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

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

Author information

1
Laboratory of Protein Structure, International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland
2
Institute of Biophysics, Academy of Sciences of the Czech Republic, 612 65 Brno, Czech Republic
3
Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacky University Olomouc, 771 46 Olomouc, Czech Republic
4
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, Maryland 21205, USA
5
Institute of Biochemistry and Biophysics Polish Academy of Sciences, 02-106 Warsaw, Poland
6
Biophysics Core Facility, International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland
7
Howard Hughes Medical Institute, Baltimore, Maryland 21205, USA
8
Department of Biophysics, Johns Hopkins University, Baltimore, Maryland 21205, USA
9
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland 21205, USA

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 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

PMID:
29122886
PMCID:
PMC5766924
DOI:
10.1074/jbc.M117.798256
[Indexed for MEDLINE]
Free PMC Article

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