Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA

Abstract

We have determined the 3.0 A resolution structure of wild-type HIV-1 reverse transcriptase in complex with an RNA:DNA oligonucleotide whose sequence includes a purine-rich segment from the HIV-1 genome called the polypurine tract (PPT). The PPT is resistant to ribonuclease H (RNase H) cleavage and is used as a primer for second DNA strand synthesis. The 'RNase H primer grip', consisting of amino acids that interact with the DNA primer strand, may contribute to RNase H catalysis and cleavage specificity. Cleavage specificity is also controlled by the width of the minor groove and the trajectory of the RNA:DNA, both of which are sequence dependent. An unusual 'unzipping' of 7 bp occurs in the adenine stretch of the PPT: an unpaired base on the template strand takes the base pairing out of register and then, following two offset base pairs, an unpaired base on the primer strand re-establishes the normal register. The structural aberration extends to the RNase H active site and may play a role in the resistance of PPT to RNase H cleavage.

Fig. 1. Process of reverse transcription of the HIV-1 genome. (A) Minus strand DNA synthesis (DNA strand in red) is initiated using a cellular tRNA annealed to the PBS. The RNA strand of the RNA:DNA duplex is degraded by RNase H of HIV-1 RT. (B) First strand transfer allows annealing of the newly formed DNA to the 3′ end of the viral genome. Transfer is mediated by identical repeated (R) sequences. (C) Minus strand DNA synthesis resumes, accompanied by RNase H digestion of all template RNA except PPT. (D) PPT is used as a primer for second strand DNA synthesis. (E) RNase H removes the tRNA and the PPT. In HIV-1, a single RNA nucleotide (from tRNA) is left by RNase H at the RNA/DNA PBS junction. (F) During second strand transfer (not shown) the newly formed PBS DNA (second strand) anneals to the PBS DNA from the first strand. Completion of second strand synthesis results in a linear DNA duplex with LTRs at both ends.

Fig. 2. Top: HIV genome sequence at the PPT (underlined) and U3 region. The minus strand synthesis initiation site is marked with an asterisk; +1 is the first nucleotide of U3. Bottom: sequence of the RNA:DNA oligonucleotide in our RT–RNA:DNA complex.

Fig. 3. Stereo view of a ribbon representation of the structure of HIV-1 RT in complex with the polypurine RNA:DNA. The fingers, palm, thumb, connection and RNase H subdomains of p66 are colored blue, red, green, yellow and orange, respectively. The p51 subunit is colored gray. The RNA template and DNA primer strands are shown in magenta and blue, respectively.

Fig. 4. The sequence and numbering scheme of the RNA:DNA PPT and the interactions between the nucleic acid and amino acid residues of HIV-1 RT (≤3.8 Å). The RNA (orange) and DNA (cyan) strands are designated Tem and Pri, respectively. The nucleotide site positions are labeled with ascending numbers from the polymerase domain toward the RNase H domain. Amino acids of the p51 subunit are designated by an asterisk following the residue number; all others are in p66. RNase H nucleotide site positions are designated positive (+1 to +4) for positions 3′ to, and negative (–1 to –9) for positions 5′ to, the scissile phosphate, where the 3′ and 5′ orientations are for the RNA strand. Hydrogen bonds are shown in red dashed lines and other types of interaction are shown in solid black lines. 2′-OH groups of RNA and phosphate groups are shown in red and gray spheres. Weakly paired (distance ≥3.6 Å), mismatched and unpaired bases are shown filled with stripes, spheres and empty, respectively. Residues Gly359 and Ala360 of the RNase H primer grip interact with the nucleic acid through their main-chain atoms. Arg284 was modeled as Ala because of weak density for the side chain. N474 interacts with Pri15-Thy through a water molecule (not shown).

Fig. 5. Simulated annealing (F_o – F_c) omit electron density maps contoured at the 2σ level at the polymerase active site (1) (omitting nucleic acid) and of the unpaired residue of template (2) (omitting unpaired residue Tem-15-Ade).

Fig. 6. Molecular surface representation of HIV-1 RT showing the nucleic acid binding cleft and the RNase H primer grip. Residues colored in cyan or magenta are amino acids within 3.8 Å of the 2′-OH of RNA template nucleotides (magenta) or any other part of nucleic acid (cyan). The RNA template is shown as red ribbon and the DNA primer in blue ribbon. Minor groove widths proximal to the thumb area or at the RNase H active site are indicated (∼10 and ∼8 Å, respectively). The trajectory and minor groove width of a hypothetical RNA strand that can be cleaved efficiently by RNase H are shown in red. The RNase H primer grip region is shown in ball and stick representation in the figure inset.

Fig. 7. Stereo view of structures of the nucleic acid template-primers in the RT–DNA:DNA (Ding et al., 1998) and RT–RNA:DNA complexes. The 19mer DNA and 31mer RNA templates are shown in yellow and magenta, respectively. The 18mer DNA and 29mer DNA primers are cyan and blue. Region I contains the 4 bp near the polymerase active site. Region II consists of the next 4 bp at the bend of the nucleic acid. The next 5 bp compose region III, followed by region IV that contains residues of the ‘unzipped’ part of PPT.

Fig. 8. Variation in minor groove width of the RNA:DNA template-primer. The four regions of nucleic acid are defined in the legend of Figure 8. The minor groove width values for canonical A- and B-type DNA are 11 and 6 Å, respectively.