Biology subjects

Pal, S.

Publications and source records attributed to Pal, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

O-mannosylation and protein maturation check-points represent therapeutic opportunities in BRAF fusion protein oncogenesis

Fusions between protein-coding genes are common oncogenic drivers across cancers, typically pairing a proto-oncogene with partner that does not independently drive cancer. In all therapeutically actionable fusions, the proto-oncogene is the drug target, the contributions to oncogenicity of the fusion partner have largely been ignored. We studied the role of BRAF fusion partners and found that they are necessary for transformation. In the setting of KIAA1549::BRAF, the most common fusion protein across brain tumors, we found that KIAA1549 is necessary for the oncogenicity of KIAA1549::BRAF and engenders a striking and specific dependency on the protein O-mannosyltransferase complex (POMT1/2). Specifically, we show that genetic silencing or pharmacologic inhibition of the protein O-mannosyltransferase complex (POMT1/2) reverses fusion-induced transformation, thereby representing a novel and MAPK independent therapeutic target. Furthermore, POMT1/2 is required to glycosylate and enable maturation of the K::B fusion protein. These findings represent a proof-of-concept for targeting the partners in oncogenic fusions as a potential cancer therapeutic strategy.

cancer biology↗

bioRxiv2025

PEGylation strategies for enhanced nanoparticle delivery to tumor associated immune cells

Barriers to nanoparticle drug delivery to the tumor microenvironment such as ECM deposition and clearance by the mononuclear phagocyte system have necessitated strategies for more effective tumor penetration. Adding polyethylene glycol (PEG) chains to the surface of nanoparticles (PEGylation) has been widely used to both enhance accumulation at the tumor site and increase blood circulation time. Recent work has also shown that immune cells (e.g. macrophages, dendritic cells, neutrophils) play an important role in the ability of NPs to effectively target and spread within a tumor. PEG chain characteristics such as size and branching affects how nanoparticles interact with tissues; however, it is unclear how PEGylation type affects NP uptake and cellular distribution in the tumor microenvironment. In this study, we evaluated the influence of both linear and branched PEGylation on nanoparticle biodistribution and uptake in tumor cells as well as tumor-infiltrating immune cells. As compared to conventional surface coatings with linear PEG, we show that modifying PEG structure to a branched conformation increases nanoparticle accumulation in the spleen of tumor-bearing mice, primarily due to significantly enhanced uptake by leukocytes. As compared to uncoated particles, we also found that nanoparticles densely coated with linear or branched PEG accumulated to a greater extent in tumors showing [≥]8-fold increases in uptake by tumor-associated macrophages and dendritic cells. These studies provide insight into PEG architecture as a design parameter in nanomedicine that can facilitate the design of more effective cancer therapies. Translational ImpactThis work uncovers immune-mediated mechanisms and design strategies to enhance NP delivery to tumors via PEGylation which provide a foundation for clinical development of cancer therapeutics. The PEGylation strategies described could be readily integrated into clinically relevant nanoparticle delivery systems (e.g. lipid nanoparticles) with minimal effort making this a highly appealing approach to address the current limitations of cancer nanomedicine.

bioengineering↗

bioRxiv2025

Optimized large-scale longitudinal biorepository of gastroesophageal adenocarcinoma patient-derived organoids: High-fidelity models for personalized treatment to overcome resistance.

