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-κB 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.

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-B) family. The p65:p50 NF-B heterodimer that predominates in many cell types can undergo stimulated movement, following degradation of the I B inhibitor, as well as shuttling in the absence of stimulation with I B 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-B. A differential dependence of these two modes of transport on the dynein molecular motor and dynactin was found. NF-B 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-B-dependent gene expression showed a dependence on endogenous dynein in a variety of cell types and in response to diverse activating stimuli, suggesting that dyneindependent transport of NF-B may be a conserved mechanism in the NF-B activation pathway and could represent a potential point of regulation.NF-kappaB ͉ nuclear transport ͉ transcription ͉ molecular motor ͉ active transport T he NF-B or Rel transcription factors are ubiquitouslyexpressed regulators of gene expression that are essential for physiological processes ranging from innate and adaptive immunity to learning and memory. NF-B functions as a dimer and is retained in a latent form in the cytoplasm, bound to the inhibitor of NF-B (I B). In the canonical pathway, incoming stimuli trigger activation of the I B-kinase complex (IKK) that phosphorylates I B␣ and leads to its degradation. The subsequent translocation of active I B-free NF-B from cytoplasm to nucleus is critical for NF-B-dependent gene expression. In the absence of I B degradation, NF-B can undergo stimulusindependent movement back and forth between the nucleus and cytoplasm in a process termed shuttling (1). Shuttling of p65:p50 NF-B dimers is believed to result from incomplete masking of the p50 nuclear localization sequence (NLS) by bound I B␣ and can be observed by blocking exportin1/crm1-mediated nuclear export under unstimulated conditions (2).While NF-B requires importin family members to cross the nuclear pore (3), the molecular mechanisms underlying NF-B cytoplasmic movement remain poorly understood. Studies examining the role of microtubules are conflicting, possibly because the pharmacological agents that disrupt microtubules result in a confounding activation of NF-B (4-10). Microtubule-dependent transport can also be probed by disrupting the multisubunit dynactin complex, and it was recently reported that dynactin could play a role in the nuclear accumulation of neuronal NF-B (10). Dynactin regulates the processivity of molecular motors using microtubules, including cytoplasmic dynein and kinesin (11,12). Dynein is a retrograde m...

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