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. 2008 Dec 5;283(49):34218-28.
doi: 10.1074/jbc.M806797200. Epub 2008 Oct 20.

Delayed Chain Termination Protects the Anti-Hepatitis B Virus Drug Entecavir From Excision by HIV-1 Reverse Transcriptase

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Delayed Chain Termination Protects the Anti-Hepatitis B Virus Drug Entecavir From Excision by HIV-1 Reverse Transcriptase

Egor P Tchesnokov et al. J Biol Chem. .
Free PMC article

Abstract

Entecavir (ETV) is a potent antiviral nucleoside analogue that is used to treat hepatitis B virus (HBV) infection. Recent clinical studies have demonstrated that ETV is also active against the human immunodeficiency virus type 1 (HIV-1). Unlike all approved nucleoside analogue reverse transcriptase RT) inhibitors (NRTIs), ETV contains a 3'-hydroxyl group that allows further nucleotide incorporation events to occur. Thus, the mechanism of inhibition probably differs from classic chain termination. Here, we show that the incorporated ETV-monophosphate (MP) can interfere with three distinct stages of DNA synthesis. First, incorporation of the next nucleotide at position n + 1 following ETV-MP is compromised, although DNA synthesis eventually continues. Second, strong pausing at position n + 3 suggests a long range effect, referred to as "delayed chain-termination." Third, the incorporated ETV-MP can also act as a "base pair confounder" during synthesis of the second DNA strand, when the RT enzyme needs to pass the inhibitor in the template. Enzyme kinetics revealed that delayed chain termination is the dominant mechanism of action. High resolution foot-printing experiments suggest that the incorporated ETV-MP "repels" the 3'-end of the primer from the active site of HIV-1 RT, which, in turn, diminishes incorporation of the natural nucleotide substrate at position n + 4. Most importantly, delayed chain termination protects ETV-MP from phosphorolytic excision, which represents a major resistance mechanism for approved NRTIs. Collectively, these findings provide a rationale and important tools for the development of novel, more potent delayed chain terminators as anti-HIV agents.

