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.
Highlights
Introduction of a covalent tether betweenHuman immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and the RNA/DNA substrate should define the cleavage position 18 bp from the polymerase active site
We found that this unique recognition is based on two complementary elements that rely on the polypurine tract (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 precise mechanism by which the PPT primer is generated by HIV-1 RT has remained elusive despite many years of extensive studies of this enzyme
Summary
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 integrated into the DNA of the infected cell. Two copies (3Ј and central) of an important region of the genome, termed the polypurine tract (PPT), are refractory to RNase H cleavage They are left intact so they can be used as primers in the synthesis of the second DNA strand, termed (ϩ)-DNA [10, 11]. The PPT RNA primer was unique because the RNA/DNA hybrid it formed could be bound by the enzyme in both orientations, the one compatible with RNase H cleavage and the one conducive to polymerization. This explained why the PPT can be extended and serve to initiate the synthesis of the (ϩ)-DNA strand, playing a key role in HIV-1 proliferation [11]. Our results revealed an unexpected mechanism of PPT generation that utilizes a unique indirect readout of the homopolymeric nucleic acid sequence that relies on specific dynamic properties of the poly(rA/dT) segment
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