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.

Biophysics Core Facility International Institute of Molecular and Cell Biology 02 109 Warsaw Poland

Department of Biophysics and Biophysical Chemistry Johns Hopkins University Baltimore Maryland 21205 USA

Department of Biophysics and Biophysical Chemistry Johns Hopkins University Baltimore Maryland 21205 USA Howard Hughes Medical Institute Baltimore Maryland 21205 USA Department of Biophysics Johns Hopkins University Baltimore Maryland 21205 USA Department of Biomedical Engineering Johns Hopkins University Baltimore Maryland 21205 USA

Institute of Biochemistry and Biophysics Polish Academy of Sciences 02 106 Warsaw Poland

Institute of Biophysics Academy of Sciences of the Czech Republic 612 65 Brno Czech Republic Regional Centre of Advanced Technologies and Materials Department of Physical Chemistry Faculty of Science Palacky University Olomouc 771 46 Olomouc Czech Republic

Laboratory of Protein Structure International Institute of Molecular and Cell Biology 02 109 Warsaw Poland

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