Enzymology
Mechanism of polypurine tract primer generation by HIV-1 reverse transcriptasePPT primer generation by HIV-1 RT

https://doi.org/10.1074/jbc.M117.798256Get rights and content
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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.

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

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This work was initially supported by a grant from the Polish National Science Center (Contract N N301 439738); later by an International Early Career Scientist grant from the Howard Hughes Medical Institute (both to M. N.); and by a Foundation for Polish Science “Ideas for Poland” award (to M. N.), Czech Science Foundation Grant P305/12/G034 (to J. Š. and M. K.), Ministry of Education, Youth and Sports of the Czech Republic Project LO1305 (to J. Š.), Praemium Academiae (to J. Š.), and National Institutes of Health Grant GM065367 and United States National Science Foundation Grant PHY 1430124 (to S. P. and T. H.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This article contains supporting results and discussion, Figs. S1–S9, and Tables S1 and S2.

1

An investigator of the Howard Hughes Medical Institute.