The Reverse Transcriptase of the Human Immunodeficiency Virus (HIV) is distinguished by its high rate of homologous recombination. A less-studied consequence of this phenomenon is the increased occurrence of non-homologous recombination, which results in length polymorphism. While most of these genome-wide variations are sporadic, some provide a replication advantage to variant strains, such as those in the Long Terminal Repeat (LTR) and p6-Gag regions. By analyzing sequences from these two regions in the HIV-1 databases, we categorize all types of non-homologous recombination into four groups based on the presence or absence of two molecular features. Additionally, drawing on established models of homologous recombination, we propose a model that describes the process of sequence duplication. This model can also be applied to explain non-homologous recombination in different types of HIV and other viruses.

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.

Genetic diversification of HIV-1 is driven largely by the error-prone activity of reverse transcriptase (RT) and the high frequency of template switching during reverse transcription. A rare consequence of nonhomologous recombination is sequence motif duplication, which can alter viral gene regulation and protein function. Previous studies have shown that such duplications occur at higher frequencies in HIV-1C, particularly within the long terminal repeat and p6-Gag regions, where they can confer replication advantages. However, the mechanistic basis for this subtype-specific bias has remained unclear. Here, we investigated whether intrinsic biochemical properties of HIV-1C RT contribute to its elevated duplication frequency. Bioinformatic analysis of 6,877 full-length HIV-1 genomes identified four duplication hotspots across the viral genome, with the highest frequencies in HIV-1C. Comparative sequence analysis revealed several subtype-specific residues in RT, including a conserved threonine at position 359 (T359) in the connection domain. Structural modeling suggested that T359 can form an additional hydrogen bond with the nascent cDNA, potentially stabilizing the RT–template complex. Biochemical characterization of recombinant RT variants demonstrated that residue 359 modulates polymerase activity and maintains subtype-specific optimal catalytic function. Functional assays further showed that HIV-1C RT exhibits enhanced template strand transfer compared with HIV-1B RT. In addition, next-generation sequencing–based primer extension assays revealed that HIV-1C RT extends mismatched 3′ termini more efficiently across multiple mismatch types. Together, these findings indicate that subtype-specific biochemical properties of HIV-1C RT, mediated in part by T359, promote nonhomologous recombination events that generate sequence motif duplications. IMPORTANCE: High-frequency sequence motif duplications are a distinctive feature of HIV-1C, yet the molecular mechanisms underlying their higher prevalence have remained unclear. Here, we demonstrate that intrinsic biochemical properties of HIV-1C reverse transcriptase (RT) promote these events through enhanced template strand transfer and improved extension of mismatched 3′ termini during reverse transcription. These properties increase the probability of nonhomologous recombination events that generate these duplications across the viral genome. Importantly, such duplications have major biological consequences at hotspots. In the long terminal repeat, duplication of transcription factor binding motifs produces promoter variants that alter transcriptional strength, influencing both latency establishment and reversal. In the p6-Gag region, duplication of the PTAP motif is linked to compensatory adaptation in drug resistance and may enhance viral fitness under antiretroviral pressure. Together, our findings provide a mechanistic explanation for the emergence of clinically relevant viral variants and highlight how subtle subtype-specific RT polymorphisms can shape HIV-1 evolution and persistence.

Efficient expression of functional proteins in heterologous hosts has become the pivotal focus of modern biotechnology and biomedical research. To this end, multiple alternatives to E. coli are being explored for recombinant protein expression. L. lactis , being a gram-positive organism, circumvents the need for an endotoxin removal step during protein purification. We report here the optimisation of the expression of HIV-1 Tat, a notoriously difficult protein, in Lactococcus lactis system. We evaluated five different promoters in two different Lactococcus lactis strains and examined the effect of pH, glucose, and induction time on the yield and purity of Tat. Finally, the recombinant Tat was functionally competent in transactivating the HIV-1 promoter in HLM-1 reporter cells. Our work provides a scaffold for future work on the expression of toxic proteins in Lactococcus lactis .

Genetic diversification of HIV-1 is driven largely by the error-prone activity of reverse transcriptase (RT) and frequent template switching during reverse transcription. A rare outcome of nonhomologous recombination is sequence motif duplication, which can alter viral gene regulation and protein function. Previous studies have shown that such duplications occur at significantly higher frequencies in HIV-1 subtype C (HIV-1C), particularly within the long terminal repeat (LTR) and p6-Gag regions, where they can confer replication advantages. However, the mechanistic basis for this subtype-specific bias remains unclear. We therefore investigated whether intrinsic biochemical properties of HIV-1C RT contribute to its elevated duplication frequency. Bioinformatic analysis of 6,877 full-length HIV-1 genomes identified four duplication hotspots, with the highest frequencies in HIV-1C. Comparative sequence analysis of RT revealed several subtype-specific residues, including a highly conserved threonine at position 359 (T359) in the connection domain of HIV-1C RT. Structural modeling suggested that T359 can form an additional hydrogen bond with the nascent cDNA, potentially stabilizing the RT-template complex. Biochemical characterization of recombinant RT variants demonstrated that residue 359 modulates polymerase activity and maintains subtype-specific optimal catalytic function. Functional assays further revealed that HIV-1C RT exhibits enhanced template strand transfer compared with HIV-1B RT. In addition, next-generation sequencing-based primer extension assays showed that HIV-1C RT extends mismatched 3′ termini more efficiently across multiple mismatch types. Together, these findings indicate that subtype-specific biochemical properties of HIV-1C RT, particularly enhanced strand transfer and mismatch extension mediated in part by T359, promote nonhomologous recombination events that generate sequence motif duplications. This work provides a mechanistic explanation for the elevated duplication frequency characteristic of HIV-1C and highlights how subtle RT polymorphisms can shape viral evolutionary trajectories.

The Reverse Transcriptase of the Human Immunodeficiency Virus (HIV) is distinguished for its high rate of homologous recombination. A less-studied consequence of this phenomenon is the increased occurrence of non-homologous recombination, which results in length polymorphism. While most of these genome-wide variations are sporadic, some provide a replication advantage to variant strains, such as those in the Long Terminal Repeat (LTR) and p6-Gag regions. By analyzing sequences from these two regions in the HIV-1 databases, we categorize all non-homologous recombination into four groups based on the presence or absence of two molecular features. Additionally, drawing on established models of homologous recombination, we propose a model that describes the process of sequence duplication. This model can also be applied to explain non-homologous recombination in different types in HIV and other viruses.