used to investigate the nuclear translocation requirements for stimulated and shuttling movement of NF-kappaB. A differential dependence of these two modes of transport on the dynein molecular motor and dynactin was found. NF-kappaB used active dynein-dependent transport following stimulation while translocation during shuttling was mediated by a dynein-independent pathway that could be potentiated by dynactin disruption, consistent with a process of facilitated diffusion. Nuclear translocation and activation of NF-kappaB-dependent gene expression showed a dependence on endogenous dynein in a variety of cell types and in response to diverse activating stimuli, suggesting that dynein-dependent transport of NF-kappaB may be a conserved mechanism in the NF-kappaB activation pathway and could represent a potential point of regulation.