Structural Basis of Polypurine Track Strand Displacement by HIV-1 Reverse Transcriptase

To complete reverse transcription, HIV-1 reverse transcriptase (RT) must displace the RNase H-resistant polypurine tract (PPT) primers. This enables synthesis of the long terminal repeats and formation of the central cDNA flap. However, the molecular mechanism of this PPT strand displacement (SD) has remained unknown, and no structural data exist on how a retroviral polymerase execute these reactions. We report the first cryo-EM structures of HIV-1 RT bound to nucleic acid substrates containing either a PPT_RNA or PPT_DNA displacement strand, with incoming dATP positioned at the polymerase catalytic site. These structures reveal key features of the PPT displacement mechanism by RT. Specifically, we observed a binding mode where the template nucleotide (T₁) base-paired to the first displacement nucleotide (D₁) undergoes a 90° rotation relative to the preceding template base (T₀). This sharp template flip positions D₁ ~30 Å away from the primer's 3'-end and is coordinated by RT_p66 residues at the SD interface: F61 and R78 contact T₁/T₀ to drive template translocation, while W24 engages both T₁ and D₁ to stabilize the displacement strand. Biochemical and virological mutagenesis experiments confirm that interactions with F61 and R78 are essential for both canonical cDNA polymerization and SD, whereas the W24-nucleotide interactions are required exclusively for SD but are dispensable for standard cDNA synthesis. These results contribute to the structural and functional understanding of PPT strand displacement by HIV-1 RT and reveal a distinct mechanistic vulnerability for the design of next-generation antiretrovirals.