A major limitation in studying gastroesophageal adenocarcinoma (GEA) has been the lack of reliable models that represent the diseases complexity. We present lessons learned from a comprehensive large-scale biobanking effort combining traditional sample collection with several in vitro models including 3-dimensional patient-derived organoids (PDOs), 2-dimensional cancer-associated fibroblasts (CAFs), tumor-infiltrating lymphocytes (TILs) and/or in vivo xenografts. This initiative started in 2018, integrating multiple advanced ex-vivo models such as PDOs, patient-derived xenografts (PDXs) and organoids (PDXOs). This unique resource now includes tumor avatars from over 380 consented patients, making it the largest living GEA biobank in the world. We achieved > 90% success rate in creating per-patient models, including 227 tumor-derived and 203 neighboring normal PDOs. These organoids accurately mirror key features of the original tumors, such as their histology (e.g. microsatellite instability), mutations, and drug response, across treatment points. Notably, PDOs can predict individual patient responses to chemotherapy within five weeks, underscoring their clinical relevance. Furthermore, high-throughput drug screening on PDO subsets generates personalized chemosensitivity profiles for 22 drugs. Through a process of continued refinement of culture techniques and tumor sampling approach, our large-scale comprehensive collection of GEA avatars represents a unique and valuable preclinical experimental resource for precision oncology. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/663874v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@f8a032org.highwire.dtl.DTLVardef@dd6fa3org.highwire.dtl.DTLVardef@1cc558corg.highwire.dtl.DTLVardef@492a5_HPS_FORMAT_FIGEXP M_FIG Schematic depiction of GEA live-banking workflow C_FIG

cancer biology↗

bioRxiv2025

Induction of Moderate DNA Damage Enhances Megakaryopoiesis and Platelet Production

A common side effect of poly-ADP ribose polymerase (PARP) inhibitors is low platelet counts, or thrombocytopenia, presumably mediated through platelet progenitors, megakaryocytes (MKs). MKs are large, hematopoietic cells with a polyploid, multi-lobulated nucleus. While DNA replication in MKs (endomitosis) is well studied, limited investigations have examined the impact of DNA damage and repair inhibition on megakaryopoiesis. To explore PARP inhibitor-induced thrombocytopenia, we treated mice with PARP inhibitors (niraparib and olaparib), which are approved for the treatment of solid tumors. While high-dose niraparib treatment led to thrombocytopenia, consistent with clinical observations, treatment at a lower dosage led to a significant, >1.5-fold increase in both the number of bone marrow MKs and circulating platelets. This increase was accompanied by elevated DNA damage in both MKs and MK progenitors, as measured by both {gamma}H2AX accumulation and comet assays of MKs. Notably, platelets from niraparib-treated mice were functionally normal in their response to ADP, TRAP, and collagen. Gamma-irradiation treatment similarly increased MK and platelet counts in mice, suggesting that moderate DNA damage enhances megakaryopoiesis and increases platelet counts. These data reveal a previously unknown relationship between MKs and DNA damage and present a novel target for triggering enhanced platelet production in vivo. Key PointsO_LITreatment of mice with low dose PARP inhibitors or gamma-irradiation enhances platelet counts. C_LIO_LILow dose PARP inhibitor treatment leads to increased DNA damage in MKs and MK progenitors and enhances bone marrow megakaryopoiesis. C_LI

cell biology↗

bioRxiv2025

VARS-fUSI: Variable Sampling for Fast and Efficient Functional Ultrasound Imaging using Neural Operators

Functional ultrasound imaging (fUSI) is a promising neuroimaging method that infers neural activity by detecting cerebral blood volume changes. It offers high sensitivity and spatial resolution relative to fMRI and is an epidural alternative to electrophysiology for medical and neuroscience applications, including brain-computer interfaces. However, current fUSI methods require hundreds of compounded images and ultrasound pulse emissions, leading to high computational costs, memory demands, and potential probe heating. We propose VARiable Sampling fUSI (VARS-fUSI), the first deep learning fUSI method to allow for different sampling durations and rates during training and inference by using neural operators. VARS-fUSI reconstructs high-quality fUSI images using 10 - 15% of the time or sampling rate needed per image while preserving decodable behavior-correlated signals. Additionally, VARS-fUSI offers efficient finetuning for generalization to new animals and humans. Demonstrated across mouse, monkey, and human data, VARS-fUSI achieves state-of-the-art performance, enhancing imaging efficiency by significantly reducing storage and processing needs.

bioengineering↗

bioRxiv2025

Range and elevation predict responses to climate change in frogs and lizards in the Western Ghats biodiversity hotspot of peninsular India

