Nef is secreted in exosomes from Nef.GFP-expressing and HIV-1-infected human astrocytes.

HIV-1 infection of the central nervous system causes HIV-associated neurocognitive disorders, even in aviremic patients. Although astrocyte malfunction was associated to these disorders, their implication is overshadowed by contributions of microglia and macrophages. Astrocytes are infected with HIV-1 in vivo and express a relevant amount of viral protein Nef. Nef was shown to stimulate its own release in exosomes from diverse cell types, which in turn have damaging effects on neighboring cells. Using immunoblotting and electron microscopy, we showed that human astrocytes expressing Nef.GFP similarly release Nef in exosomes. Importantly, Nef.GFP expression increases the secretion of exosomes from human astrocytes up to 5.5-fold, as determined by total protein content and nanoparticle tracking analysis. Protein analysis of exosomes and viruses separated on iodixanol gradient further showed that native or pseudotyped HIV-1-infected human astrocytes release exosomes, which contain Nef. Our results provide the basis for future studies of the damaging role of Nef-exosomes produced by HIV-infected astrocytes on the central nervous system.

Inhibition of a NEDD8 Cascade Restores Restriction of HIV by APOBEC3G.

Cellular restriction factors help to defend humans against human immunodeficiency virus (HIV). HIV accessory proteins hijack at least three different Cullin-RING ubiquitin ligases, which must be activated by the small ubiquitin-like protein NEDD8, in order to counteract host cellular restriction factors. We found that conjugation of NEDD8 to Cullin-5 by the NEDD8-conjugating enzyme UBE2F is required for HIV Vif-mediated degradation of the host restriction factor APOBEC3G (A3G). Pharmacological inhibition of the NEDD8 E1 by MLN4924 or knockdown of either UBE2F or its RING-protein binding partner RBX2 bypasses the effect of Vif, restoring the restriction of HIV by A3G. NMR mapping and mutational analyses define specificity determinants of the UBE2F NEDD8 cascade. These studies demonstrate that disrupting host NEDD8 cascades presents a novel antiretroviral therapeutic approach enhancing the ability of the immune system to combat HIV.

Reversible phosphorylation of cyclin T1 promotes assembly and stability of P-TEFb.

The positive transcription elongation factor b (P-TEFb) is a critical coactivator for transcription of most cellular and viral genes, including those of HIV. While P-TEFb is regulated by 7SK snRNA in proliferating cells, P-TEFb is absent due to diminished levels of CycT1 in quiescent and terminally differentiated cells, which has remained unexplored. In these cells, we found that CycT1 not bound to CDK9 is rapidly degraded. Moreover, productive CycT1:CDK9 interactions are increased by PKC-mediated phosphorylation of CycT1 in human cells. Conversely, dephosphorylation of CycT1 by PP1 reverses this process. Thus, PKC inhibitors or removal of PKC by chronic activation results in P-TEFb disassembly and CycT1 degradation. This finding not only recapitulates P-TEFb depletion in resting CD4+ T cells but also in anergic T cells. Importantly, our studies reveal mechanisms of P-TEFb inactivation underlying T cell quiescence, anergy, and exhaustion as well as proviral latency and terminally differentiated cells.

Reactivation of latent HIV-1 by new semi-synthetic ingenol esters.

The ability of HIV to establish long-lived latent infection is mainly due to transcriptional silencing of viral genome in resting memory T lymphocytes. Here, we show that new semi-synthetic ingenol esters reactivate latent HIV reservoirs. Amongst the tested compounds, 3-caproyl-ingenol (ING B) was more potent in reactivating latent HIV than known activators such as SAHA, ingenol 3,20-dibenzoate, TNF-α, PMA and HMBA. ING B activated PKC isoforms followed by NF-κB nuclear translocation. As virus reactivation is dependent on intact NF-κB binding sites in the LTR promoter region ING B, we have shown that. ING B was able to reactivate virus transcription in primary HIV-infected resting cells up to 12 fold and up to 25 fold in combination with SAHA. Additionally, ING B promoted up-regulation of P-TEFb subunits CDK9/Cyclin T1. The role of ING B on promoting both transcription initiation and elongation makes this compound a strong candidate for an anti-HIV latency drug combined with suppressive HAART.