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.
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.
In a multicentric, observational, investigator-blinded, and longitudinal clinical study of 764 ART-naïve subjects, we identified nine different promoter variant strains of HIV-1 subtype C (HIV-1C) ...emerging in the Indian population, with some of these variants being reported for the first time. Unlike several previous studies, our work here focuses on the evolving viral regulatory elements, not the coding sequences. The emerging viral strains contain additional copies of the existing transcription factor binding sites (TFBS), including TCF-1α/LEF-1, RBEIII, AP-1, and NF-κB, created by sequence duplication. The additional TFBS are genetically diverse and may blur the distinction between the modulatory region of the promoter and the viral enhancer. In a follow-up analysis, we found trends, but no significant associations between any specific variant promoter and prognostic markers, probably because the emerging viral strains might not have established mono infections yet. Illumina sequencing of four clinical samples containing a coinfection indicated the domination of one strain over the other and establishing a stable ratio with the second strain at the follow-up time points. Since a single promoter regulates viral gene expression and constitutes the master regulatory circuit with Tat, the acquisition of additional and variant copies of the TFBS may significantly impact viral latency and latent reservoir characteristics. Further studies are urgently warranted to understand how the diverse TFBS profiles of the viral promoter may modulate the characteristics of the latent reservoir, especially following the initiation of antiretroviral therapy.
The administration of antiretroviral therapy (ART) leads to a rapid reduction in plasma viral load in HIV-1 seropositive subjects. However, when ART is suspended, the virus rebounds due to the ...presence of a latent viral reservoir. Several techniques have been developed to characterize this latent viral reservoir. Of the various assay formats available presently, the Tat/Rev induced limiting dilution assay (TILDA) offers the most robust and technically simple assay strategy. The TILDA formats reported thus far are limited by being selective to one or a few HIV-1 genetic subtypes, thus, restricting them from a broader level application. The novel TILDA, labelled as U-TILDA ('U' for universal), can detect all the major genetic subtypes of HIV-1 unbiasedly, and with comparable sensitivity of detection. U-TILDA is well suited to characterize the latent reservoirs of HIV-1 and aid in the formulation of cure strategies.
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Efficient expression of functional proteins in heterologous hosts has become the pivotal focus of modern biotechnology and biomedical research. To this end, multiple alternatives to E. coli are being ...explored for recombinant protein expression. L. lactis, being a gram-positive organism, circumvents the need for an endotoxin removal step during protein purification. We report here the optimisation of the expression of HIV-1 Tat, a notoriously difficult protein, in Lactococcus lactis system. We evaluated five different promoters in two different Lactococcus lactis strains and examined the effect of pH, glucose, and induction time on the yield and purity of Tat. Finally, the recombinant Tat was functionally competent in transactivating the HIV-1 promoter in HLM-1 reporter cells. Our work provides a scaffold for future work on the expression of toxic proteins in Lactococcus lactis.
•Lactococcus lactis is a useful alternative to E. coli for expressing toxic proteins such as HIV-1 Tat.•P2 promoter is the best choice among PepN, NisA, P11, P170 and P2 promoters for HIV-1 Tat expression.•Expression through the P2 promoter demonstrates the least proteolytic processing in the MG1363 strain of L. lactis.•0.25% Glucose without pH adjustment serves as the best nutritional condition for HIV-1 Tat expression.
With growing awareness of eco-sustainability, health hazards, and material degradation linked to ultraviolet (UV) exposure, UV shielding films have garnered importance across industries like ...packaging, cosmetics, and photothermal materials. Consequently, the demand for utilizing renewable biobased resources to create sustainable, environmentally friendly, and economically viable materials has intensified. Responding to this, we have developed a novel composite film through the synergic combination of biopolymers: sodium alginate (SA), κ-carrageenan (kC), lignin (LG), and a bioionic liquid (IL). Choline indole-3-acetate, the used IL, has been strategically employed to confer antioxidant, antibacterial, UV shielding, and plasticizing properties to the SAkC composite films. The native SAkC film exhibits only 28% radical scavenging activity and lacks antibacterial and UV shielding capabilities. Intriguingly, IL doping significantly enhances antioxidant activity to 65%, completely inhibits bacterial growth, and increases the UV-blocking ability to 92%. The addition of a low loading of lignin (0.9 wt % with respect to polymer) proves sufficient for achieving complete UV blocking without compromising other properties. A 50 wt % IL loading with respect to the polymer matrix enhanced the mechanical strength of the film with elongation (69%) and tensile strength (32.5 MPa), obviating the need for additional cross-linkers and plasticizers. The antibacterial properties of the synthesized IL and developed SAkC film were ascertained using E. coli and S. aureus bacteria. The film shows excellent UV shielding in a wide temperature range (20–80 °C) and remains stable during continuous exposure to UV–A radiation of 390 nm for 24 h. UV–A blocking was also confirmed by testing the shielding of Rhodamine dye (RhB) degradation covered with the SAkC-IL-LG film where only 4.1% of RhB degradation was observed for 1 h of exposure.
For large-scale operations, membrane flux and fouling present a critical barrier to the economic sustainability of the water treatment process. To achieve high permeability, anti-fouling and ...anti-bacterial properties, we report a novel scalable and simple approach for the membrane preparation involving functionalization of polyphenylene oxide by di-quaternization and chemical grafting of aliphatic carbon chain spacer for fine-tuned hydrophobic-hydrophilic balance and comb-shaped morphology. Five aliphatic carbon chains (three, four, six, eight, and ten carbon atoms) were chemically grafted on the di-quaternized polyphenylene oxide (QPPO) to comprehensively study the impact of functional groups on membrane surface properties, water flux, anti-fouling and anti-bacterial characteristics. 3C-SUL-g-QPPO membrane showed high pure water flux (875 ± 10.02 L m−2 h−1) and bovine serum albumin rejection (98.04 ± 1.02 %), attributed to hydration between water and zwitterionic surface and well-architected porous structure. The surface roughness and water contact angle for the 3C-SUL-g-QPPO membrane were minimal. The 3C-SUL-g-QPPO membrane showed good anti-fouling characteristics, as confirmed by the flux recovery and decline ratio data. The anti-bacterial activity against Gram-negative E. coli and Gram-positive S. aureus was attributed to the di-quaternized nitrogen of grafted polymer. The present work suggests quaternization of PPO for improved hydrophilicity and enhanced membrane permeability, while chemical grafting of aliphatic carbon chain balanced hydrophobic-hydrophilic character, improving the membrane anti-fouling properties.
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•Side-chain grafted PPO-based membranes for anti-fouling/bacterial applications.•Quaternization of PPO enhanced hydrophilicity and permeability of membranes.•Carbon chain balanced hydrophobicity-hydrophilicity, improved anti-fouling nature.•Membranes showed antibacterial activity against Gram-negative/positive bacteria.
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