Anthropogenic climate change is altering the environment at unprecedented rates with severe consequences for most living organisms. As a result, species may extend, truncate, or shift their ranges in order to adapt to changing conditions. Species Distribution Models (SDMs) provide a data driven approach to predict future distributions under climate change and prioritize areas for conservation. Here, using a 10-year dataset with 4049 occurrence records of frogs from eight families and 27 genera as well as 733 occurrences of lizards from two families and 11 genera across the Western Ghats, we built SDMs to assess the changes in species distributions due to climate change. As expected, the temperature gradient across elevation and seasonality gradient across latitude contribute most to the climatic limits of species distributions. Latitudinal extents of most species were narrower in future predictions compared to the present, but there was little shift in latitudinal positions. On the other hand, most species shifted their distributions towards higher elevations, but the elevational range sizes remained the same. A total of 75 species of frogs (55%) and 15 species of lizards (45%) lose more than half of the suitable area, with few exceptions in both taxa that show an increase. In cases where a shift or increase in distribution was observed, the ability of the species to access and survive in these areas remains uncertain due to discontinuous topography and the presence of sister species. Overall, the frog and lizard fauna of the Western Ghats will be severely affected by climate change in the future due to a loss in climatic suitability.

ecology↗

bioRxiv2024

Cell-free screening, production and animal testing of a STI-related chlamydial major outer membrane protein supported in nanolipoproteins.

Vaccine development against Chlamydia, a prevalent sexually transmitted infection (STI), is imperative due to its global public health impact. However, significant challenges arise in the production of effective subunit vaccine based on recombinant protein antigens, particularly with membrane proteins like the Major Outer Membrane Protein (MOMP). Cell-free protein synthesis (CFPS) technology is an attractive method to address these challenges as a method of high-throughput membrane protein and protein complex production coupled with nanolipoprotein particles (NLPs). NLPs provides a supporting scaffold while allowing easy adjuvant addition during formulation. Over the last decade we have been working toward production and characterization of MOMP-NLP complexes for vaccine testing. The work presented here highlights the expression and biophysical analyses, including transmission electron microscopy (TEM) and dynamic light scattering (DLS), confirm formation and functionality of MOMP-NLP complexes for use in animal studies. Moreover, immunization studies in preclinical models compare the past and present protective efficacy of MOMP-NLP formulations, particularly when co-adjuvanted with CpG and FSL1. Ex vivo assessments further highlight the immunomodulatory effects of MOMP-NLP vaccinations, emphasizing their potential in eliciting robust immune responses. However, further research is warranted to further optimize vaccine formulations, validate efficacy against Chlamydia trachomatis, and better understand underlying mechanisms of immune response.

immunology↗

bioRxiv2024

Diverse somatic Transformer and sex chromosome karyotype pathways regulate gene expression in Drosophila gonad development

The somatic sex determination gene transformer (tra) is required for the highly sexually dimorphic development of most somatic cells, including those of the gonads. In addition, somatic tra is required for the germline development even though it is not required for sex determination within germ cells. Germ cell autonomous gene expression is also necessary for their sex determination. To understand the interplay between these signals, we compared the phenotype and gene expression of larval wild-type gonads and the sex-transformed tra gonads. XX larval ovaries transformed into testes were dramatically smaller than wild-type, with significant reductions in germ cell number, likely due to altered geometry of the stem cell niche. Additionally, there was a defect in progression into spermatocyte stages. XY larval testes transformed into ovaries had excessive germ cells, possibly due to the earlier onset of cell division. We suggest that germ cells are neither fully female nor male following somatic sex transformation, with certain pathways characteristic of each sex expressed in tra mutants. We found multiple patterns of somatic and germline gene expression control exclusively due to tra, exclusively due to sex chromosome karyotype, but usually due to a combination of these factors showing tra and sex chromosome karyotype pathways regulate gene expression during Drosophila gonad development.

