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, 75 (10), 4832-42

Selective Excision of AZTMP by Drug-Resistant Human Immunodeficiency Virus Reverse Transcriptase

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Selective Excision of AZTMP by Drug-Resistant Human Immunodeficiency Virus Reverse Transcriptase

P L Boyer et al. J Virol.

Abstract

Two distinct mechanisms can be envisioned for resistance of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to nucleoside analogs: one in which the mutations interfere with the ability of HIV-1 RT to incorporate the analog, and the other in which the mutations enhance the excision of the analog after it has been incorporated. It has been clear for some time that there are mutations that selectively interfere with the incorporation of nucleoside analogs; however, it has only recently been proposed that zidovudine (AZT) resistance can involve the excision of the nucleoside analog after it has been incorporated into viral DNA. Although this proposal resolves some important issues, it leaves some questions unanswered. In particular, how do the AZT resistance mutations enhance excision, and what mechanism(s) causes the excision reaction to be relatively specific for AZT? We have used both structural and biochemical data to develop a model. In this model, several of the mutations associated with AZT resistance act primarily to enhance the binding of ATP, which is the most likely pyrophosphate donor in the in vivo excision reaction. The AZT resistance mutations serve to increase the affinity of RT for ATP so that, at physiological ATP concentrations, excision is reasonably efficient. So far as we can determine, the specificity of the excision reaction for an AZT-terminated primer is not due to the mutations that confer resistance, but depends instead on the structure of the region around the HIV-1 RT polymerase active site and on its interactions with the azido group of AZT. Steric constraints involving the azido group cause the end of an AZT 5'-monophosphate-terminated primer to preferentially reside at the nucleotide binding site, which favors excision.

