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

0486050 - BFÚ 2018 RIV US eng J - Journal Article
Figiel, M. - Krepl, Miroslav - Park, S. - Poznanski, J. - Skowronek, K. - Golab, A. - Ha, T. - Šponer, Jiří - Nowotny, M.
Mechanism of polypurine tract primer generation by HIV-1 reverse transcriptase.
Journal of Biological Chemistry. Roč. 293, č. 1 (2018), s. 191-202. ISSN 0021-9258. E-ISSN 1083-351X
R&D Projects: GA ČR(CZ) GBP305/12/G034
Institutional support: RVO:68081707
Keywords : human-immunodeficiency-virus * strand dna-synthesis * retroviral rnases-h * rna/dna hybrid
OECD category: Biochemistry and molecular biology
Impact factor: 4.106, year: 2018

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 slip-page 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.
Permanent Link: http://hdl.handle.net/11104/0280938