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Background The HIV-1 Subtype C, among other viral subtypes, is responsible for nearly 50% of the global infections including approximately 99% of the infections in India (UNAIDS 2012). HIV-1 subtype C is characterized by the presence of three NF-B binding sites in the enhancer as opposed to one or two such elements in other viral subtypes. Additionally, NF- Bs ite proximal to the Sp1 motifs, referred to here as the C-B site, is genetically distinct from the other two canonical NF- Bs ites (H-B sites). Furthermore, the Sp1III site proximal to the C-B motif also demonstrates subtype-specific genetic variations. Here, we examined the functional significance of the two unique regulatory elements, the C-B and the Sp1III motifs, for gene expression regulation from the viral promoter (Perkins et al. 1997). Methods and results Using several panels of reporter vectors expressing luciferase under the control of variant viral promoters, we examined gene expression in different mammalian cells under diverse activation conditions. The data confirm that the proximity and orientation of two sites, the C- Ba nd Sp1III motifs, are critical for optimal gene expression from the viral promoter. We found that a functional cooperation among the three NF-B sites is necessary for maximal promoter activity. Collectively, the data appeared to suggest that in addition to conferring a quantitative gain-offunction on the viral promoter, the presence of C-B site may have catalyzed associated genetic variation in the proximal Sp1III site through a positive selection pressure. To understand the functional association between these two sites under more relevant conditions, we constructed viral promoters consisting of only the C-B and the Sp1III sites, in the absence of other NF- Ba nd Sp1 sites. The subtype C specific Sp1III site showed higher magnitude of gene expression with the H- but not with the C- Bs ite. Experiments, using infectious viruses and the chromatin immunoprecipitation analysis, have also suggested the functional association between the two sites. In gel-shift assays the C-B site recruited the p50 and p65 heteroduplexes at a level comparable to that of the H- Bs ite. Importantly, given that the C- Bs ite is a G/C-centric motif, we asked and found that the C-B site is responsive to the p52:Bcl3 complex in contrast to the A/T-centric H-B site known to be more responsive to the p50:p65 complex promoter. Conclusions Collectively our data are suggestive of co-evolution of the two unique regulatory elements, the C- Ba nd the C-Sp1III motifs, that confer qualitative gain-of-function on the subtype C promoter thus probably playing a critical role towards the global predominance of the viral subtype. We further hypothesize that the C- Bs ite being aG /Ccentric motif, in combination with the two upstream H-B sites, confers a sustained and prolonged gene expression on HIV-1 subtype C viral promoter.

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.