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. 2011 Aug 26;286(34):29575-83.
doi: 10.1074/jbc.M111.268235. Epub 2011 Jul 7.

Impact of Primer-Induced Conformational Dynamics of HIV-1 Reverse Transcriptase on Polymerase Translocation and Inhibition

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Free PMC article

Impact of Primer-Induced Conformational Dynamics of HIV-1 Reverse Transcriptase on Polymerase Translocation and Inhibition

Anick Auger et al. J Biol Chem. .
Free PMC article

Abstract

The rapid emergence and the prevalence of resistance mutations in HIV-1 reverse transcriptase (RT) underscore the need to identify RT inhibitors with novel binding modes and mechanisms of inhibition. Recently, two structurally distinct inhibitors, phosphonoformic acid (foscarnet) and INDOPY-1 were shown to disrupt the translocational equilibrium of RT during polymerization through trapping of the enzyme in the pre- and the post-translocation states, respectively. Here, we show that foscarnet and INDOPY-1 additionally display a shared novel inhibitory preference with respect to substrate primer identity. In RT-catalyzed reactions using RNA-primed substrates, translocation inhibitors were markedly less potent at blocking DNA polymerization than in equivalent DNA-primed assays; i.e. the inverse pattern observed with marketed non-nucleoside inhibitors that bind the allosteric pocket of RT. This potency profile was shown to correspond with reduced binding on RNA·DNA primer/template substrates versus DNA·DNA substrates. Furthermore, using site-specific footprinting with chimeric RNA·DNA primers, we demonstrate that the negative impact of the RNA primer on translocation inhibitor potency is overcome after 18 deoxyribonucleotide incorporations, where RT transitions primarily into polymerization-competent binding mode. In addition to providing a simple means to identify similarly acting translocation inhibitors, these findings suggest a broader role for the primer-influenced binding mode on RT translocation equilibrium and inhibitor sensitivity.

Figures

FIGURE 1.
FIGURE 1.
NNTIs potently inhibit DNA-primed but not RNA-primed polymerization reaction. Compound potency (IC50 value) was established using an RNA or a DNA PPT primer annealed to the PPT37 template. RNA-primed reactions were performed for 30 min, while the DNA-primed reactions were quenched after 5 min to establish compound potency during the linear phase of the polymerization reaction. IC50 values were calculated by a four-parameter logistic fit.
FIGURE 2.
FIGURE 2.
INDOPY-1 and PFA are unable to stabilize RT complexed with RNA·DNA substrate. Preformed RT-hybrid (RNA·DNA or DNA·DNA) complexes were incubated with increasing concentrations of INDOPY-1 or PFA or with a fixed concentration (50 μm) of the next complementary nucleotide (dTTP for the PPT+2D or dGTP for the PPT+3D). These complexes were challenged for 15 min at room temperature with heparin to trap dissociated RT molecules before the samples were analyzed on 6% non-denaturating gel.
FIGURE 3.
FIGURE 3.
Template length affects NNTIs potency only on RNA-primed polymerization reaction. Compound potency (IC50 value) was determined for each set of primer/template substrates described under “Experimental Procedures.” Polymerization reaction times for each set of primer/template substrates (RNA-primed = 30 min and DNA-primed = 5 min) were derived from supplemental Fig. S1 to establish IC50 values within the linear phase of the polymerization reaction.
FIGURE 4.
FIGURE 4.
Extending the RNA PPT primer by more than 18 DNA residues sensitizes the NNTIs while decreasing the potency of the NNRTIs. A, diagram of the chimeric primer/template substrates. B, time course of the different chimeric primer/template substrates. C, compound potency was established for each chimeric primer using the optimal reaction time derived from the time-course experiment showed in B (DNA PPT and RNA PPT+24D = 5 min; RNA PPT, RNA PPT+12D, and RNA PPT+18D = 30 min). D, -fold change in potency of RT inhibitors (IC50 using the chimeric primer/IC50 using the pure RNA PPT primer) upon addition of DNA residues at the 3′-end of the RNA PPT primer.
FIGURE 5.
FIGURE 5.
HIV-1 RT binds in a polymerase-competent binding mode only on the RNA PPT primer extended by more than 18 DNA residues. A, nucleic acid sequences used in the footprint. Arrows indicate the position of the post-translocated KOONO-induced cleavages on the PPT37 DNA template. The asterisk indicates the position of the radiolabel. B, site-specific KOONO footprint using the RT mutant E478Q. Control lanes in the absence of KOONO are indicated. Lane 1 contains unliganded RT. Lane 2 contains 100 μm PFA. Lane 3 contains the next templated dideoxynucleotide (20 μm) to act as a chain terminator. Lane 4 is identical to lane 3 except for the presence of the next templated nucleotide after the chain terminator (50 μm); e.g. for DNA PPT, lane 3 contains ddATP (20 μm) and lane 4 contains ddATP (20 μm) and dCTP (50 μm). Arrows indicate the position (or expected position) of the footprint: the top arrow represents a pre-translocated cleavage, the middle arrow represents a post-translocated cleavage, and the bottom arrow represents the post-translocated +1 cleavage after incorporation of a ddNTP.
FIGURE 6.
FIGURE 6.
Model of the sensitivity of RT to NNRTIs and NNTIs dictated by primer-guided binding dynamics. Top, DNA polymerization from an RNA-primed substrate is sensitive to NNRTIs. For RNA-primed substrates, and for substrates in which less than 18 nt of DNA have been incorporated, RT predominantly is in equilibrium between RNase H (RNH) and polymerization (Pol) modes. NNRTIs are able to potently inhibit RT in this regime by favoring the non-polymerization competent RNH mode. In contrast, NNTIs show a reduced ability to inhibit polymerization, which corresponds to a decreased ability to stabilize ternary complexes and an inability to trap RT in a particular polymerization-incompetent translocation state. Bottom, DNA polymerization from a DNA-primed substrate is sensitive to NNTIs. For DNA-primed substrates and for on-going polymerization, RT is primarily in polymerization mode, transitioning between the pre- and post-translocational states facilitating trapping by NNTIs, and disfavoring inhibition by NNRTIs.

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