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

Abstract

In a multicentric, observational, investigator-blinded, and longitudinal clinical study of 764 ART-naïve 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-1α/LEF-1, RBEIII, AP-1, and NF-κB, 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.

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

“…The outcome that enhanced transcriptional strength is inversely correlated with transcriptional noise, although anticipated, is surprising since gene expression noise of the LTR is expected to play a crucial role in the ON/OFF decision-making (14,20) (PMID: 16051143, 18344999). The cost of reduced transcriptional noise of the LTR, the consequence of the enhanced transcriptional strength, may lead to the loss of replication fitness of viral strains that contain more NF-κB motifs in the enhancer, as exemplified by our recent report (36) (PMID: 34899661). The proportion of 4-κB viral strains was found to be 1-2% (n= 607 samples) in 2003 in India (38) (PMID: 15184461), which increased to 20-30% (n= 159 samples) in ten years (34).…”

“…The distance between the two motifs is not optimal for cooperative binding of NF-κB, as was experimentally demonstrated (58) (PMID: 19683540). In contrast, the canonical 3- and 4-κB LTRs of HIV-1C not only contain a larger number of the κB-motifs in the LTR, but the spacer length separating the motifs is only three base pairs, closer to the optimal distance of two base pairs estimated optimal for cooperative binding of NF-κB (see discussion) (34,36) (PMID: 23132857, 34899661). Given these important variations in the organization of the homotypic clusters of NF-κB motifs in HIV-1C LTR, we wanted to understand if some level of cooperative binding would be possible in HIV-1C.…”

“…The distance between the two motifs is not optimal for cooperative binding of NF-κB, as was experimentally demonstrated (58) (PMID: 19683540). In contrast, the canonical 3-and 4-κB LTRs of HIV-1C not only contain a larger number of the κB-motifs in the LTR, but the spacer length separating the motifs is only three base pairs, closer to the optimal distance of two base pairs estimated optimal for cooperative binding of NF-κB (see discussion) (34,36)…”

“…Over the years, our laboratory monitored the evolution of HIV-1C in India and identified two major hotspots of genetic variation in the viral genome -in the LTR and p6-Gag (34)(35)(36)(37)(38) (PMID: 15184461, 23132857, 24960272, 29773649, 34899661). The variations in the LTR are centered around the creation of additional copies of certain TFBS by sequence motif duplication.…”

“…The variations in the LTR are centered around the creation of additional copies of certain TFBS by sequence motif duplication. A recent study from our laboratory identified the emergence of at least nine different promoter variant viral strains of HIV-1C containing duplications of TFBS belonging to TCFα/LEF, RBE III, and/or NF-κB (36) (PMID: 34899661). Of note, the LTR variations are characterized by not only copy number difference but also sequence variation and the co-duplication of the TFBS motifs.…”