Evaluating a New Class of AKT/mTOR Activators for HIV Latency Reversing Activity Ex Vivo and In Vivo.

An ability to activate latent HIV-1 expression could benefit many HIV cure strategies, but the first generation of latency reversing agents (LRAs) has proven disappointing. We evaluated AKT/mTOR activators as a potential new class of LRAs. Two glycogen synthase kinase-3 inhibitors (GSK-3i's), SB-216763 and tideglusib (the latter already in phase II clinical trials) that activate AKT/mTOR signaling were tested. These GSK-3i's reactivated latent HIV-1 present in blood samples from aviremic individuals on antiretroviral therapy (ART) in the absence of T cell activation, release of inflammatory cytokines, cell toxicity, or impaired effector function of cytotoxic T lymphocytes or NK cells. However, when administered in vivo to SIV-infected rhesus macaques on suppressive ART, tideglusib exhibited poor pharmacodynamic properties and resulted in no clear evidence of significant SIV latency reversal. Whether alternative pharmacological formulations or combinations of this drug with other classes of LRAs will lead to an effective in vivo latency-reversing strategy remains to be determined.IMPORTANCE If combined with immune therapeutics, latency reversing agents (LRAs) have the potential to reduce the size of the reservoir sufficiently that an engineered immune response can control the virus in the absence of antiretroviral therapy. We have identified a new class of LRAs that do not induce T-cell activation and that are able to potentiate, rather than inhibit, CD8+ T and NK cell cytotoxic effector functions. This new class of LRAs corresponds to inhibitors of glycogen synthase kinase-3. In this work, we have also studied the effects of one member of this drug class, tideglusib, in SIV-infected rhesus monkeys. When tested in vivo, however, tideglusib showed unfavorable pharmacokinetic properties, which resulted in lack of SIV latency reversal. The disconnect between our ex vivo and in vivo results highlights the importance of developing next generation LRAs with pharmacological properties that allow systemic drug delivery in relevant anatomical compartments harboring latent reservoirs.

FOXO1 promotes HIV latency by suppressing ER stress in T cells.

Quiescence is a hallmark of CD4+ T cells latently infected with human immunodeficiency virus 1 (HIV-1). While reversing this quiescence is an effective approach to reactivate latent HIV from T cells in culture, it can cause deleterious cytokine dysregulation in patients. As a key regulator of T-cell quiescence, FOXO1 promotes latency and suppresses productive HIV infection. We report that, in resting T cells, FOXO1 inhibition impaired autophagy and induced endoplasmic reticulum (ER) stress, thereby activating two associated transcription factors: activating transcription factor 4 (ATF4) and nuclear factor of activated T cells (NFAT). Both factors associate with HIV chromatin and are necessary for HIV reactivation. Indeed, inhibition of protein kinase R-like ER kinase, an ER stress sensor that can mediate the induction of ATF4, and calcineurin, a calcium-dependent regulator of NFAT, synergistically suppressed HIV reactivation induced by FOXO1 inhibition. Thus, our studies uncover a link of FOXO1, ER stress and HIV infection that could be therapeutically exploited to selectively reverse T-cell quiescence and reduce the size of the latent viral reservoir.

Metabolic reprogramming of human CD8+ memory T cells through loss of SIRT1.

The expansion of CD8+CD28- T cells, a population of terminally differentiated memory T cells, is one of the most consistent immunological changes in humans during aging. CD8+CD28- T cells are highly cytotoxic, and their frequency is linked to many age-related diseases. As they do not accumulate in mice, many of the molecular mechanisms regulating their fate and function remain unclear. In this paper, we find that human CD8+CD28- T cells, under resting conditions, have an enhanced capacity to use glycolysis, a function linked to decreased expression of the NAD+-dependent protein deacetylase SIRT1. Global gene expression profiling identified the transcription factor FoxO1 as a SIRT1 target involved in transcriptional reprogramming of CD8+CD28- T cells. FoxO1 is proteasomally degraded in SIRT1-deficient CD8+CD28- T cells, and inhibiting its activity in resting CD8+CD28+ T cells enhanced glycolytic capacity and granzyme B production as in CD8+CD28- T cells. These data identify the evolutionarily conserved SIRT1-FoxO1 axis as a regulator of resting CD8+ memory T cell metabolism and activity in humans.

SMYD2-Mediated Histone Methylation Contributes to HIV-1 Latency.

Transcriptional latency of HIV is a last barrier to viral eradication. Chromatin-remodeling complexes and post-translational histone modifications likely play key roles in HIV-1 reactivation, but the underlying mechanisms are incompletely understood. We performed an RNAi-based screen of human lysine methyltransferases and identified the SET and MYND domain-containing protein 2 (SMYD2) as an enzyme that regulates HIV-1 latency. Knockdown of SMYD2 or its pharmacological inhibition reactivated latent HIV-1 in T cell lines and in primary CD4+ T cells. SMYD2 associated with latent HIV-1 promoter chromatin, which was enriched in monomethylated lysine 20 at histone H4 (H4K20me1), a mark lost in cells lacking SMYD2. Further, we find that lethal 3 malignant brain tumor 1 (L3MBTL1), a reader protein with chromatin-compacting properties that recognizes H4K20me1, was recruited to the latent HIV-1 promoter in a SMYD2-dependent manner. We propose that a SMYD2-H4K20me1-L3MBTL1 axis contributes to HIV-1 latency and can be targeted with small-molecule SMYD2 inhibitors.

Identification and functional characterization of the distinct plant pectin esterases PAE8 and PAE9 and their deletion mutants.

MAIN CONCLUSION: PAE8 and PAE9 have pectin acetylesterase activity and together remove one-third of the cell wall acetate associated with pectin formation in Arabidopsis leaves. In pae8 and pae9 mutants, substantial amounts of acetate accumulate in cell walls. In addition, the inflorescence stem height is decreased. Pectic polysaccharides constitute a significant part of the primary cell walls in dicotyledonous angiosperms. This diverse group of polysaccharides has been implicated in several physiological processes including cell-to-cell adhesion and pathogenesis. Several pectic polysaccharides contain acetyl-moieties directly affecting their physical properties such as gelling capacity, an important trait for the food industry. In order to gain further insight into the biological role of pectin acetylation, a reverse genetics approach was used to investigate the function of genes that are members of the Pectin AcetylEsterase gene family (PAE) in Arabidopsis. Mutations in two members of the PAE family (PAE8 and PAE9) lead to cell walls with an approximately 20 % increase in acetate content. High-molecular-weight fractions enriched in pectic rhamnogalacturonan I (RGI) extracted from the mutants had increased acetate content. In addition, the pae8 mutant displayed increased acetate content also in low-molecular-weight pectic fractions. The pae8/pae9-2 double mutant exhibited an additive effect by increasing wall acetate content by up to 37 %, suggesting that the two genes are not redundant and act on acetyl-substituents of different pectic domains. The pae8 and pae8/pae9-2 mutants exhibit reduced inflorescence growth underscoring the role of pectic acetylation in plant development. When heterologously expressed and purified, both gene products were shown to release acetate from the corresponding mutant pectic fractions in vitro. PAEs play a significant role in modulating the acetylation state of pectic polymers in the wall, highlighting the importance of apoplastic metabolism for the plant cell and plant growth.