developmental biology↗

bioRxiv2024

Acinetobacter baumannii DacC influences cell shape, biofilm formation, and physiological fitness by manifesting DD-carboxypeptidase and β-lactamase dual-enzyme activities

With the growing threat of drug-resistant Acinetobacter baumannii, there is an urgent need to comprehensively understand the physiology of this nosocomial pathogen. As penicillin-binding proteins are attractive targets for antibacterial therapy, herein we have tried to explore the physiological roles of two putative DD-carboxypeptidases, viz., dacC and dacD in A. baumannii. Surprisingly, the deletion of dacC resulted in a reduced growth rate, loss of rod-shaped morphology, reduction in biofilm-forming ability, and enhanced susceptibility towards {beta}-lactams, whereas, the deletion of dacD had no such effect. Interestingly, ectopic expression of dacC restored the lost phenotypes. The double deletion mutant in which both dacC and dacD were absent showed properties similar to the dacC single knockout. On the other hand, cell-shape reverting efficiency in septuple PBP deleted E. coli and in vitro enzyme kinetics assessments reveal that dacD is a stronger DD-CPase as compared to dacC. The expression of dacC was in the log phase whereas dacD expression takes place in the stationary phase. In summary, we conclude that dacC encodes a dual enzyme, possessing activities of DD-CPase and {beta}-lactamase, which significantly affects the physiology of A. baumannii in various ways whereas dacD encodes a strong DD-CPase and plays a role in cell morphology, though it exerts negligible impact on other physiological aspects like intrinsic antibiotic resistance or biofilm formation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/603720v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@18820c9org.highwire.dtl.DTLVardef@208baeorg.highwire.dtl.DTLVardef@b2673eorg.highwire.dtl.DTLVardef@983aae_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Evaluation in mice of cell-free produced CT584 as a Chlamydia vaccine antigen

Chlamydia trachomatis is the most prevalent bacterial sexually transmitted pathogen worldwide. Since chlamydial infection is largely asymptomatic with the potential for serious complications, a preventative vaccine is likely the most viable long-term answer to this public health threat. Cell-free protein synthesis (CFPS) utilizes the cellular protein manufacturing machinery decoupled from the requirement for maintaining cellular viability, offering the potential for flexible, rapid, and de-centralized production of recombinant protein vaccine antigens. Here, we use CFPS to produce the putative chlamydial type three secretion system (T3SS) needle-tip protein, CT584, for use as a vaccine antigen in mouse models. High-speed atomic force microscopy (HS-AFM) imaging and computer simulations confirm that CFPS-produced CT584 retains a native-like structure prior to immunization. Female mice were primed with CT584 adjuvanted with CpG-1826 intranasally (i.n.) or CpG-1826 + Montanide ISA 720 intramuscularly (i.m.), followed four-weeks later by an i.m. boost before respiratory challenge with 104 inclusion forming units (IFU) of Chlamydia muridarum. Immunization with CT584 generated robust antibody responses but weak cell mediated immunity and failed to protect against i.n. challenge as demonstrated by body weight loss, increased lungs weights and the presence of high numbers of IFUs in the lungs. While CT584 alone may not be the ideal vaccine candidate, the speed and flexibility with which CFPS can be used to produce other potential chlamydial antigens makes it an attractive technique for antigen production.

immunology↗

bioRxiv2024

Stochastic modelling of single-cell gene expression adaptation reveals non-genomic contribution to evolution of tumor subclones

Cancer progression is an evolutionary process driven by the selection of cells adapted to gain growth advantage. We present the first formal study on the adaptation of gene expression in subclonal evolution. We model evolutionary changes in gene expression as stochastic Ornstein-Uhlenbeck processes, jointly leveraging the evolutionary history of subclones and single-cell expression data. Applying our model to sublines derived from single cells of a mouse melanoma revealed that sublines with distinct phenotypes are underlined by different patterns of gene expression adaptation, indicating non-genetic mechanisms of cancer evolution. Interestingly, sublines previously observed to be resistant to anti-CTLA-4 treatment showed adaptive expression of genes related to invasion and non-canonical Wnt signaling, whereas sublines that responded to treatment showed adaptive expression of genes related to proliferation and canonical Wnt signaling. Our results suggest that clonal phenotypes emerge as the result of specific adaptivity patterns of gene expression.

