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Background. The degree to which human immunodeficiency virus (HIV) continues to replicate during antiretroviral therapy (ART) is controversial. We conducted a randomized, double-blind, placebo-controlled study to assess whether raltegravir intensification reduces low-level viral replication, as defined by an increase in the level of 2-long terminal repeat (2-LTR) circles. Methods. Thirty-one subjects with an ART-suppressed plasma HIV RNA level of <40 copies/mL and a CD4⁺ T-cell count of ≥350 cells/mm³ for ≥1 year were randomly assigned to receive raltegravir 400 mg twice daily or placebo for 24 weeks. 2-LTR circles were analyzed by droplet digital polymerase chain reaction at weeks 0, 1, 2, and 8. Results. The median duration of ART suppression was 3.8 years. The raltegravir group had a significant increase in the level of 2-LTR circles, compared to the placebo group. The week 1 to 0 ratio was 8.8-fold higher (P = .0025) and the week 2 to 0 ratio was 5.7-fold higher (P = .023) in the raltegravir vs. placebo group. Intensification also led to a statistically significant decrease in the D-dimer level, compared to placebo (P = .045). Conclusions. Raltegravir intensification resulted in a rapid increase in the level of 2-LTR circles in a proportion of subjects, indicating that low-level viral replication persists in some individuals even after long-term ART. Intensification also reduced the D-dimer level, a coagulation biomarker that is predictive of morbidity and mortality among patients receiving treatment for HIV infection.

Polyploidization is an evolutionary event leading to structural changes of the genome(s), particularly allopolyploidization, which combines different genomes of distinct species. The tetraploid species, Sorghum halepense , is assumed an allopolyploid species formed by hybridization between diploid S. bicolor and S. propinquum . The repeat profiles of S. bicolor , S. halepense , and their relatives were compared to elucidate the repeats’ role in shaping their genomes. The repeat frequencies and profiles of the three diploid accessions ( S. bicolor , S. bicolor ssp. verticilliflorum , and S. bicolor var. technicum ) and two tetraploid accessions ( S. halepense ) are similar. However, the polymorphic distribution of the subtelomeric satellites preferentially enriched in the tetraploid S. halepense indicates drastic genome rearrangements after the allopolyploidization event. Verified by CENH3 chromatin immunoprecipitation (ChIP)-sequencing and fluorescence in situ hybridization (FISH) analysis the centromeres of S. bicolor are mainly composed of the abundant satellite SorSat137 (CEN38) and diverse CRMs, Athila of Ty3_gypsy and Ty1_copia-SIRE long terminal repeat (LTR) retroelements. A similar centromere composition was found in S. halepense . The potential contribution of S. bicolor in the formation of tetraploid S. halepense is discussed.

The HIV-1 Tat protein interacts with TAR RNA and recruits CDK9/cyclin T1 and other host factors to induce HIV-1 transcription. Thus, Tat–TAR RNA interaction, which is unique for HIV-1, represents an attractive target for anti-HIV-1 therapeutics. To target Tat–TAR RNA interaction, we used a crystal structure of acetylpromazine bound to the bulge of TAR RNA, to dock compounds from the Enamine database containing over two million individual compounds. The docking procedure identified 173 compounds that were further analyzed for the inhibition of HIV-1 infection. The top ten inhibitory compounds with IC50 ≤ 6 µM were selected and the three least toxic compounds, T6780107 (IC50 = 2.97 μM), T0516-4834 (IC50 = 0.2 μM) and T5628834 (IC50 = 3.46 μM), were further tested for HIV-1 transcription inhibition. Only the T0516-4834 compound showed selective inhibition of Tat-induced HIV-1 transcription, whereas the T6780107 compound inhibited equally basal and Tat-induced transcription and the T5628834 compound only inhibited basal HIV-1 transcription. The compounds were tested for the inhibition of translation and showed minimal (<25%) effect. The T0516-4834 compound also showed the strongest inhibition of HIV-1 RNA expression and p24 production in CEM T cells and peripheral blood mononuclear cells infected with HIV-1 IIIB. Of the three compounds, only the T0516-4834 compound significantly disrupted Tat–TAR RNA interaction. Additionally, of the three tested compounds, T5628834 and, to a lesser extent, T0516-4834 disrupted Tat–CDK9/cyclin T1 interaction. None of the three compounds showed significant inhibition of the cellular CDK9 and cyclin T1 levels. In silico modelling showed that the T0516-4834 compound interacted with TAR RNA by binding to the bulge formed by U23, U25, C39, G26,C39 and U40 residues. Taken together, our study identified a novel benzoxazole compound that disrupted Tat–TAR RNA interaction and inhibited Tat-induced transcription and HIV-1 infection, suggesting that this compound might serve as a new lead for anti-HIV-1 therapeutics.

