Biology subjects

Ranga, U.

Publications and source records attributed to Ranga, U..

8 recordsLinked to original sources

bioRxiv2026

Enhanced strand transfer and mismatch extension by HIV-1C reverse transcriptase promote sequence motif duplication

Genetic diversification of HIV-1 is driven largely by the error-prone activity of reverse transcriptase (RT) and frequent template switching during reverse transcription. A rare outcome of nonhomologous recombination is sequence motif duplication, which can alter viral gene regulation and protein function. Previous studies have shown that such duplications occur at significantly higher frequencies in HIV-1 subtype C (HIV-1C), particularly within the long terminal repeat (LTR) and p6-Gag regions, where they can confer replication advantages. However, the mechanistic basis for this subtype-specific bias remains unclear. We therefore investigated whether intrinsic biochemical properties of HIV-1C RT contribute to its elevated duplication frequency. Bioinformatic analysis of 6,877 full-length HIV-1 genomes identified four duplication hotspots, with the highest frequencies in HIV-1C. Comparative sequence analysis of RT revealed several subtype-specific residues, including a highly conserved threonine at position 359 (T359) in the connection domain of HIV-1C RT. Structural modeling suggested that T359 can form an additional hydrogen bond with the nascent cDNA, potentially stabilizing the RT-template complex. Biochemical characterization of recombinant RT variants demonstrated that residue 359 modulates polymerase activity and maintains subtype-specific optimal catalytic function. Functional assays further revealed that HIV-1C RT exhibits enhanced template strand transfer compared with HIV-1B RT. In addition, next-generation sequencing-based primer extension assays showed that HIV-1C RT extends mismatched 3' termini more efficiently across multiple mismatch types. Together, these findings indicate that subtype-specific biochemical properties of HIV-1C RT, particularly enhanced strand transfer and mismatch extension mediated in part by T359, promote nonhomologous recombination events that generate sequence motif duplications. This work provides a mechanistic explanation for the elevated duplication frequency characteristic of HIV-1C and highlights how subtle RT polymorphisms can shape viral evolutionary trajectories.

microbiology↗

bioRxiv2026

A highly stringent high-throughput screening assay for identifying transcription-modulating agents of HIV-1 latency

HIV-1 latency remains a central obstacle to curing infection, and current latency-modulating agents (LMAs) suffer from poor specificity and inconsistent efficacy. To enable discovery of small molecules (SMs) that directly target the viral master transcriptional regulatory circuit (MTRC), we developed a highly stringent, dual-reporter high-throughput screening (HTS) assay based on a natural HIV-1 subtype C long terminal repeat (LTR) variant, LRhR-HC, which exhibits markedly reduced transcriptional noise and a high activation threshold. We engineered Jurkat cells to stably harbour two independent reporter cassettes driven by the tough-to-activate LRhR-HC-LTR and the canonical LR-HHC-LTR, enabling simultaneous detection of latency-promoting and latency-reversing activities through both fluorescent and secreted enzymatic reporters. This dual-reporter line responded robustly and predictably to conventional latency-reversal agents (LRAs) and latency-promoting agents (LPAs), validating assay responsiveness. Z'-factor measurements demonstrated excellent assay performance, with values ranging from approximately 0.7 across different formats, confirming a strong dynamic range and reproducibility. Screening of the 1,520-compound Prestwick chemical library (PCL), which includes FDA-approved drugs, identified 27 candidate LPAs, including known agents such as Spironolactone and Aminacrine, thereby validating the assay specificity while revealing several novel inhibitory molecules. A mechanistically focused panel of 10 additional compounds yielded two putative LPAs and one LRA, with secondary analyses confirming their latency-modulating activities and cytotoxicity profiles. Collectively, this HIV-1C-derived dual-reporter platform provides a stringent and flexible HTS system for identifying LMAs with potential applications in both block-and-lock and shock-and-kill cure strategies.

molecular biology↗

bioRxiv2025

Promoter evolution in HIV-1C establishes latent reservoirs highly resistant to reversal

Latent viral reservoirs remain a major barrier to curing HIV-1, with the long-terminal repeat (LTR) and Tat playing crucial roles in regulating viral transcription. Subtype-specific transcription factor binding site (TFBS) variations within the LTR significantly influence latency and reservoir stability. In earlier work, we identified HIV-1C LTR variants with duplicated TFBS motifs, including NF-{kappa}B, AP1, RBEIII, and TCF-1/LEF-1. Using five cell models, including Jurkat and primary CD4 T cells, we compared canonical R-LTR and variant R2-LTR strains. Across sub-genomic reporters, single-round infections, and full-length viral vectors, we found that the balance between RBEIII and NF-{kappa}B motifs governs stability of latency. The two-viruses-one-cell system that normalized confounding environmental factors further revealed that latency is primarily controlled by intrinsic transcriptional circuits rather than external stimuli. In longitudinal studies of HIV-1 individuals from acute and chronic infection phases, we observed dominant R strains during early infection and the spontaneous emergence of R2 strains in nearly half of chronic-phase subjects, a process accelerated by ART. Upon CD4 T cell activation, R strains preferentially rebounded, while R2 strains showed strong resistance to reversal, even in subjects harbouring a co-infection. Together, these findings establish the clinical significance of LTR variation in latency regulation and identify the R2 phenotype as a critical determinant of reservoir persistence. These results underscore the importance of addressing reservoir heterogeneity in cure strategies, particularly in HIV-1C-prevalent regions.

