The treatment of AIDS remains a serious challenge owing to high genetic variation of Human Immunodeficiency Virus type 1 (HIV-1). The use of different antiretroviral drugs (ARV) is significantly ...limited by severe side-effects that further compromise the quality of life of the AIDS patient. In the present study, we have evaluated a liposome system for the delivery of nevirapine, a hydrophobic non-nucleoside reverse transcriptase inhibitor. Liposomes were prepared from egg phospholipids using thin film hydration. The parameters of the process were optimized to obtain spherical liposomes below 200 nm with a narrow polydispersity. The encapsulation efficiency of the liposomes was optimized at different ratios of egg phospholipid to cholesterol as well as drug to total lipid. The data demonstrate that encapsulation efficiency of 78.14% and 76.25% were obtained at egg phospholipid to cholesterol ratio of 9:1 and drug to lipid ratio of 1:5, respectively. We further observed that the size of the liposomes and the encapsulation efficiency of the drug increased concomitantly with the increasing ratio of drug and lipid and that maximum stability was observed at the physiological pH. Thermal analysis of the drug encapsulated liposomes indicated the formation of a homogenous drug-lipid system. The magnitude of drug release from the liposomes was examined under different experimental conditions including in phosphate buffered saline (PBS), Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum or in the presence of an external stimulus such as low frequency ultrasound. Within the first 20 minutes 40, 60 and 100% of the drug was released when placed in PBS, DMEM or when ultrasound was applied, respectively. We propose that nevirapine-loaded liposomal formulations reported here could improve targeted delivery of the anti-retroviral drugs to select compartments and cells and alleviate systemic toxic side effects as a consequence.
Acetylation of histones and non-histone proteins is an important post-translational modification involved in the regulation of gene expression in eukaryotes and all viral DNA that integrates into the ...human genome (e.g. the human immunodeficiency virus). Dysfunction of histone acetyltransferases (HATs) is often associated with the manifestation of several diseases. In this respect, HATs are the new potential targets for the design of therapeutics. In this study, we report that curcumin (diferuloylmethane), a major curcumanoid in the spice turmeric, is a specific inhibitor of the p300/CREB-binding protein (CBP) HAT activity but not of p300/CBP-associated factor, in vitro and in vivo. Furthermore, curcumin could also inhibit the p300-mediated acetylation of p53 in vivo. It specifically represses the p300/CBP HAT activity-dependent transcriptional activation from chromatin but not a DNA template. It is significant that curcumin could inhibit the acetylation of HIV-Tat protein in vitro by p300 as well as proliferation of the virus, as revealed by the repression in syncytia formation upon curcumin treatment in SupT1 cells. Thus, non-toxic curcumin, which targets p300/CBP, may serve as a lead compound in combinatorial HIV therapeutics.
Using cell‐associated DNA and cell‐free RNA of human immunodeficiency virus type‐1 (HIV‐1), we investigated the role of drug‐resistant viral variants that emerged during early antiretroviral therapy ...(ART) in determining virological outcome. This case‐control study compared virologic nonresponder children (two viral loads VLs ≥ 200 copies/mL within 2 years of ART) and responder children (two VLs < 200 copies/mL after six months of ART) infected with HIV‐1 initiated on nonnucleoside reverse‐transcriptase inhibitor (NNRTI)‐based ART. The partial reverse‐transcriptase gene of HIV‐1 in cell‐associated DNA was genotyped using next‐generation sequencing (NGS; Illumina; threshold 0.5%; at baseline and month six of ART) and in cell‐free RNA (concurrently and at virological failure; VL > 1000 copies/mL at ≥ 12 months of ART) using the Sanger method. Among 30 nonresponders and 37 responders, baseline differences were insignificant while adherence, VL, and drug resistance mutations (DRMs) observed at month six differed significantly (
P ≥ 0.05). At month six, NGS estimated a higher number of DRMs compared with Sanger (50% vs 33%;
P = 0.001). Among the nonresponders carrying a resistant virus (86.6%) at virological failure, 26% harbored clinically relevant low‐frequency DRMs in the cell‐associated DNA at month six (0.5%‐20%; K103N, G190A, Y181C, and M184I). Plasma VL of > 3 log
10copies/mL (AOR, 30.4; 95% CI, 3.3‐281;
P = 0.003) and treatment‐relevant DRMs detected in the cell‐associated DNA at month six (AOR, 24.2; 95% CI, 2.6‐221;
P = 0.005) were independently associated with increased risk for early virological failure. Our findings suggest that treatment‐relevant DRMs acquired in cell‐associated DNA during the first six months of ART can predict virological failure in children initiated on NNRTI‐based ART.
