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

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.

Figure 1.

RNase H cleavage in cross-linked complexes of HIV-1 RT with substrates that comprise the PPT sequence. A, sequence of HIV-1 RNA region that contains the PPT (marked in bold). The upstream U-tract and downstream U3 element are indicated. B, schematic of the cross-linked complexes of PPT1 and PPT2 hybrid substrates. HIV-1 RT is shown in schematic representation (light gray for polymerase domain, dark gray for RNase H domain, and white for p51). The cross-links are marked with wavy lines. The positions of terminal fluorescent dyes (Cy5 and fluorescein (FAM)) in the RNA strands are indicated. The expected positions of the RNase H active site and cleavage in the cross-linked complex are indicated with arrows. The sequence alteration that is required to introduce thiol-modified guanine in the DNA strand is indicated in italics. Numbering of the substrate residues relative to the scissile phosphate (used in all of the analyses) is shown in the left panel. C, RNase H cleavage within cross-linked complexes with PPT substrates. Reaction products were separated in urea-polyacrylamide gels and visualized with Cy5 (upper panels) or fluorescein (FAM) (lower panels) fluorescence. M, marker (Cy5-labeled 24-mer RNA corresponding to the product of the cleavage 18 nt from the 3′ end of the primer); D, reaction in the presence of DTT.

Figure 2.

Effect of substrate sequence on RNase H cleavage within cross-linked complexes. A, substrates used for the analysis of PPT cleavage specificity and sequence determinants of cleavage. Only RNA strands of the hybrid substrates are shown for clarity. All of the sequences are presented from 5′ to 3′. PPT segments are marked in bold. For substrates CL4, CL5, and CL6, the preferred residues at the key consensus positions are shown in green, and the non-preferred residues are shown in red. For A/U-box substrates, the sequences that were replaced in the CL4 substrate are shown in blue. The expected cleavage sites are indicated with arrows. B, cleavage rates of the substrates listed in A within cross-linked complexes. Error bars represent S.D. of three independent measurements. Lines represent the result of global fitting of the data using a pseudo-zero-order reaction. C, t_½ values calculated from global fitting of the data. Detailed results with statistical analyses are shown in Table S1, and the global fitting is shown in Fig. S6. n.d., not determined.

Figure 3.

Flexibility of RNA/DNA hybrids (looping assay). A, schematic representation of the substrates that were used in the assay. Blue and red bars represent DNA and RNA, respectively. Fluorophores on 5′ termini of the strands are indicated. B, sequences of the 6-nt RNA fragments in the substrates. Abasic sites are indicated with underscores. C, looping of substrates with 6-bp RNA/DNA hybrid segments. Error bars represent S.D. of three independent measurements. C4, black; A box, magenta; C box, blue; G box, orange; U box, green; AbG, cyan; Aba, red; Gba, gray. D, looping times of the analyzed substrates.

Figure 4.

Base-pair slippage of poly(rA/dT) tracts. A and B, example of transient base-pair slippage observed in the molecular dynamics simulation of the PPT2 substrate. The protein is in gray. The RNA and DNA strands are in red and blue, respectively. The A-T base pairs are in color: −2 (green), −3 (yellow), −4 (orange), and −5 (cyan). A, structure before the simulation. B, shift in base pairing (slippage) that is observed when the catalytic interaction between the RNase H domain and PPT2 substrate is imposed.

Figure 5.

Model of PPT recognition by HIV-1 RT. A, two elements are involved in PPT recognition: sequence preference in positions −4, −2, and +1 (magenta) for cleavage by the RNase H domain and conformational changes of the substrate that are required for RNase H cleavage to occur. B, cleavage at the expected site (PPT-U3 junction) involves both preferred residues at the cleavage consensus positions (green) and the ability of the PPT sequence to undergo the conformational change without distortion. C, cleavage in the middle of the PPT body (A-tract) is inhibited by three elements: (i) non-preferred residues in the consensus positions (red), (ii) rigidity of the poly(rA/dT) sequence that makes the conformational change less likely (gray arrow), and (iii) deformations of the substrate (poly(A)-sequence slippage) when the deformation is enforced, leading to misalignment of the RNA at the RNase H active site. Ranges of PPT residues that are included in each schematic are indicated with colored frames at the bottom.