Figures

FIGURE 1.
FIGURE 1.
Chemical structures of selected NRTIs and ETV.
FIGURE 2.
FIGURE 2.
Effects of incorporation of ETV-MP at position n on subsequent nucleotide incorporation events. A, DNA/DNA primer-template substrates used in this assay. B, multiple nucleotide incorporation events in the presence of a constant concentrations of dNTPs and increasing concentrations of ETV-TP. We used primer-template T45/P1b that provides a single site of incorporation for the inhibitor. ETV-mediated pausing is observed at the site of incorporation and at position n + 3. The asterisk shows inhibitor independent pausing. C, DNA synthesis was here limited to position n + 4 with primer-template T50A6/P1 in the presence of a constant concentration of ETV-TP, dCTP, and dTTP and increasing concentrations of dGTP. Lane C, a control experiment where ETV-TP was omitted while dCTP and dTTP were present at 0.5 μm in order to control for possible misincorporation events at position n. The arrows serve the same purpose as in panel B. D, DNA synthesis as in C except that a constant concentration of ETV-TP and dTTP and increasing concentrations of dCTP were present in the reaction mixture. Lane c1, a control experiment where Mg2+ was omitted. Lane c2, a control experiment where ETV-TP was omitted in order to control for possible misincorporation events at position n. Lane c3, a control experiment where ETV-TP was substituted with 0.5 μm dGTP in the presence of 0.5 μm dTTP and dCTP. The arrows serve the same purpose as in B. The product fraction was calculated as the ratio of product at position n + 4 over the sum of remaining substrate and intermediate products in the same lane.
FIGURE 3.
FIGURE 3.
Effect of ETV-MP in the template strand. A, DNA/DNA primer-template substrate used in the reaction. E illustrates ETV-MP incorporation. B, DNA synthesis along a template containing either dGMP (left) or ETV-MP (right). Lane c1, a control experiment where MgCl2 was omitted. Lane c2, a control experiment where dATP, dTTP, and ddCTP were added in order to terminate DNA synthesis opposite ETV-MP. Lane c3, a control experiment where dATP and ddTTP were added as an additional marker. The star on the left shows pausing in the absence of inhibitor, whereas the asterisks illustrate ETV-mediated inhibition of DNA synthesis.
FIGURE 4.
FIGURE 4.
Site-specific footprinting of HIV-1 RT-DNA substrate complexes containing ddGMP or ETV-MP at the 3′-end of the primer. A, reaction scheme. Complexes were treated with Fe2+ following incorporation of ETV-MP or ddGMP. The additional presence of PFA or dTTP provides conditions to trap the pre- or post-translocated complex, respectively. Fe2+/RNase H cleavage at position -18 or -17 distinguishes between the conformations. B, footprinting patterns with ddGMP or ETV-MP terminated primers. -Fe lane, a control experiment in the absence of Fe2+. +Fe lane, treatment of the binary complexes with Fe2+ prior to nucleotide incorporation. +Fe/+PFA, footprint in the presence of 100 μm PFA that shows a bias toward pretranslocation. C, rate of dissociation of RNA/DNA template-primer. The rate constant koff was determined with an RNA/DNA version of the template-primer shown in A. The primers were terminated with ETV-MP or ddGMP at position n, and RNase H cleavage products were quantified at different time points following the addition of trap. D, inhibition of DNA synthesis with primer-templates containing dGMP (G), ddGMP (ddG), or ETV-MP (E) at the 3′-end of the primer.
FIGURE 5.
FIGURE 5.
RNase H activity on RNA/DNA template-primer substrates containing dGMP or ETV-MP 4 nucleotides upstream the 3′-end of the primer. A, RNase H activity monitored with 3′-end-labeled RNA/DNA template-primer following incubation with heparin for variable time. Lane c1, a control experiment where heparin was omitted. Lane c2, a control experiment where MgCl2 was omitted. Lane c3, a control experiment where trap was added prior to the addition of HIV-1 RT in order to assess the efficiency of the trap. The arrows point to the specific polymerase-dependent RNase H cleavage products that are indicative for pre- and post-translocated conformations. Lane c4, a control experiment where RNA template was subjected to alkaline hydrolysis to produce a latter. Asterisks show polymerase-independent RNase H cleavage. B, RNA/DNA substrate used in the reaction. Position -19 illustrates the distance in nucleotides between the polymerase and RNase H active sites of HIV-1 RT with respect to the 3′-end of the primer. ETV-MP or dGMP is incorporated at position n. The arrows and asterisks assign the RNase H cuts to the sequence of the template. C, rate of the dissociation of the RNA/DNA template-primer was determined as described in the legend to Fig. 4.
FIGURE 6.
FIGURE 6.
ATP-dependent excision on dGMP- or ETV-MP-containing primers. A, time course of ATP-dependent excision of dGMP (left) or ETV-MP (right) at the 3′-end of the primer (position n). The gel shows reactions with HIV-1 RT containing the TAMs cluster used in this study. Lane c, a control experiment where MgCl2 was omitted. The asterisk shows side reactions that reflect misincorporation events. B, graphic representation of data shown in A. C, time course experiments with primers containing dGMP and ETV-MP, respectively, at position n and three additional nucleotides at positions n + 1 to n + 3. D, graphic representation of data shown in C. WT, wild type.
FIGURE 7.
FIGURE 7.
Model of ETV-mediated delayed chain termination. Green cylinders show the template strand, whereas blue cylinders show the primer strand DNA. A red cylinder represents the incorporated ETV-MP. The larger blue-lined cylinder points to the nucleotide binding site of HIV-1 RT, and the arrow represents the RNase H active site schematically. The RNase H mapping studies of Fig. 5 suggest that the RT enzyme can bind its nucleic acid substrate at various positions. Nucleotide binding can only occur when the complex exists in its post-translocated configuration, whereas the pyrophosphate analogue PFA stabilizes the pretranslocated complex. The data show that these polymerase-dependent conformations are in equilibrium with various polymerase-independent conformations. A primer containing ETV-MP at position n followed by three natural nucleotides affects this equilibrium and favors polymerase- or excision-independent binding, which is indicated by the larger arrows.

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