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by claude@2026-07, 2026-07-04
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The paper investigates the molecular mechanism by which HIV-1 reverse transcriptase displaces the RNase H-resistant polypurine tract (PPT) primers, a required step in completing reverse transcription and forming the central cDNA flap. Using the first cryo-EM structures of HIV-1 RT bound to nucleic acid substrates designed to model either PPTRNA or PPTDNA displacement, the authors report that the incoming dATP occupies the catalytic site and that strand displacement proceeds via a template nucleotide (T1) that flips about 90° relative to the prior template base (T0). This template flip relocates the first displacement nucleotide (D1) ~30 Å from the primer’s 3’-end and is coordinated by RTp66 residues F61 and R78 at the strand-displacement interface, while W24 engages both T1 and D1 to stabilize the displacement strand. The paper’s explicit limitation is that it studies this mechanism using specific structured nucleic-acid substrates and structural snapshots, complemented by mutagenesis and biochemical/virological assays rather than directly visualizing full-length transcription in vivo. The paper does not explicitly discuss endometriosis or adenomyosis; it was included in the corpus via a keyword match in the upstream search index.
Abstract
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 (T 1 ) base-paired to the first displacement nucleotide (D 1 ) undergoes a 90° rotation relative to the preceding template base (T 0 ). This sharp template flip positions D 1 ∼30 Å away from the primer’s 3’-end and is coordinated by RT p66 residues at the SD interface: F61 and R78 contact T 1 /T 0 to drive template translocation, while W24 engages both T 1 and D 1 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.
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Abstract
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 PPTRNA or PPTDNA 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 RTp66 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.
Competing Interest Statement
The authors have declared no competing interest.
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