Pre-steady-state kinetic studies establish entecavir 5'-triphosphate as a substrate for HIV-1 reverse transcriptase

Abstract

The novel 2'-deoxyguanosine analog Entecavir (ETV) is a potent inhibitor of hepatitis B virus (HBV) replication and is recommended for treatment in human immunodeficiency virus type 1 (HIV-1) and HBV-co-infected patients because it had been reported that ETV is HBV-specific. Recent clinical observations, however, have suggested that ETV may indeed demonstrate anti-HIV-1 activity. To investigate this question at a molecular level, kinetic studies were used to examine the interaction of 5'-triphosphate form of ETV with wild type (WT) HIV-1 reverse transcriptase (RT) and the nucleoside reverse transcriptase inhibitor-resistant mutation M184V. Using single turnover kinetic assays, we found that HIV-1 WT RT and M184V RT could use the activated ETV triphosphate metabolite as a substrate for incorporation. The mutant displayed a slower incorporation rate, a lower binding affinity, and a lower incorporation efficiency with the 5'-triphosphate form of ETV compared with WT RT, suggesting a kinetic basis for resistance. Our results are supported by cell-based assays in primary human lymphocytes that show inhibition of WT HIV-1 replication by ETV and decreased susceptibility of the HIV-1 containing the M184V mutation. This study has important therapeutic implications as it establishes ETV as an inhibitor for HIV-1 RT and illustrates the mechanism of resistance by the M184V mutant.

FIGURE 1.. Incorporation of ETVTP by HIV-1 RT.

A, the structure of ETVTP. B, the 24-nucleotide (nt) DNA primer annealed to a 36-nt DNA template that was used for all experiments. The arrow denotes the position of the next dNTP to be incorporated. C, products from single-turnover incorporation of ETVTP alone (100 μm) or with the natural dNTPs (100 μm of each) into the D24/D36 primer/template (50 nm) by WT RT (250 nm). Lanes represent the increasing reaction time; Lane 1, 0 min; lane 2, 1 min; lane 3, 2 min; lane 4, 3 min; lane 5, 5 min; lane 6, 10 min. The sizes of the DNA primer (24-mer) and the extended products are indicated. D, products from single-turnover incorporation of dGTP alone (100 μm), all four natural dNTPs (100 μm each) and CBVTP alone (200 μm) into the D24/D36 primer/template (50 nm) by WT RT (250 nm). Lanes represent the increasing reaction times as described above. The sizes of the DNA primer (24-mer) and the extended products are indicated.

FIGURE 2.. Concentration dependence of the observed rate on ETVTP concentration for WT HIV-1 RT.

A, single-turnover incorporation was measured by mixing a preincubated solution of RT (250 nm) and DNA/DNA primer/template (50 nm) with various concentrations of ETVTP (curves displayed represent 0.5 μm (○), 1 μm (□), 5 μm (◇), 20 μm (●) and 50 μm (△)) and MgCl₂ (10 mm) at 37 °C (all concentrations are final after mixing). B, K_d,ETVTP curve was generated from all ETVTP concentrations tested (500 nm to 50 μm) where the observed rate (k_obsd) was plotted against ETVTP concentration. The fit to the data gives an equilibrium binding constant (K_d,ETVTP) of 2.23 ± 0.67 μm and a maximum rate of incorporation (k_pol) of 0.107 ± 0.007 s⁻¹. C, pre-steady-state burst kinetics of incorporation of ETVMP into a DNA/DNA primer/template by WT RT were measured by mixing a preincubated solution of RT (100 nm) and primer/template (300 nm) with ETVTP (50 μm) and Mg²⁺ (10 mm) under rapid quench conditions (all concentrations are final after mixing). The reaction was quenched at indicated times and analyzed by 20% sequencing gel electrophoresis. The solid line represents a fit to a burst equation with an amplitude (A) equal to 79 ± 12 nm, an observed first-order rate constant for the burst phase (k_obsd) equal to 0.125 ± 0.031 s⁻¹, and an observed rate for the linear phase (k_ss) equal to 0.020 ± 0.006 s⁻¹.

FIGURE 3.. Effect of ETVTP versus dGTP incorporation on primer/template binding to WT HIV-1 RT.

Burst amplitudes were measured at varying concentrations of D24/D36 using dGTP (■) or ETVTP (●) as the substrate for incorporation. Data were fit to the quadratic equation (see “Experimental Procedures”). The K_d,DNA for D24/D36 during dGTP incorporation was 31.3 ± 9.3 nm, whereas during ETVTP incorporation the K_d,DNA was 127.5 ± 4.9 nm.

FIGURE 4.. Overlap between ETV and the next 2′-deoxyribose in the primer chain.

The surface of the methylene moiety is colored orange, and the closest atoms in the next 2′-deoxyribose are in gray. The green arrow near the lower left corner shows the largest overlap found when ETV is in position +5 in the primer.