The natural cysteine to serine variation at position 31 of Tat in HIV-1C disrupts the dicysteine motif attenuating the chemokine function of Tat. We ask if there exists a trade-off in terms of a gain ...of function for HIV-1C Tat due to this natural variation. We constructed two Tat-expression vectors encoding Tat proteins discordant for the serine 31 residue (CS-Tat vs. CC-Tat), expressed the proteins in Jurkat cells under doxycycline control, and performed the whole transcriptome analysis to compare the early events of Tat-induced host gene expression. Our analysis delineated a significant enrichment of pathways and gene ontologies associated with the angiogenic signaling events in CS-Tat stable cells. Subsequently, we validated and compared angiogenic signaling events induced by CS- vs. CC-Tat using human umbilical vein endothelial cells (HUVEC) and the human cerebral microvascular endothelial cell line (hCMEC/D3). CS-Tat significantly enhanced the production of CCL2 from HUVEC and induced an activated phenotype in endothelial cells conferring on them enhanced migration, invasion, and
morphogenesis potential. The ability of CS-Tat to induce the activated phenotype in endothelial cells could be of significance, especially in the context of HIV-associated cardiovascular and neuronal disorders. The findings from the present study are likely to help appreciate the functional significance of the SAR (signature amino acid residues) influencing the unique biological properties.
We observe the emergence of several promoter-variant viral strains in India during recent years. The variant viral promoters contain additional copies of transcription factor binding sites present in ...the viral modulatory region or enhancer, including RBEIII, LEF-1, Ap-1 and/or NF-κB. These sites are crucial for governing viral gene expression and latency. Here, we infer that one variant viral promoter R2N3-LTR containing two copies of RBF-2 binding sites (an RBEIII site duplication) and three copies of NF-κB motifs may demonstrate low levels of gene expression noise as compared to the canonical RN3-LTR or a different variant R2N4-LTR (a duplication of an RBEIII site and an NF-κB motif). To demonstrate this, we constructed a panel of sub-genomic viral vectors of promoter-variant LTRs co-expressing two reporter proteins (mScarlet and Gaussia luciferase) under the dual-control of Tat and Rev. We established stable pools of CEM.NKR-CCR5 cells (CEM-CCR5
reporter cells) and evaluated reporter gene expression under different conditions of cell activation.
The R2N3-LTR established stringent latency that was highly resistant to reversal by potent cell activators such as TNF-α or PMA, or even to a cocktail of activators, compared to the canonical RN3- or the variant R2N4-LTR. The R2N3-LTR exhibited low-level basal gene expression in the absence of cell activation that enhanced marginally but significantly when activated. In the presence of Tat and Rev, trans-complemented in the form of an infectious virus, the R2N3-LTR demonstrated gene expression at levels comparable to the wild-type viral promoter. The R2N3-LTR is responsive to Tat and Rev factors derived from viral strains representing diverse genetic subtypes.
With extremely low-level transcriptional noise, the R2N3-LTR can serve as an excellent model to examine the establishment, maintenance, and reversal of HIV-1 latency. The R2N3-LTR would also be an ideal viral promoter to develop high-throughput screening assays to identify potent latency-reversing agents since the LTR is not affected by the usual background noise of the cell.
To compare the markers of inflammation and immune activation in virally suppressed HIV-infected children on antiretroviral therapy, who practiced regular structured exercise comprising running and ...yoga to those who did not over a 2-year period.
This retrospective cohort study included 72 children aged 8 to 16 years divided into 2 groups, exercisers (n = 36) and the nonexercisers (n = 36) based on their intentional physical activity. The analyses were carried out at baseline and after 2 years (Y2) for the soluble biomarkers of inflammation and immune activation (tumor necrosis factor alpha, interleukin-6, interleukin-10, interferon gamma, sCD14, and sCD163). In addition, cell-associated biomarker (CD38), lipopolysaccharides, and the gene expression of interleukin-2 and brain-derived neurotrophic factor were also measured at Y2.
Reduction in levels of sCD14 (effect size ES, -0.6; 95% confidence interval CI, -1.08 to -0.14), tumor necrosis factor alpha (ES, -0.7; 95% CI, -1.18 to -0.23), interferon gamma (ES, -0.7; 95% CI, -1.17 to -0.22), and interleukin-10 (ES, -0.6; 95% CI, -1.08 to -0.14) was observed among exercisers as compared with nonexercisers at Y2. In addition, CD38+ expressing CD4+ T cells were found to be lower among exercisers (P = .01) at Y2. However, the differences in levels of interleukin-6, sCD163, lipopolysaccharides, interleukin-2, and brain-derived neurotrophic factor were not significantly different among the 2 groups.
The study result suggests that regular structured physical activity improves the inflammatory profile of antiretroviral therapy-treated HIV-infected children.