Figures

FIG. 1
FIG. 1
The low dNTP extension assay tests the ability of wild-type and variant HIV-1 RTs to extend a primer using low concentrations of all four dNTPs. The strong pause site at approximately 350 nucleotides is probably due to a stem structure in the DNA which is used by the M13 bacteriophage for replication. When HIV-1 RT is polymerizing through this stem structure, the RT tends to pause.
FIG. 2
FIG. 2
Models showing the binding of ATP and PPi to AZT-resistant HIV-1 RT. van der Waals surfaces are drawn for polymerase active site residues (magenta) and residues involved in ATP binding and AZT resistance (yellow). Mutated amino acids M41L, K70R, L210W, and T215Y are shown with black labels, and amino acids that could be involved with ATP binding but that are not mutated (E44, K46) are shown with magenta labels. The two terminal nucleotide base pairs of the template-primer are shown. The 3′ end of the primer is AZTMP; the azido group is labeled. AZT-21 has the amino acid substitutions M41L, D67N, K70R, T215Y, and K219Q. The amino acid substitution T215Y has been modeled here in order to show potential aromatic interactions with this residue. The wild-type amino acids at K219 and D67 were retained in the figure to show a potential salt bridge between the residues. As described in the text, the AZT resistance mutations at these residues will destroy this salt bridge and may increase the ability of the pyrophosphate donor to bind. K219 and D67 are shown as stick diagrams to avoid obscuring the pyrophosphate binding site. The presumptive salt bridge between Lys219 and Asp67 is shown as a dotted line (see text). Panel A shows the model with PPi bound, and panel B shows the model with ATP bound.
FIG. 2
FIG. 2
Models showing the binding of ATP and PPi to AZT-resistant HIV-1 RT. van der Waals surfaces are drawn for polymerase active site residues (magenta) and residues involved in ATP binding and AZT resistance (yellow). Mutated amino acids M41L, K70R, L210W, and T215Y are shown with black labels, and amino acids that could be involved with ATP binding but that are not mutated (E44, K46) are shown with magenta labels. The two terminal nucleotide base pairs of the template-primer are shown. The 3′ end of the primer is AZTMP; the azido group is labeled. AZT-21 has the amino acid substitutions M41L, D67N, K70R, T215Y, and K219Q. The amino acid substitution T215Y has been modeled here in order to show potential aromatic interactions with this residue. The wild-type amino acids at K219 and D67 were retained in the figure to show a potential salt bridge between the residues. As described in the text, the AZT resistance mutations at these residues will destroy this salt bridge and may increase the ability of the pyrophosphate donor to bind. K219 and D67 are shown as stick diagrams to avoid obscuring the pyrophosphate binding site. The presumptive salt bridge between Lys219 and Asp67 is shown as a dotted line (see text). Panel A shows the model with PPi bound, and panel B shows the model with ATP bound.
FIG. 3
FIG. 3
Steric hindrance when an AZT-terminated primer is bound to RT at the P site. The figure, based on the structure of the ternary RT-DNA-dNTP complex (8), shows that the distance between the azido of AZT and D185 would cause steric conflict; the distance between D185 and the first and second azido nitrogens is less than the sum of the van der Waals radii. The P and N sites are marked.
FIG. 4
FIG. 4
(A) The PBS primer was 5′ end labeled and annealed to the template as described in Materials and Methods. The 3′ end of the primer was blocked by the addition of an AZT residue. The ability of wild-type HIV-1 RT and the RT variants to remove the blocking AZT residue (deblocking) and to extend the freed end of the primer was tested in the presence of 10.0 μM concentrations of each dNTP, 1.0 μM AZTTP, and varying concentrations of ATP (1.0, 2.0, 5.0, and 10.0 mM) as the pyrophosphate donor. In the cell, nucleoside analogs will be present in the triphosphate form, and after a primer is deblocked there is a possibility that HIV-1 RT will add another nucleoside analog back on to the 3′ end of the primer rather than the normal dNTP, which in this case is dTTP. The addition of AZTTP to the reaction mixture is meant to reflect what can occur within the cell. A control lane with no added wild-type RT shows the pattern of the starting template-primer. A control lane to which has been added HIV-1 RT but no ATP indicates the amount of extendable primer. This could result from primer which did not get blocked by an AZT residue or from a low level of deblocking by the enzyme using the dNTPs as the pyrophosphate donor or a combination of both processes. The locations of the starting PBS primer and the fully extended primer are marked. (B) The gel in panel 4A was scanned by a PhosphorImager. In each lane, the amount of radioactivity in the full-length product was divided by the total amount of radioactivity to determine the percentage of full-length product. This value was plotted versus the level of ATP present in the reaction mixture. The percentage of full-length product in the No ATP control lane indicates that the background level is very low (<1.0%). (C) The 3′ end of the primer was blocked by the addition of an AZT residue, and the ability of wild-type HIV-1 RT and the RT variants to remove the blocking AZT residue and extend the freed end of the primer was tested in the presence of 10.0 μM concentrations of each dNTP, 1.0 μM AZTTP, and varying concentrations of NaPPi (25.0, 50.0, 100.0, and 200.0 μM) as the pyrophosphate donor. The locations of the starting PBS primer and the fully extended primer are marked. (D) The gel in panel C was scanned by a PhosphorImager. In each lane, the amount of radioactivity in the full-length product was divided by the total amount of radioactivity to determine the percentage of full-length product. This value was plotted versus the level of NaPPi present in the reaction mixture. The percentage of full-length product in the no NaPPi control lane indicates that the background level is very low compared to that for the reactions where NaPPi is present.
FIG. 5
FIG. 5
(A) The PBS primer was 5′ end labeled and annealed to the template as described in Materials and Methods. The 3′ end of the primer was blocked by the addition of a ddT residue. The ability of wild-type HIV-1 RT and the RT variants to remove the blocking ddT residue (deblocking) and extend the freed end of the primer was tested in the presence of 10.0 μM concentrations of each dNTP, 1.0 μM ddTTP, and varying concentrations of ATP (1.0, 2.0, 5.0, and 10.0 mM) as the pyrophosphate donor. Experiments using D4T as the blocking group gave similar results. (B) The gel in panel A was scanned by a PhosphorImager. In each lane, the amount of radioactivity in the full-length product was divided by the total amount of radioactivity to determine the percentage of full-length product. This value was plotted versus the level of ATP present in the reaction mixture. (C) The 3′ end of the primer was blocked by the addition of a ddT residue, and the ability of wild-type HIV-1 RT and the RT variants to remove the blocking ddT residue and extend the freed end of the primer was tested in the presence of 10.0 μM concentrations of each dNTP, 1.0 μM ddTTP, and varying concentrations of NaPPi (25.0, 50.0, 100.0, and 200.0 μM) as the pyrophosphate donor. The locations of the starting PBS primer and the fully extended primer are marked. (D) The gel in panel C was scanned by a PhosphorImager. In each lane, the amount of radioactivity in the full-length product was divided by the total amount of radioactivity to determine the percentage of full-length product. This value was plotted versus the level of NaPPi present in the reaction mixture.
FIG. 6
FIG. 6
(A) The 3′ end of the primer was blocked by the addition of an AZT residue. The ability of wild-type HIV-1 RT and the RT variants to remove the blocking AZT residue (deblocking) and extend the freed end of the primer was tested in the presence of 100.0 μM concentrations of each dNTP, 10.0 μM AZTTP, and varying concentrations of ATP (1.0, 2.0, 5.0, and 10.0 mM) as the pyrophosphate donor. The ratio of dTTP:AZTTP remained at 10:1. The locations of the starting PBS primer and the fully extended primer are marked. (B) The gel in panel A was scanned by a PhosphorImager. In each lane, the amount of radioactivity in the full-length product was divided by the total amount of radioactivity to determine the percentage of full-length product. This value was plotted versus the level of ATP present in the reaction mixture. (C) The 3′ end of the primer was blocked by the addition of a ddT residue. The ability of wild-type HIV-1 RT and the RT variants to remove the blocking ddT residue (deblocking) and extend the freed end of the primer was tested in the presence of 100.0 μM concentrations of each dNTP, 10.0 μM ddTTP, and varying concentrations of ATP (1.0, 2.0, 5.0, and 10.0 mM) as the pyrophosphate donor. The ratio of dTTP:ddTTP remained at 10:1. The locations of the starting PBS primer and the fully extended primer are marked. (D) The gel in panel C was scanned by a PhosphorImager. In each lane, the amount of radioactivity in the full-length product was divided by the total amount of radioactivity to determine the percentage of full-length product. This value was plotted versus the level of ATP present in the reaction mixture. Experiments using D4TTP as the blocking group gave similar results.

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