Mechanism of polypurine tract primer generation by HIV-1 reverse transcriptase

J Biol Chem. 2018 Jan 5;293(1):191-202. doi: 10.1074/jbc.M117.798256. Epub 2017 Nov 9.


HIV-1 reverse transcriptase (RT) possesses both DNA polymerase activity and RNase H activity that act in concert to convert single-stranded RNA of the viral genome to double-stranded DNA that is then integrated into the DNA of the infected cell. Reverse transcriptase-catalyzed reverse transcription critically relies on the proper generation of a polypurine tract (PPT) primer. However, the mechanism of PPT primer generation and the features of the PPT sequence that are critical for its recognition by HIV-1 RT remain unclear. Here, we used a chemical cross-linking method together with molecular dynamics simulations and single-molecule assays to study the mechanism of PPT primer generation. We found that the PPT was specifically and properly recognized within covalently tethered HIV-1 RT-nucleic acid complexes. These findings indicated that recognition of the PPT occurs within a stable catalytic complex after its formation. We found that this unique recognition is based on two complementary elements that rely on the PPT sequence: RNase H sequence preference and incompatibility of the poly(rA/dT) tract of the PPT with the nucleic acid conformation that is required for RNase H cleavage. The latter results from rigidity of the poly(rA/dT) tract and leads to base-pair slippage of this sequence upon deformation into a catalytically relevant geometry. In summary, our results reveal an unexpected mechanism of PPT primer generation based on specific dynamic properties of the poly(rA/dT) segment and help advance our understanding of the mechanisms in viral RNA reverse transcription.

Keywords: cysteine-mediated cross-linking; human immunodeficiency virus (HIV); molecular dynamics; nucleic acid structure; protein-nucleic acid interaction; reverse transcriptase; ribonuclease H.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Sequence
  • Crystallography, X-Ray / methods
  • DNA Primers / biosynthesis*
  • DNA Primers / chemistry
  • DNA, Viral
  • HIV Reverse Transcriptase / metabolism*
  • HIV Reverse Transcriptase / physiology*
  • HIV-1 / genetics
  • Nucleic Acid Conformation
  • Nucleic Acids
  • Poly A
  • Poly U
  • Polynucleotides
  • Purines / chemistry
  • RNA, Viral / chemistry
  • Ribonuclease H / metabolism


  • DNA Primers
  • DNA, Viral
  • Nucleic Acids
  • Polynucleotides
  • Purines
  • RNA, Viral
  • Poly A
  • Poly U
  • reverse transcriptase, Human immunodeficiency virus 1
  • HIV Reverse Transcriptase
  • Ribonuclease H

Associated data

  • PDB/4PQU
  • PDB/1HYS