Enhanced strand transfer and mismatch extension by HIV-1C reverse transcriptase promote sequence motif duplication

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.IMPORTANCEHigh-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.