cancer biology↗

bioRxiv2024

Valproic Acid Treatment Enhances Chromosome Flexibility and Electron Transport in MCF7 Breast Cancer Cells

The structural integrity of the chromosomes is essential to every functional process within the eukaryotic nuclei. Chromosomes are DNA-histone complexes essential for the inheritance of genetic information to the offspring and any defect in it is linked to mitotic errors, cancer growth, and cellular aging. Changes in the mechanical properties of a chromosome could lead to its compromised function and stability, leading to chromosome breaks. Here, we studied the changes in chromosome physical properties using metaphase chromosomes isolated from human breast cancer cells (MCF7) exposed to Valproic Acid (VPA), a known epigenetic modifier drug involved in histone hyperacetylation and DNA demethylation. Due to chromosomal structural intricacy, preparative and technical limitations of analytical tools, we employed a label-free atomic force microscopy approach for simultaneously visualizing and mapping single chromosome elasticity. Additionally, we performed electron transport characteristics of metaphase chromosomes to elucidate the effect of VPA. Our multi-parametric strategy of probing physical properties of chromosomes offers a new scope in terms of analytical tools for studying chromosomal structural changes/aberrations and associated structure-function relationships pertinent to cancer.

biophysics↗

bioRxiv2024

Brain activation during the N-back working memory task in individuals with spinal cord injury: a functional near-infrared spectroscopy study

Cognitive impairments have frequently been reported in individuals with spinal cord injury (SCI) across different domains such as working memory, attention, and executive function. The mechanism of cognitive impairment after SCI is not well understood due to the heterogeneity of SCI sample populations, and may possibly be due to factors such as cardiovascular dysfunction, concomitant traumatic brain injury (TBI), hypoxia, sleep disorders, and body temperature dysregulation. In this study, we implement the Neuropsychiatric Unit Cognitive Assessment Tool (NUCOG) to assess cognitive differences between individuals with SCI and age-matched able-bodied (AB) controls. We then use an N-back working memory task and functional near-infrared spectroscopy (fNIRS) to elucidate the neurovascular correlates of cognitive function in individuals with SCI. We observed significant differences between the SCI and AB groups on measures of executive function on the NUCOG test. On the N-back task, across the three levels of difficulty: 0-back, 2-back, and 3-back, no significant differences were observed between the SCI and AB group; however, both groups performed worse as the level of difficulty increased. Although there were no significant differences in N-back performance scores between the two groups, functional brain hemodynamic activity differences were observed between the SCI and AB groups, with the SCI group exhibiting higher maximum oxygenated hemoglobin concentration in the right inferior parietal lobe. These findings support the use of fNIRS to study cognitive function in individuals with SCI and may provide a useful tool during rehabilitation to obtain quantitative functional brain activity metrics.

neuroscience↗

bioRxiv2024

Neuropilin-1 controls vascular permeability through juxtacrine regulation of endothelial adherens junctions

Neuropilin-1 (NRP1) regulates endothelial cell (EC) biology through modulation of vascular endothelial growth factor receptor 2 (VEGFR2) signalling by presenting VEGFA to VEGFR2. How NRP1 impacts VEGFA-mediated vascular hyperpermeability has however remained unresolved, described as exerting either a positive or a passive function. Using EC-specific Nrp1 knock-out mice, we discover that EC-expressed NRP1 exerts an organotypic role. In the ear skin, VEGFA/VEGFR2-mediated vascular leakage was increased following loss of EC NRP1, implicating NRP1 in negative regulation of VEGFR2 signalling. In contrast, in the back skin and trachea, loss of EC NRP1 decreased vascular leakage. In accordance, phosphorylation of vascular endothelial (VE)-cadherin was increased in the ear skin but suppressed in the back skin of Nrp1 iECKO mice. NRP1 expressed on perivascular cells has been shown to impact VEGF-mediated VEGFR2 signalling. Importantly, expression of NRP1 on perivascular cells was more abundant in the ear skin than in the back skin. Global loss of NRP1 resulted in suppressed VEGFA-induced vascular leakage in the ear skin, implicating perivascular NRP1 as a juxtacrine co-receptor of VEGFA in this compartment. Altogether, we demonstrate that perivascular NRP1 is an active participant in EC VEGFA/VEGFR2 signalling and acts as an organotypic modifier of EC biology.

