Abstract
Rilpivirine (RPV) is a second generation nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) that efficiently inhibits HIV-1 resistant to first generation NNRTIs. Virological failure during therapy with RPV and emtricitabine is associated with the appearance of E138K and M184I mutations in RT. Here we investigate the biochemical mechanism of RT inhibition and resistance to RPV. We used two transient kinetics approaches (quench-flow and stopped-flow) to determine how subunit-specific mutations in RT p66 or p51 affect association and dissociation of RPV to RT as well as their impact on binding of dNTP and DNA and the catalytic incorporation of nucleotide. We compared WT with four subunit-specific RT mutants, p66(M184I)/p51(WT), p66(E138K)/p51(E138K), p66(E138K/M184I)/p51(E138K), and p66(M184I)/p51(E138K). Ile-184 in p66 (p66(184I)) decreased the catalytic efficiency of RT (k(pol)/K(d)(.dNTP)), primarily through a decrease in dNTP binding (K(d)(.dNTP)). Lys-138 either in both subunits or in p51 alone abrogated the negative effect of p66(184I) by restoring dNTP binding. Furthermore, p51(138K) reduced RPV susceptibility by altering the ratio of RPV dissociation to RPV association, resulting in a net reduction in RPV equilibrium binding affinity (K(d)(.RPV) = k(off.RPV)/k(on.RPV)). Quantum mechanics/molecular mechanics hybrid molecular modeling revealed that p51(E138K) affects access to the RPV binding site by disrupting the salt bridge between p51(E138) and p66(K101). p66(184I) caused repositioning of the Tyr-183 active site residue and decreased the efficiency of RT, whereas the addition of p51(138K) restored Tyr-183 to a WT-like conformation, thus abrogating the Ile-184-induced functional defects.
Highlights
Reverse transcriptase mutations E138K and M184I emerged most frequently in Human immunodeficiency virus type 1 (HIV-1) patients who failed rilpivirine/emtricitabine/tenofovir combination therapy
We did not pursue the characterization of the p66E138K/M184I/ p51E138K/M184I variant of HIV-1 reverse transcriptase (RT) (M184I mutation in p51) because p51184I did not show any change in activity profile (53)
We found that the dNTP binding affinity of p66M184I/p51E138K is very similar to that of WT (Kd.dNTP values of 5.6 versus 6.5 M measured by rapid quench-flow (RQF) or 3.4 versus 3.4 M measured by SF), clearly demonstrating that E138K in p51 alone is sufficient to compensate for the defect imparted by Susceptibility of WT and Mutant RTs to RPV—We used a PicoGreen dsDNA quantitation assay to determine the susceptibility of WT and mutant RTs to RPV under steady state conditions
Summary
Reverse transcriptase mutations E138K and M184I emerged most frequently in HIV-1 patients who failed rilpivirine/emtricitabine/tenofovir combination therapy. Rilpivirine (RPV) is a second generation nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) that efficiently inhibits HIV-1 resistant to first generation NNRTIs. Virological failure during therapy with RPV and emtricitabine is associated with the appearance of E138K and M184I mutations in RT. Pre-steady state kinetics approaches have shown that mutations remote from the NNIBP reduce binding of the first generation NNRTI NVP primarily by decreasing the association rate of the inhibitor (36 – 41). Wainberg and colleagues (51, 53) carried out early biochemical experiments using homopolymeric substrates and reported that the addition of E138K to the M184I background increases the processivity of DNA synthesis (53) Their steady state kinetic constant determination showed that E138K mutation in both subunits is required for restoring Km.dNTP to levels comparable with those of WT RT (51, 53). The M184I mutation decreased the efficiency of DNA synthesis primarily by reducing the dNTP binding affinity, whereas the E138K mutation in p51 restored this defect by restoring the binding affinity (Kd.dNTP) for the nucleotide substrate
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