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We demonstrate that at least three different promoter variant strains of HIV-1 subtype C have been gradually expanding and replacing the standard subtype C viruses in India, and possibly in South Africa and other global regions, over the past decade. The new viral strains contain an additional NF-κB, NF-κB-like, or RBEIII site in the viral promoter. Although the acquisition of an additional RBEIII site is a property shared by all the HIV-1 subtypes, acquiring an additional NF-κB site remains an exclusive property of subtype C. The acquired κB site is genetically distinct, binds the p50-p65 heterodimer, and strengthens the viral promoter at the levels of transcription initiation and elongation. The 4-κB viruses dominate the 3-κB "isogenic" viral strains in pairwise competition assays in T-cell lines, primary cells, and the ecotropic human immunodeficiency virus mouse model. The dominance of the 4-κB viral strains is also evident in the natural context when the subjects are coinfected with κB-variant viral strains. The mean plasma viral loads, but not CD4 counts, are significantly different in 4-κB infection suggesting that these newly emerging strains are probably more infectious. It is possible that higher plasma viral loads underlie selective transmission of the 4-κB viral strains. Several publications previously reported duplication or deletion of diverse transcription factor-binding sites in the viral promoter. Unlike previous reports, our study provides experimental evidence that the new viral strains gained a potential selective advantage as a consequence of the acquired transcription factor-binding sites and importantly that these strains have been expanding at the population level.

After screening a large number of clinical samples of HIV-1 subtype C in India, a subset of viral strains containing sequence insertions upstream of the viral enhancer has been identified. The sequence insertions contained binding sites for at least two different transcription factors NF-κB and RBEIII, importantly, in a mutually exclusive fashion. Furthermore, while some of the viral strains contained insertions of κB-like sites, a few others contained dual insertions of the RBEIII and κB sites together but only one of the two was intact. NF-κB acquisition appears to be the most common phenotype unique for subtype C with nearly half of the variant strains containing such insertions. Given that subtype C already contains three functional NF-κB sites in the viral enhancer, acquisition of a fourth NF-κB motif in some variant viral strains is intriguing. Further investigation is warranted to examine the significance of the sequence insertions for the replicative fitness of the variant viral strains.

Subtype-C strains of HIV-1, among the various viral subtypes, are responsible for ~50% of the global and 85-99% of Indian infections. Among others, the most significant molecular feature differentially conserved in the subtype-C promoter is the polymorphism within the enhancer region constituted by NF-κB sites. While the viral promoter of majority of the subtypes contains two NF-κB sites, subtype-C promoter consists of three canonical motifs. Notably, a minority of clade-C primary isolates contain κB or κB-like sites, in addition to the canonical κB sites. Previous studies from our laboratory identified nearly 6% (34/609) of the primary isolates from India to demonstrate κB-site polymorphism in C-LTR. The functional importance of additional κB or κB-like sites in C-LTR has not been evaluated. We confirmed the subtype nature of the viral isolates by sequencing and phylogenetic analysis of LTR, Tat and Env in 20 of 34 samples demonstrating κB-site polymorphism. Sequence analysis of the additional κB- or κB-like sites identified extensive variation among the viral isolates. Among them, a particular sequence variation, constituting the κB-like site GGGACTTTCT, with a C-T variation at position 10, was found to be the most common. The functional importance of the κB-like site in C-LTR has been evaluated by reporter gene expression from isogenic LTR promoters and EMSA. We compared gene expression pattern of LTR constructs in Jurkat cells under different conditions of cell activation including TNF-α, PMA, PHA and combinations that activate NF-κB pathway. The data demonstrate the strongest reporter gene expression from the LTRs containing the additional κB-like sites especially under synergistic activation conditions. Gel shift experiments with TNF-α activated Jurkat cell nuclear extracts showed the recruitment of p50/p65 heterodimer to the variant κB-like site. Our data for the first time provide experimental evidence that the κB-like site in C-LTR is an authentic κB-site and this site confers quantitative and qualitative gain of function. Furthermore, we hypothesize that this gain of function may augment viral fitness and hence contribute to global predominance of subtype-C infections.