cell biology↗

bioRxiv2023

Ribosome demand links transcriptional bursts to protein expression noise

Stochastic variation in protein expression generates phenotypic heterogeneity in a cell population and has an important role in antibiotic persistence, mutation penetrance, tumor growth and therapy resistance. Studies investigating molecular origins of noise have predominantly focused on the transcription process. However, the noise generated in the transcription process is further modulated by translation. This influences the expression noise at the protein level which eventually determines the extent of phenotypic heterogeneity in a cell population. Studies across different organisms have revealed a positive association between translational efficiency and protein noise. However, the molecular basis of this association has remained unknown. In this work, through stochastic modeling of translation in single mRNA molecules and empirical measurements of protein noise, we show that ribosome demand associated with high translational efficiency in a gene drives the correlation between translational efficiency and protein noise. We also show that this correlation is present only in genes with bursty transcription. Thus, our work reveals the molecular basis of how coding sequence of genes, along with their promoters, can regulate noise. These findings have important implications for investigating protein noise and phenotypic heterogeneity across biological systems.

systems biology↗

bioRxiv2023

Extracellular matrix scaffold-assisted tumor vaccines induce tumor regression and long-term immune memory

Injectable scaffold delivery is an immune engineering strategy to enhance the efficacy and reliability of cancer vaccine immunotherapy. The composition and structure of the biomaterial scaffold determines both vaccine release kinetics and inherent immune stimulation via the scaffold host response. Extracellular matrix (ECM) scaffolds prepared from decellularized tissues initiate an acute alternative inflammatory response following implantation, which facilitates wound healing following tumor resection and promotes local cancer immune surveillance. However, it remains unknown whether this environment is compatible with generating protective anti-tumor cytotoxic immunity with local immunotherapy delivery. Here, we engineered an ECM scaffold-assisted therapeutic cancer vaccine that maintained an immune microenvironment consistent with tissue reconstruction. Immune adjuvants MPLA, GM-CSF, and CDA were screened in a cancer vaccine formulated for decellularized small intestinal submucosa (SIS) ECM scaffold co-delivery. Though MPLA and GM-CSF showed the greatest increase in local myeloid cell infiltration, we found that the STING pathway adjuvant CDA was the most potent inducer of cytotoxic immunity with SIS-ECM scaffold delivery. Further, CDA did not diminish hallmark ECM immune responses needed in wound healing such as high Il4 cytokine expression. SIS scaffold delivery enhanced therapeutic vaccine efficacy using CDA and the antigen ovalbumin, curing greater than 50% of established EG.7 tumors in young mice and 75% in 24-week-old mature mice, compared to soluble components alone (0% cured). SIS-ECM scaffold assisted vaccination extended antigen exposure, was dependent on CD8+ cytotoxic T cells, and generated long term anti-tumor memory at least 7 months post-vaccination in both young and mature-aged mice. This study shows that an ECM scaffold is a promising delivery vehicle to enhance cancer vaccine efficacy while being orthogonal to characteristics of pro-healing immune hallmarks.

