Hypersusceptibility mechanism of Tenofovir-resistant HIV to EFdA

Abstract

Background: The K65R substitution in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is the major resistance mutation selected in patients treated with first-line antiretroviral tenofovir disoproxil fumarate (TDF). 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), is the most potent nucleoside analog RT inhibitor (NRTI) that unlike all approved NRTIs retains a 3'-hydroxyl group and has remarkable potency against wild-type (WT) and drug-resistant HIVs. EFdA acts primarily as a chain terminator by blocking translocation following its incorporation into the nascent DNA chain. EFdA is in preclinical development and its effect on clinically relevant drug resistant HIV strains is critically important for the design of optimal regimens prior to initiation of clinical trials.

Results: Here we report that the K65R RT mutation causes hypersusceptibility to EFdA. Specifically, in single replication cycle experiments we found that EFdA blocks WT HIV ten times more efficiently than TDF. Under the same conditions K65R HIV was inhibited over 70 times more efficiently by EFdA than TDF. We determined the molecular mechanism of this hypersensitivity using enzymatic studies with WT and K65R RT. This substitution causes minor changes in the efficiency of EFdA incorporation with respect to the natural dATP substrate and also in the efficiency of RT translocation following incorporation of the inhibitor into the nascent DNA. However, a significant decrease in the excision efficiency of EFdA-MP from the 3' primer terminus appears to be the primary cause of increased susceptibility to the inhibitor. Notably, the effects of the mutation are DNA-sequence dependent.

Conclusion: We have elucidated the mechanism of K65R HIV hypersusceptibility to EFdA. Our findings highlight the potential of EFdA to improve combination strategies against TDF-resistant HIV-1 strains.

Figure 1

Inhibition of WT and K65R RT-catalyzed DNA synthesis by EFdA-TP. T_d31/P_d18-P0 was incubated with WT or K65R HIV-1 RT for 50 minutes in the presence of 1 μM dNTPs, MgCl₂ and increasing concentrations of EFdA-TP (0–1,500 nM). The experiment was carried out in the (A) absence or (B) presence of 3.5 mM ATP. The template sequence is shown next to the gels and the numbers indicate the points of EFdA-TP incorporation (+1, +6 and +10).

Figure 2

Effect of K65R mutation on the translocation state of RT bound to T/P_EFdA-MP. (A) The translocation state of HIV-1 RT after EFdA-MP incorporation was determined using site-specific Fe²⁺ footprinting. T_d43/P_d30-EFdA-MP (100 nM) with 5'-Cy3-label on the DNA template was incubated with WT or K65R HIV-1 RT (600 nM) and various concentrations of the next incoming nucleotide (dTTP). The complexes were treated for 5 minutes with ammonium iron sulphate (1 mM) and resolved on a polyacrylamide 7 M urea gel. An excision at position −18 indicates a pre-translocation complex, while the one at position −17 represents a post-translocation complex. (B) The post-translocated complexes were determined from the gels and plotted using GraphPad Prism. Light blue indicates the physiological dNTP concentrations. (C) Schematic representation of the position of EFdA-MP-terminated primers at the pre- and post-translocated sites.

Figure 3

ATP- and PPi-dependent rescue of EFdA-MP terminated primers by WT and K65R RTs. (A) ATP-dependent rescue of T_d31/P_{d18-P0-EFdA-MP}. Purified T_d31/P_{d18-P0-EFdA-MP} was incubated with WT or K65R RT in the presence of 10 mM MgCl₂, 3.5 mM ATP, 100 μM dATP, 0.5 μM dTTP, and 10 μM ddGTP at 37°C. Aliquots of the reaction were stopped at the indicated time points (0–90 min). The results of at four independent experiments were plotted using one site hyperbola in Graphpad Prism 4. (B) PPi-dependent rescue of T_d31/P_{d18-P0-EFdA-MP}. Purified T_d31/P_{d18-P0-EFdA-MP} was incubated with WT or K65R RT in the presence of 6 mM MgCl₂, 150 μM PPi, 100 μM dATP, 0.5 μM dTTP, and 10 μM ddGTP at 37°C. Aliquots of the reaction were stopped at the indicated time points (0–40 min). The results of two independent experiments were plotted using one site hyperbola in Graphpad Prism 4.

Figure 4

Molecular models of dATP and EFdA-TP in the active sites of WT and K65R HIV RT. dATP (yellow sticks, A and C) and EFdA-TP (cyan sticks, B and D) are shown at the active sites of WT HIV RT, (A and B) or K65R HIV RT (C and D). The fingers and palm subdomains are shown in blue and red cartoon, respectively. The primer and template strands are shown in dark gray and light gray sticks, respectively. Figures were made using PyMOL (The PyMOL Molecular Graphics System, Version 1.3 Schrödinger, LLC).