The primary reason why AIDS cannot be completely cured is the existence of a latent HIV-1 reservoir. Currently, the facts of HIV-1 latency, including its establishment and maintenance, are incomplete. Human immunodeficiency virus type 1 (HIV-1) cannot be completely eliminated because of existence of the latent HIV-1 reservoir. However, the facts of HIV-1 latency, including its establishment and maintenance, are incomplete. FKBP3, encoded by the FKBP3 gene, belongs to the immunophilin family of proteins and is involved in immunoregulation and such cellular processes as protein folding. In a previous study, we found that FKBP3 may be related to HIV-1 latency using CRISPR screening. In this study, we knocked out the FKBP3 gene in multiple latently infected cell lines to promote latent HIV-1 activation. We found that FKBP3 could indirectly bind to the HIV-1 long terminal repeat through interaction with YY1, thereby recruiting histone deacetylase 1/2 to it. This promotes histone deacetylation and induces HIV-1 latency. Finally, in a primary latent cell model, we confirmed the effect of FKBP3 knockout on the latent activation of HIV-1. Our results suggest a new mechanism for the epigenetic regulation of HIV-1 latency and a new potential target for activating latent HIV-1. IMPORTANCE The primary reason why AIDS cannot be completely cured is the existence of a latent HIV-1 reservoir. Currently, the facts of HIV-1 latency, including its establishment and maintenance, are incomplete. Using a CRISPR library in our earlier screening of genes related to HIV-1 latency, we identified FBKP3 as a candidate gene related to HIV-1 latency. Therefore, in this mechanistic study, we first confirmed the HIV-1 latency-promoting effect of FKBP3 and determined that FKBP3 promotes histone deacetylation by recruiting histone deacetylase 1/2 to the HIV-1 long terminal repeat. We also confirmed, for the first time, that FKBP3 can act as a transcription factor (TF) recruitment scaffold and participate in epigenetic regulation of HIV-1 latency. These findings suggest a new mechanism for the epigenetic regulation of HIV-1 latency and a new potential target for activating latent HIV-1.

Persistence of human immunodeficiency virus 1 (HIV-1) latency and residual immune activation remain major barriers to treatment in patients receiving highly active antiretroviral therapy (HAART). In the present study, we investigated the molecular mechanisms of persistent HIV infection and residual immune activation in HAART-treated patients. We showed that the expression level of B-cell CLL/lymphoma 11B (BCL11B) was significantly increased in CD4+T cells from HIV-infected patients undergoing HAART, and this was accompanied by increased expression of BCL11B-associated chromatin modifiers and inflammatory factors in comparison to healthy controls and untreated patients with HIV. In vitro assays showed that BCL11B significantly inhibited HIV-1 long terminal repeat (LTR)-mediated transcription. Knockdown of BCL11B resulted in the activation of HIV latent cells, and dissociation of BCL11B and its related chromatin remodeling factors from the HIV LTR. Our findings suggested that increased expression of BCL11B and its related chromatin modifiers contribute to HIV-1 transcriptional silencing, and alteration of BCL11B levels might lead to abnormal transcription and inflammation.

Attempts to eradicate HIV have been thwarted by the persistence of a small pool of quiescent memory CD4 T cells that harbor a transcriptionally silent, integrated form of the virus that can produce infectious virions following an anamnestic immune response. Transcription factors downstream of T-cell receptor activation, such as NF-κB/Rel and nuclear factor of activated T cells (NFAT) transcription members, are considered important regulators of HIV transcription during acute HIV infection. We now report studies exploring their precise role as antagonists of HIV latency using cell and primary CD4 T cell models of HIV-1 latency. Surprisingly, RNA interference studies performed in J-Lat CD4 T cells suggested that none of the NFATs, including NFATc1, NFATc2, NFATc3, and NFAT5, played a key role in the reactivation of latent HIV. However, cyclosporin A markedly inhibited the reactivation response. These results were reconciled when calcium signaling through calcineurin was shown to potentiate prostratin induced activation of NF-κB that in turn stimulated the latent HIV long terminal repeat (LTR). Similar effects of calcineurin were confirmed in a primary CD4 T cell model of HIV latency. These findings highlight an important role for calcineurin in NF-κB-dependent induction of latent HIV transcription. Innovative approaches exploiting the synergistic actions of calcineurin and prostratin in the absence of generalized T-cell activation merit exploration as a means to attack the latent viral reservoir.