microbiology↗

bioRxiv2024

Neurovirulent and non-neurovirulent strains of HIV-1 and their Tat proteins induce differential cytokine-chemokine profiles

HIV-1 enters the central nervous system (CNS) early in infection, and a significant proportion of people with HIV experience CNS complications despite anti-retroviral therapy. Chronic immune dysfunction, inflammatory cytokines and chemokines, and viral proteins like Tat and gp120 released by HIV-1-infected immune cells are implicated in the pathogenesis of HIV-1-associated neurocognitive disorders (HAND). To elucidate the contribution of non-viral factors to CNS complications in people with HIV-1, a comparative analysis of neurovirulent subtype B (HIV-1ADA) and non-neurovirulent subtype C (HIV-1Indie-C1) isolates was performed. Culture supernatants from HIV-1-infected PBMCs, either with or without immunodepletion of Tat and gp120, were used to treat SH-SY5Y neuroblastoma cells. HIV-1ADA-infected PBMC media showed significantly higher cytotoxicity than HIV-1IndieC1-infected PBMC media, notwithstanding the depletion of Tat and gp120, highlighting the role of non-viral factors contributing to neurotoxicity. A comparison of inflammatory profiles revealed that HIV-1ADA media contained elevated levels of cytokines (IL-1, IL-1{beta}, IL-6, TNF) and chemokines (CCL2, CCL3, CCL4, IP10). Given the involvement of Tat in upregulating immune mediators, PBMCs from healthy subjects were treated with recombinant purified Tat from subtype B or C. Subtype B Tat induced a stronger inflammatory response than subtype C Tat. These results confirm that both viral and non-viral immune factors mediate neuronal damage in people with HIV.

microbiology↗

bioRxiv2023

The Expression of HIV-1 Tat in Lactococcus lactis

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.

microbiology↗

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↗

bioRxiv2021

The evolution of regulatory elements in the emerging promoter variant strains of HIV-1

In a multicentric, observational, investigator-blinded, and longitudinal clinical study of 764 ART-naive 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 coding sequences. The emerging viral strains contain additional copies of the existing transcription factor binding sites (TFBS), including TCF-1/LEF-1, RBEIII, AP-1, and NF-{kappa}B, 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 not 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 co-infection 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. Significance StatementA unique conglomeration of TFBS enables the HIV-1 promoter to accomplish two diametrically opposite functions - transcriptional activation and transcriptional silencing. The various phases of viral latency -establishment, maintenance, and reversal -collectively determine the replication fitness of individual viral strains. A profound variation in the TFBS composition of the viral promoter may significantly alter the viral latency properties and the latent reservoir characteristics. Although the duplication of certain TFBS remains a quality unique to HIV-1C, the high-level genetic recombination of HIV-1 may promote the transfer of such molecular properties to the other HIV-1 subtypes. The emergence of several promoter-variant viral strains may make the task of a functional cure more challenging in HIV-1C.

microbiology↗

bioRxiv2020

A stronger transcription regulatory circuit of HIV-1C drives rapid establishment of latency with implications for the direct involvement of Tat

The magnitude of transcription factor binding site variation emerging in HIV-1C, especially the addition of NF-{kappa}B motifs by sequence duplication, makes the examination of transcriptional silence challenging. How can HIV-1 establish and maintain latency despite having a strong LTR? We constructed panels of sub-genomic reporter viral vectors with varying copy numbers of NF-{kappa}B motifs (0 to 4 copies) and examined the profile of latency establishment in Jurkat cells. We found surprisingly that the stronger the viral promoter, the faster the latency establishment. Importantly, at the time of commitment to latency and subsequent points, Tat levels in the cell were not limiting. Using highly sensitive strategies, we demonstrate the presence of Tat in the latent cell, recruited to the latent LTR. Our data allude, for the first time, to Tat establishing a negative feedback loop during the late phases of viral infection, leading to the rapid silencing of the viral promoter. ImportanceOver the past 10-15 years, HIV-1C has been evolving rapidly towards gaining stronger transcriptional activity by sequence duplication of major transcription factor binding sites. The duplication of NF-{kappa}B motifs is unique and exclusive for HIV-1C, a property not shared with any of the other eight HIV-1 genetic families. What mechanism(s) does HIV-1C employ to establish and maintain transcriptional silence despite the presence of a strong promoter and a concomitant strong, positive transcriptional feedback is the primary question we attempted to address in the present manuscript. The role Tat plays in latency reversal is well established. Our work with the most common HIV-1 subtype C (HIV-1C) offers crucial leads towards Tat possessing a dual-role in serving both as transcriptional activator and repressor at different phases of the viral infection of the cell. The leads we offer through the present work have significant implications for HIV-1 cure research.

microbiology↗