Commonly used opioids, such as morphine have been implicated in augmented SIV/HIV persistence within the central nervous system (CNS). However, the extent of myeloid cell polarization and viral ...persistence in different brain regions remains unclear. Additionally, the additive effects of morphine on SIV/HIV dysregulation of gut-brain crosstalk remain underexplored. Therefore, studies focused on understanding how drugs of abuse such as morphine affect immune dynamics, viral persistence and gut-brain interrelationships are warranted.
For a total of 9 weeks, rhesus macaques were ramped-up, and twice daily injections of either morphine (
= 4) or saline (
= 4) administered. This was later followed with infection with SHIVAD8EO variants. At necropsy, mononuclear cells were isolated from diverse brain frontal lobe, cerebellum, medulla, putamen, hippocampus (HIP) and subventricular zone (SVZ) and gut lamina propria (LP) and muscularis (MUSC) of ascending colon, duodenum, and ileum regions. Multiparametric flow cytometry was used to were profile for myeloid cell polarity/activation and results corroborated with indirect immunofluorescence assays. Simian human immunodeficiency virus (SHIV) DNA levels were measured with aid of the digital droplet polymerase chain reaction (PCR) assay. Luminex assays were then used to evaluate soluble plasma/CSF biomarker levels. Finally, changes in the fecal microbiome were evaluated using 16S rRNA on the Illumina NovaSeq platform.
Flow Cytometry-based semi-supervised analysis revealed that morphine exposure led to exacerbated M1 (CD14/CD16)/M2 (CD163/CD206) polarization in activated microglia that spanned across diverse brain regions. This was accompanied by elevated SHIV DNA within the sites of neurogenesis-HIP and SVZ. HIP/SVZ CD16+ activated microglia positively correlated with SHIV DNA levels in the brain (
= 0.548,
= 0.042). Simultaneously, morphine dependence depleted butyrate-producing bacteria, including
(
= 0.05),
(
= 0.068) genera and
(
= 0.068). Finally, morphine also altered the regulation of CNS inflammation by reducing the levels of IL1 Receptor antagonist (IL1Ra).
These findings are suggestive that morphine promotes CNS inflammation by altering receptor modulation, increasing myeloid brain activation, distorting gut-brain crosstalk, and causing selective enhancement of SHIV persistence in sites of neurogenesis.
Tat/Rev Induced Limiting Dilution Assay (TILDA) is instrumental in estimating the size of latent reservoirs of HIV-1. Here, we report an optimized TILDA containing a broader detection range compared ...to the reported methods and high sensitivity. Giving priority to sequence conservation, we positioned the two forward primers and the probe in exon-1 of HIV-1. The reverse primers are positioned in highly conserved regions of exon-7. The optimized TILDA detected eight molecular clones belonging to five major genetic subtypes of HIV-1 with a comparable detection sensitivity. Using the optimized assay, we show that only a minor proportion of CD4
T cells of primary clinical samples can spontaneously generate multiply spliced viral transcripts. A significantly larger proportion of the cells produced viral transcripts following activation. The optimized TILDA is suitable to characterize HIV-1 latent reservoirs and the therapeutic strategies intended to target the reservoir size.
Positive coactivator 4 (PC4), a human transcriptional coactivator, is involved in diverse processes like chromatin organization and transcription regulation. It is hyperphosphorylated during mitosis, ...with unknown significance. For the first time, we demonstrate the function of PC4 outside the nucleus upon nuclear envelope breakdown. A fraction of PC4 associates with Aurora A and Aurora B and undergoes phosphorylation, following which PC4 activates both Aurora A and B to sustain optimal kinase activity to maintain the phosphorylation gradient for the proper functioning of the mitotic machinery. This mitotic role is evident in PC4 knockdown cells where the defects are rescued only by the catalytically active Aurora kinases, but not the kinase‐dead mutants. Similarly, the PC4 phosphodeficient mutant failed to rescue such defects. Hence, our observations establish a novel mitotic function of PC4 that might be dependent on Aurora kinase‐mediated phosphorylation.
Human transcriptional coactivator PC4 is involved in many processes including chromatin organisation and transcription regulation. PC4 is a highly abundant non‐histone chromatin‐associated protein that is hyperphosphorylated during mitosis. Here, the authors demonstrate that PC4 functions outside the nucleus upon mitotic nuclear envelope breakdown, acting as both a novel substrate and activator of the Aurora kinases. It interacts with Aurora kinase A on centrosomes and with Aurora kinase B at the midbody during mitosis to maintain optimal activity of both kinases, ensuring proper fidelity of mitosis and cytokinesis. This finding could have applications in pathophysiological conditions where fine regulation of Aurora kinase activity is required.
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