Enhanced Transcriptional Strength of HIV-1 Subtype C Minimizes Gene Expression Noise and Confers Stability to the Viral Latent State.

Stochastic fluctuations in gene expression emanating from the 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 the NF-κB 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 different copy numbers of the NF-κB motif (ranging from 0 to 4), we used 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-κB levels were regulated pharmacologically. Further, we show that strong LTRs containing at least two copies of the NF-κB motif in the enhancer establish a more stable latent state and demonstrate more rapid latency reversal than weak LTRs containing fewer motifs. We also demonstrate a cooperative binding of NF-κB to the motif cluster in HIV-1C LTRs containing two, three, or four NF-κB motifs (Hill coefficient [H] = 2.61, 3.56, and 3.75, respectively). The present work alludes to a possible evolution of the HIV-1C LTR toward gaining transcriptional strength associated with attenuated gene expression noise with implications for viral latency. IMPORTANCE Over the past two consecutive decades, HIV-1 subtype C (HIV-1C) has been undergoing directional evolution toward augmenting the transcriptional strength of the long terminal repeat (LTR) by adding more copies of the existing transcription factor binding site (TFBS) by sequence duplication. Additionally, the duplicated elements are genetically diverse, suggesting broader-range signal receptivity by variant LTRs. The HIV-1 promoter is inherently noisy, and the stochastic fluctuations in gene expression of variant LTRs may influence the active transcription (ON)/transcriptional silence (OFF) latency decisions. The evolving NF-κB 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 the HIV-1C LTR leads to concomitantly reduced gene expression noise, consequently leading to stabler latency maintenance and rapid latency reversal. The present work offers a novel lead toward appreciating the molecular mechanisms governing HIV-1 latency.

Validation of CRISPR activation system in Aedes cells using multicistronic plasmid vectors.

Aedes mosquitoes transmit several pathogens including flaviviruses to humans which result in high morbidity and mortality. Owing to adaptability and climate change, these mosquito vectors are predicted to establish in new geographical areas thus exposing larger populations to the risk of infection. Therefore, control of Aedes vector is necessary to prevent disease transmission. Recently, genetic approaches to vector control have shown promise; however, the tools and methods for manipulating the mosquito genome are rather limited. While CRISPR-Cas9 system has been adapted for gene editing purposes in Aedes mosquito, the dCas9-based transcription control of genes remain unexplored. In this study we report implementation of the CRISPR activation system in Aedes cells. For this we designed, constructed and tested a bi-partite plasmid-based strategy that allows expression of the dCas9-VPR and targeting guide RNA together with a reporter cassette. Quantitative analysis of the fluorescent reporter gene levels showed a robust over-expression validating CRISPR activation in Aedes cells. This strategy and the biological parts will be useful resource for synthetic transcription factor-based robust upregulation of Aedes genes for application of synthetic biology approaches for vector control.