bioengineering↗

bioRxiv2023

Identifying Selectivity Filters in Protein Biosensor for Ligand Screening

Specialized sensing mechanisms in bacteria enable the identification of cognate ligands with remarkable selectivity in highly xenobiotic-polluted environments, where these ligands are utilized as energy sources. Here, via an integrated all-atom computer simulation, biochemical assay and isothermal titration calorimetry approaches we determine the molecular basis of MopR, a phenol biosensors complex selection process of ligand entry. Our results reveal a set of strategically placed selectivity filters along the ligand entry pathway of MopR. These filters act as checkpoints, screening diverse aromatic ligands at the protein surface based on their chemical features and sizes. Ligands meeting specific criteria are allowed to enter the sensing site in an orientation-dependent manner. Sequence and structural analyses demonstrate the conservation of this ligand entry mechanism across the sensor class, with individual amino acids along the selectivity filter path playing a critical role in ligand selection. Together, this investigation highlights the importance of interactions with the ligand entry pathway, in addition to interactions within the binding pocket, for achieving ligand selectivity in biological sensing. The findings enhance our understanding of ligand selectivity in bacterial phenol biosensors and provide insights for the rational expansion of the biosensor repertoire, particularly for the biotechnologically relevant class of aromatic pollutants.

biochemistry↗

bioRxiv2022

Enhanced transcriptional strength of HIV-1 subtype C minimizes gene expression noise and confers stability to the viral latent state

The stochastic fluctuations in gene expression emanating from HIV-1 long terminal repeat (LTR), amplified by the Tat positive feedback circuit, determine the choice between viral infection fates: active transcription (ON) or transcriptional silence (OFF). The emergence of several transcription factor binding site (TFBS) variant strains in HIV-1 subtype C (HIV-1C), especially those containing the duplication of NF-{kappa}B motif, mandates the evaluation of the effect of enhanced transcriptional strength on gene expression noise and its influence on viral fate-selection switch. Using a panel of subgenomic LTR-variant strains containing varying copy numbers of the NF-{kappa}B motif (ranging from 0 to 4), we employed flow cytometry, mRNA quantification, and pharmacological perturbations to demonstrate an inverse correlation between promoter strength and gene expression noise in Jurkat T-cells and primary CD4+ T-cells. The inverse correlation is consistent in clonal cell populations, at constant intracellular concentrations of Tat, and when NF-{kappa}B levels were regulated pharmacologically. Further, we show that strong LTRs containing at least two copies of the NF-{kappa}B motif in the enhancer establish a stabler latent state and demonstrate rapid latency reversal than weak LTRs containing fewer motifs. An engineered LTR containing three copies of the C-{kappa}B motif (CCC), an element unique for HIV-1C, demonstrated significantly higher levels of gene expression noise compared to the canonical HHC-LTR or two other engineered LTRs containing three copies of the H-{kappa}B (HHH) or F-{kappa}B (FFF) motif. This result suggests the indispensable nature of the C-{kappa}B motif for HIV-1C despite higher-level gene expression noise. We also demonstrate a cooperative binding of NF-{kappa}B to the motif cluster in HIV-1C LTRs containing two, three, or four NF-{kappa}B motifs (H = 2.61, 3.56, and 3.75, respectively). The present work alludes to a possible evolution of HIV-1C LTR towards gaining transcriptional strength associated with attenuated gene expression noise with implications for viral latency. Author SummaryOver the past two consecutive decades, HIV-1C has been undergoing directional evolution towards augmenting the transcriptional strength of the LTR by adding more copies of the existing TFBS by sequence duplication. Additionally, the duplicated elements are genetically diverse, suggesting broader-range signal receptivity by variant LTRs. HIV-1 promoter is inherently noisy, and the stochastic fluctuations in gene expression of variant LTRs may influence the ON/OFF latency decisions. The evolving NF-{kappa}B motif variations of HIV-1C offer a powerful opportunity to examine how the transcriptional strength of the LTR might influence gene expression noise. Our work here shows that the augmented transcriptional strength of HIV-1C LTR leads to a concomitantly reduced gene expression noise, consequently leading to stabler latency maintenance and rapid latency reversal. The present work offers a novel lead towards appreciating the molecular mechanisms governing HIV-1 latency.

microbiology↗