Persistent latent reservoir of replication-competent proviruses in memory CD4 T cells is a major obstacle to curing HIV infection. Pharmacological activation of HIV expression in latently infected cells is being explored as one of the strategies to deplete the latent HIV reservoir. In this study, we characterized the ability of romidepsin (RMD), a histone deacetylase inhibitor approved for the treatment of T-cell lymphomas, to activate the expression of latent HIV. In an in vitro T-cell model of HIV latency, RMD was the most potent inducer of HIV (EC50 = 4.5 nM) compared with vorinostat (VOR; EC50 = 3,950 nM) and other histone deacetylase (HDAC) inhibitors in clinical development including panobinostat (PNB; EC50 = 10 nM). The HIV induction potencies of RMD, VOR, and PNB paralleled their inhibitory activities against multiple human HDAC isoenzymes. In both resting and memory CD4 T cells isolated from HIV-infected patients on suppressive combination antiretroviral therapy (cART), a 4-hour exposure to 40 nM RMD induced a mean 6-fold increase in intracellular HIV RNA levels, whereas a 24-hour treatment with 1 µM VOR resulted in 2- to 3-fold increases. RMD-induced intracellular HIV RNA expression persisted for 48 hours and correlated with sustained inhibition of cell-associated HDAC activity. By comparison, the induction of HIV RNA by VOR and PNB was transient and diminished after 24 hours. RMD also increased levels of extracellular HIV RNA and virions from both memory and resting CD4 T-cell cultures. The activation of HIV expression was observed at RMD concentrations below the drug plasma levels achieved by doses used in patients treated for T-cell lymphomas. In conclusion, RMD induces HIV expression ex vivo at concentrations that can be achieved clinically, indicating that the drug may reactivate latent HIV in patients on suppressive cART.

Antiretroviral therapy (ART) can halt HIV-1 replication but fails to target the long-lived latent viral reservoir. Several pharmacological compounds have been evaluated for their ability to reverse HIV-1 latency, but none has demonstrably reduced the latent HIV-1 reservoir or affected viral rebound after the interruption of ART. We evaluated orally administered selective Toll-like receptor 7 (TLR7) agonists GS-986 and GS-9620 for their ability to induce transient viremia in rhesus macaques infected with simian immunodeficiency virus (SIV) and treated with suppressive ART. In an initial dose-escalation study, and a subsequent dose-optimization study, we found that TLR7 agonists activated multiple innate and adaptive immune cell populations in addition to inducing expression of SIV RNA. We also observed TLR7 agonist–induced reductions in SIV DNA and measured inducible virus from treated animals in ex vivo cell cultures. In a second study, after stopping ART, two of nine treated animals remained aviremic for more than 2 years, even after in vivo CD8+ T cell depletion. Moreover, adoptive transfer of cells from aviremic animals could not induce de novo infection in naïve recipient macaques. These findings suggest that TLR7 agonists may facilitate reduction of the viral reservoir in a subset of SIV-infected rhesus macaques.

A long-term goal of developmental biology is to understand how morphogens establish gradients that promote proper tissue patterning. A number of reports describe the formation of the Wg (Wnt1) gradient in Drosophila and have shown that Porcupine, a predicted membrane-bound O-acyl transferase, is required for the correct distribution of Wg protein. The discovery that Wnts are palmitoylated on a conserved cysteine residue suggests that porcupine activity and Wnt palmitoylation are important for the generation of Wnt gradients. To establish the role of porcupine in Wnt gradient formation in vertebrates, we tested the role of porcupine/Wnt palmitoylation in human embryonic kidney 293T cells and in the chick neural tube. Our results lead us to conclude that: (1) vertebrate Wnt1 and Wnt3a possess at least one additional site for porcupine-mediated lipid-modification; (2)porcupine-mediated lipid-modification of Wnt proteins promotes their activity in 293T cells and in the chick neural tube; and (3) porcupine-mediated lipid-modification reduces the range of activity of Wnt1 and Wnt3a in the chick neural tube. These findings highlight the importance of porcupine-mediated lipid modifications in the formation of vertebrate Wnt activity gradients.

Pyrimidinol carboxylic acids were designed as inhibitors of HIV-1 RNase H function. These molecules can coordinate to two divalent metal ions in the RNase H active site. Inhibition of enzymatic activity was measured in a biochemical assay, but no antiviral effect was observed. Binding was demonstrated via a solid state structure of the isolated p15-Ec domain of HIV-1 RT showing inhibitor and two Mn(II) ions bound to the RNase H active site.

Secreted frizzled related proteins (Sfrps) are extracellular attenuators of Wnt signaling that play important roles in both embryogenesis and oncogenesis. Although Sfrps are generally thought to bind and sequester Wnts away from active receptor complexes, very little is known about the specificity of Sfrp family members for various Wnts. In the developing chick neural tube, sfrp-1, 2, and 3 transcripts are expressed in and adjacent to the dorsal neural tube, where Wnt-1 and Wnt-3a are expressed. To better define the possible roles of Sfrp-1, 2, and 3 in the neural tube, we first tested the ability of purified Sfrps to inhibit Wnt-3a-induced accumulation of ␤-catenin in L cells. We find that both Sfrp-1 and Sfrp-2 can inhibit Wnt-3a activity while Sfrp-3 cannot. To determine where Sfrp-1 and Sfrp-2 impinge on the Wnt signaling pathway, we tested the ability of these Sfrps to inhibit Wnt signaling induced by the addition of LiCl, an inhibitor of GSK-3. Sfrp-1 and Sfrp-2 are unable to inhibit the accumulation of ␤-catenin in LiCl-treated cells, suggesting that the ability of Sfrps to inhibit the accumulation of ␤-catenin is GSK-3 dependent. We have further shown that Sfrp-2 inhibits the ability of ectopic Wnt-3a to stimulate proliferation in the developing chick neural tube. These results provide the framework for understanding how Sfrps function to regulate Wnt-3a activity in developing embryos and in cancer. Developmental Dynamics 235:681-690, 2006.