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

Malgorzata Figiel, Miroslav Krepl, Sangwoo Park, Jaroslaw Poznański, Krzysztof Skowronek, Agnieszka Golab, Taekjip Ha, Jiří Šponer, Marcin Nowotny

Research output: Contribution to journalArticle

Abstract

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 doublestranded 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-1RTremain 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.Wefound that this unique recognition is based on two complementary elements that rely on the PPT sequence: RNaseHsequence preference and incompatibility of the poly(rA/dT) tract of the PPT with the nucleic acid conformation that is required for RNaseHcleavage. The latter results from rigidity of the poly(rA/dT) tract and leads to base-pair slippageof this sequenceupondeformation 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.

Original languageEnglish (US)
Pages (from-to)191-202
Number of pages12
JournalJournal of Biological Chemistry
Volume293
Issue number1
DOIs
StatePublished - Jan 5 2018

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Figiel, M., Krepl, M., Park, S., Poznański, J., Skowronek, K., Golab, A., Ha, T., Šponer, J., & Nowotny, M. (2018). Mechanism of polypurine tract primer generation by HIV-1 reverse transcriptase. Journal of Biological Chemistry, 293(1), 191-202. https://doi.org/10.1074/jbc.M117.798256