Promoter evolution in HIV-1C establishes latent reservoirs highly resistant to reversal
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Latent viral reservoirs remain a major barrier to curing HIV-1, with the long-terminal repeat (LTR) and Tat playing crucial roles in regulating viral transcription. Subtype-specific transcription factor binding site (TFBS) variations within the LTR significantly influence latency and reservoir stability. In earlier work, we identified HIV-1C LTR variants with duplicated TFBS motifs, including NF-κB, AP1, RBEIII, and TCF-1α/LEF-1. Using five cell models, including Jurkat and primary CD4⁺ T cells, we compared canonical R-LTR and variant R2-LTR strains. Across sub-genomic reporters, single-round infections, and full-length viral vectors, we found that the balance between RBEIII and NF-κB motifs governs stability of latency. The ‘two-viruses-one-cell’ system that normalized confounding environmental factors further revealed that latency is primarily controlled by intrinsic transcriptional circuits rather than external stimuli. In longitudinal studies of HIV-1⁺ individuals from acute and chronic infection phases, we observed dominant R strains during early infection and the spontaneous emergence of R2 strains in nearly half of chronic-phase subjects, a process accelerated by ART. Upon CD4⁺ T cell activation, R strains preferentially rebounded, while R2 strains showed strong resistance to reversal, even in subjects harbouring a co-infection. Together, these findings establish the clinical significance of LTR variation in latency regulation and identify the R2 phenotype as a critical determinant of reservoir persistence. These results underscore the importance of addressing reservoir heterogeneity in cure strategies, particularly in HIV-1C-prevalent regions.