The Evolution of Regulatory Elements in the Emerging Promoter-Variant Strains of HIV-1 Subtype C

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 the 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 no 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 coinfection 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.

HIV-1 subtypes and latent reservoirs

“…First, numerous transcription factor binding sites (TFBS) are densely packed in proximity within the short span of the three regulatory domains – U3, R, and U5, possibly enabling viral gene expression in host cells of diverse lineages. Second, the various TFBS of the LTR, including GATA, c-MYB, RFB-1, RFB-2, LEF-1, USF, AP-1 NF-κB, Sp1, and others, belong to transcription factor families (TFF) typically conducive to cellular activation and/or suppressive signals [18 ▪ ]. In other words, many of these TFBS can recruit transcription-promoting or transcription-suppressive host factors, depending on the cellular activation status.…”

“…Only in the HIV-1C family the presence of additional copies of the NF-κB motif, three or even four, is observed [22,23]. The additional NF-κB motifs of HIV-1C are genetically diverse; furthermore, they may be positioned in the viral modulator region, thus blurring the distinction between the viral modulator and enhancer [18 ▪ ]. Although the presence of densely packed diverse TFBS in the LTR is believed to augment transcriptional noise significantly, thus enabling the ON/OFF fates of the virus, the existence of multiple copies of the NF-κB motif in HIV-1C LTR was shown to reduce the transcriptional noise considerably, stabilizing viral latency [5].…”

“…While the eight bases of the core RBEIII motif (5′-ACTGCTGA-3′) are highly conserved among all subtypes, the sequences flanking the RBEIII core are diverse and characterized by subtype-specific sequences [27]. Notably, the duplication of the RBEIII core sequence is accomplished with the invariable co-duplication of the flanking sequences, typically containing the binding sites for other TFF, primarily the AP-1 family [28,18 ▪ ]. The co-duplication of RBEIII and AP-1 motifs is expected to modulate the viral latency properties significantly as these two elements are independently known to be transcription-suppressive in a contextual manner.…”

“…In HIV-1C, RBEIII motif duplication is often associated with the co-duplication of sequence motifs binding TF other than AP-1 family members. The co-duplication of RBEIII and TCFα/LEF or NF-κB has been reported [18 ▪ ]. The effect of such diverse sequence motif duplications on HIV-1 latency and reservoir characteristics warrants evaluation.…”

“…The various LRA may be classified into those directly activating the NF-κB pathway, such as the agonists of the Protein Kinase C (PKC), Toll-Like-Receptors (TLR), or the Non-Canonical NF-κB signaling, and the other group indirectly modulating cellular activation signal pathways, including that of NF-κB signaling, such as STAT modulators, Epigenetic modulators, or PTEF-b releasing agents [58]. Since HIV-1 genetic families demonstrate different profiles of TFBS, including those of NF-κB motif [18 ▪ ,22,32,60], a rigorous evaluation of the LRA must be performed using reference panels of diverse viral subtypes. The LRAs belonging to certain classes, such as Prostratin, a PKC agonist [61], Vorinostat, an epigenetic modulator [20,60], or other Histone de-acetylase (HDAC) inhibitors [36,58,59,61] were evaluated against HIV-1 subtypes [21,36,55,61].…”