Clinical trials are necessary in order to develop treatments for diseases; however, they can often be costly, time consuming, and demanding to the patients. This paper summarizes several common methods used for optimal design that can be used to address these issues. In addition, we introduce a novel method for optimizing experiment designs applied to HIV 2-LTR clinical trials. Our method employs Bayesian techniques to optimize the experiment outcome by maximizing the Expected Kullback-Leibler Divergence (EKLD) between the a priori knowledge of system parameters before the experiment and the a posteriori knowledge of the system parameters after the experiment. We show that our method is robust and performs equally well if not better than traditional optimal experiment design techniques.

Green fluorescent protein (GFP) chromophore and its congeners draw significant attention mostly for bioimaging purposes. In this work we probed these compounds as antiviral agents. We have chosen LTR-III DNA G4, the major G-quadruplex (G4) present in the long terminal repeat (LTR) promoter region of the human immunodeficiency virus-1 (HIV-1), as the target for primary screening and designing antiviral drug candidates. The stabilization of this G4 was previously shown to suppress viral gene expression and replication. FRET-based high-throughput screening (HTS) of 449 GFP chromophore-like compounds revealed a number of hits, sharing some general structural features. Structure-activity relationships (SAR) for the most effective stabilizers allowed us to establish structural fragments, important for G4 binding. Synthetic compounds, developed on the basis of SAR analysis, exhibited high LTR-III G4 stabilization level. NMR spectroscopy and molecular modeling revealed the possible formation of LTR-III G4-ligand complex with one of the lead selective derivative ZS260.1 positioned within the cavity, thus supporting the LTR-III G4 attractiveness for drug targeting. Selected compounds showed moderate activity against HIV-I (EC50 1.78–7.7 μM) in vitro, but the activity was accompanied by pronounced cytotoxicity.

In HIV‐1 infected cells, the LTR promoter, once organized into chromatin, is transcriptionally inactive in the absence of stimulation. To examine the chromosomal events involved in transcriptional activation, we analyzed histone acetylation and factor recruitment at contiguous LTR regions by a quantitative chromatin immunoprecipitation assay. In chronically infected cells treated with a phorbol ester, we found that acetylation of both histones H3 and H4 occurs at discrete nucleosomal regions before the onset of viral mRNA transcription. Concomitantly, we observed the recruitment of known cellular acetyl‐transferases to the promoter, including CBP, P/CAF and GCN5, as well as that of the p65 subunit of NF‐κB. The specific contribution of the viral Tat transactivator was assayed in cells harboring the sole LTR. We again observed nucleosomal acetylation and the recruitment of specific co‐factors to the viral LTR upon activation by either recombinant Tat or a phorbol ester. Strikingly, P/CAF was found associated with the promoter only in response to Tat. Taken together, these results contribute to the elucidation of the molecular events underlying HIV‐1 transcriptional activation.

Histone deacetylase (HDAC) inhibitors present an exciting new approach to activate HIV production from latently infected cells to potentially enhance elimination of these cells and achieve a cure. M344, a novel HDAC inhibitor, shows robust activity in a variety of cancer cells and relatively low toxicity compared to trichostatin A (TSA). However, little is known about the effects and action mechanism of M344 in inducing HIV expression in latently infected cells. Using the Jurkat T cell model of HIV latency, we demonstrate that M344 effectively reactivates HIV-1 gene expression in latently infected cells. Moreover, M344-mediated activation of the latent HIV LTR can be strongly inhibited by a NF-κB inhibitor aspirin. We further show that M344 acts by increasing the acetylation of histone H3 and histone H4 at the nucleosome 1 (nuc-1) site of the HIV-1 long terminal repeat (LTR) and by inducing NF-κB p65 nuclear translocation and direct RelA DNA binding at the nuc-1 region of the HIV-1 LTR. We also found that M344 synergized with prostratin to activate the HIV-1 LTR promoter in latently infected cells. These results suggest the potential of M344 in anti-latency therapies and an important role for histone modifications and NF-κB transcription factors in regulating HIV-1 LTR gene expression.