Biology subjects

Nala, N.

Publications and source records attributed to Nala, N..

3 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↗

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↗

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↗