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

Abstract

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.