Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase

Abstract

We have examined amino acid substitutions at residues 115 and 116 in the reverse transcriptase (RT) of HIV-1. A number of properties were examined, including polymerization and processivity on both DNA and RNA templates, strand displacement, ribonucleotide misincorporation, and resistance to nucleoside analogs. The RT variants Tyr-115-Phe and Phe-116-Tyr are similar to wild-type HIV-1 RT in most, but not all, respects. In contrast, the RT variant Tyr-115-Val is significantly impaired in polymerase activity compared with wild-type RT; however, Tyr-115-Val is able to incorporate ribonucleotides as well as deoxyribonucleotides during polymerization and is resistant to a variety of nucleoside analogs.

Figure 1

Polymerase activity of the RT variants relative to wild-type RT. The level of radioactivity incorporated by wild-type RT represents 100% activity and the level of radioactivity incorporated by the RT variants is normalized to this value. The template primers used are described in Materials and Methods. (A) RDDP activities. (B) DDDP activities.

Figure 2

Processivity assays with DNA and RNA templates. End-labeled primers were annealed to template DNA and RNA and extended in the presence of a cold trap that prevents multiple rounds of polymerization. (A) Processivity with the PBS DNA oligonucleotide annealed to sense PPT-PBS DNA. (B) Processivity with the PBS DNA oligonucleotide annealed to sense PPT-PBS RNA. The locations of the 3′ ends of two known secondary-structural elements, TAR (for a review, see ref. 1) and the poly(A) hairpin (34), are shown at the right. Duplicate assays, labeled 1 and 2, were done for each sample. WT, wild type.

Figure 3

Strand-displacement activity of wild-type (WT) and mutant HIV-1 RTs. The PBS DNA oligonucleotide was end-labeled with [γ³²P]ATP and annealed to sense single-strand DNA. A 10-fold excess of unlabeled DNA oligonucleotides also was annealed to the sense-strand DNA to provide the targets to be displaced. The RT polymerization reaction was done in the presence of two different concentrations of KCl: 75 mM KCl (A) and 35 mM KCl (B). The 5′ end of the first unlabeled target DNA oligonucleotide (marked at the right) indicates the starting point for strand displacement. Duplicate assays, labeled 1 and 2, were done for each sample.

Figure 4

UTP misincorporation by wild-type (WT) and mutant HIV-1 RTs. An unlabeled sequencing primer was annealed to single-strand M13mp18 DNA. RT polymerization was done with radioactive [α³²P]UTP in the reaction mixture. Extension products with UTP incorporation were radioactively labeled and visible on x-ray film. Duplicate assays, labeled 1 and 2, were done for each sample.

Figure 5

Inhibition curves for various nucleoside analogs. The amount of radioactivity incorporated into the template-primer by wild-type and mutant HIV-1 RTs in the absence of inhibitor represents 100% enzyme activity. The amount of radioactivity incorporated at the various concentration of inhibitor is normalized to this value. The template primer for testing ddITP and ddGTP inhibition was poly(rC)⋅oligo(dG), whereas that for ddTTP and AZTTP was poly(rA)⋅oligo(dT). The template-primer for testing (−)-β-l-2′,3′-dideoxy-3′-thiacytidine triphosphate (3TCTP) inhibition was a sequencing primer annealed to single-strand M13mp18 DNA. When single-strand M13mp18 was used as a template with the −47 primer, the Tyr-115–Val mutant produced 36% as much DNA product as wild-type HIV-1 RT; with poly(rA)⋅oligo(dT) and poly(rC)⋅oligo(dT), the Tyr-115–Val mutant produced 50–75% as much DNA product.

Figure 6

View of the polymerase active site of HIV-1 RT. The sugar ring of the incoming dCTP is positioned between the hydrophobic side chains of Tyr-115 and Ala-114 at the back (Van der Waals spheres in purple), and Glu-151 and Arg-72 in front (Van der Waals spheres in cyan). Phe-116, although too far away to interact directly with the sugar of the dCTP, forms the floor of the 3′ OH binding pocket (Van der Waals spheres in purple). Yellow lines indicate interactions of the 3′ OH of dCTP with the ring of Tyr-115, the main-chain NH of Tyr-115, the main-chain NH of Ala-114, β-phosphate of dCTP, and CO of Glu-151. Bridging interactions of the NH group of Glu-151 with the guanidinium of Arg-72 and the main-chain CO of Lys-73 are shown as green dotted lines. The interactions of the Tyr-115 ring and the Glu-151 side chain with the 2′ carbon of dCTP are shown as red dotted lines.