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The G140S mutation in HIV integrases from raltegravir-resistant patients rescues catalytic defect due to the resistance Q148H mutation.

Delelis O, Malet I, Na L, Tchertanov L, Calvez V, Marcelin AG, Subra F, Deprez E, Mouscadet JF - Nucleic Acids Res. (2009)

Bottom Line: Importantly, in most cases, an additional mutation at the position G140 is associated with the Q148 pathway.We found that (i) integration is impaired for Q148H when compared with the wild-type, G140S and G140S/Q148H mutants; and (ii) the N155H and G140S mutations confer lower levels of resistance than the Q148H mutation.In conclusion, the Q148H mutation is responsible for resistance to Raltegravir whereas the G140S mutation increases viral fitness in the G140S/Q148H context.

View Article: PubMed Central - PubMed

Affiliation: LBPA, CNRS, Ecole Normale Supérieure de Cachan, 94235 Cachan, France. delelis@lbpa.ens-cachan.fr

ABSTRACT
Raltegravir (MK-0518) is the first integrase (IN) inhibitor to be approved by the US FDA and is currently used in clinical treatment of viruses resistant to other antiretroviral compounds. Virological failure of Raltegravir treatment is associated with mutations in the IN gene following two main distinct genetic pathways involving either the N155 or Q148 residue. Importantly, in most cases, an additional mutation at the position G140 is associated with the Q148 pathway. Here, we investigated the viral DNA kinetics for mutants identified in Raltegravir-resistant patients. We found that (i) integration is impaired for Q148H when compared with the wild-type, G140S and G140S/Q148H mutants; and (ii) the N155H and G140S mutations confer lower levels of resistance than the Q148H mutation. We also characterized the corresponding recombinant INs properties. Enzymatic performances closely parallel ex vivo studies. The Q148H mutation 'freezes' IN into a catalytically inactive state. By contrast, the conformational transition converting the inactive form into an active form is rescued by the G140S/Q148H double mutation. In conclusion, the Q148H mutation is responsible for resistance to Raltegravir whereas the G140S mutation increases viral fitness in the G140S/Q148H context. Altogether, these results account for the predominance of G140S/Q148H mutants in clinical trials using Raltegravir.

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Resistance of IN mutants to RAL. (A) HeLa p4 cells were infected, in triplicate, with 3 ng of each virus, in the presence of various RAL concentrations. β-Galactosidase production was quantified by the CPRG assay. Data from a representative experiment (performed three times) is shown. The IC50 was determined as the concentration of RAL inhibiting β-galactosidase production by 50% with respect to untreated infected cells. (B) MTT assay. The MTT assay was performed 48 and 72 h after infection for all viruses. For the WT and G140S/Q148H mutant, the assay was performed with and without 500 nM RAL. The data shown are the means of three independent experiments.
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Figure 2: Resistance of IN mutants to RAL. (A) HeLa p4 cells were infected, in triplicate, with 3 ng of each virus, in the presence of various RAL concentrations. β-Galactosidase production was quantified by the CPRG assay. Data from a representative experiment (performed three times) is shown. The IC50 was determined as the concentration of RAL inhibiting β-galactosidase production by 50% with respect to untreated infected cells. (B) MTT assay. The MTT assay was performed 48 and 72 h after infection for all viruses. For the WT and G140S/Q148H mutant, the assay was performed with and without 500 nM RAL. The data shown are the means of three independent experiments.

Mentions: We investigated the mechanism underlying the effects of these mutations on resistance to RAL, by determining the IC50 for each IN mutant (Figure 2A). The IC50 value (10 nM) obtained for the WT virus confirmed the potency of RAL as an inhibitor of HIV-1. At 24 or 72 h, no cytotoxic effects from the concentrations used in this experiment were observed in the MTT assay after infection (Figure 2B). G140S and E92Q mutants had slightly higher IC50 values (30 nM) than the WT. In sharp contrast, the N155H, Q148H and G140S/Q148H mutants had much higher IC50 values, at 130, 450 and >1000 nM, respectively. Thus, all the mutants identified in clinical trials using RAL were resistant to this compound, but to different extents. The same experiment was conducted with the strand transfer inhibitor L,731-988, a diketo acid which, similar to RAL, belongs to the INSTI group. The resistance profiles observed with this drug followed the same pattern as RAL (data not shown) but the values obtained were in low micromolar range for L,731-988, rather than in the nanomolar range as seen with RAL. Thus DKA, a well-characterized drug and RAL, both of which share the same mechanism of action, probably bind to the same binding site, inhibiting the strand transfer reaction by interfering with the binding of target DNA to IN.Figure 2.


The G140S mutation in HIV integrases from raltegravir-resistant patients rescues catalytic defect due to the resistance Q148H mutation.

Delelis O, Malet I, Na L, Tchertanov L, Calvez V, Marcelin AG, Subra F, Deprez E, Mouscadet JF - Nucleic Acids Res. (2009)

Resistance of IN mutants to RAL. (A) HeLa p4 cells were infected, in triplicate, with 3 ng of each virus, in the presence of various RAL concentrations. β-Galactosidase production was quantified by the CPRG assay. Data from a representative experiment (performed three times) is shown. The IC50 was determined as the concentration of RAL inhibiting β-galactosidase production by 50% with respect to untreated infected cells. (B) MTT assay. The MTT assay was performed 48 and 72 h after infection for all viruses. For the WT and G140S/Q148H mutant, the assay was performed with and without 500 nM RAL. The data shown are the means of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2651800&req=5

Figure 2: Resistance of IN mutants to RAL. (A) HeLa p4 cells were infected, in triplicate, with 3 ng of each virus, in the presence of various RAL concentrations. β-Galactosidase production was quantified by the CPRG assay. Data from a representative experiment (performed three times) is shown. The IC50 was determined as the concentration of RAL inhibiting β-galactosidase production by 50% with respect to untreated infected cells. (B) MTT assay. The MTT assay was performed 48 and 72 h after infection for all viruses. For the WT and G140S/Q148H mutant, the assay was performed with and without 500 nM RAL. The data shown are the means of three independent experiments.
Mentions: We investigated the mechanism underlying the effects of these mutations on resistance to RAL, by determining the IC50 for each IN mutant (Figure 2A). The IC50 value (10 nM) obtained for the WT virus confirmed the potency of RAL as an inhibitor of HIV-1. At 24 or 72 h, no cytotoxic effects from the concentrations used in this experiment were observed in the MTT assay after infection (Figure 2B). G140S and E92Q mutants had slightly higher IC50 values (30 nM) than the WT. In sharp contrast, the N155H, Q148H and G140S/Q148H mutants had much higher IC50 values, at 130, 450 and >1000 nM, respectively. Thus, all the mutants identified in clinical trials using RAL were resistant to this compound, but to different extents. The same experiment was conducted with the strand transfer inhibitor L,731-988, a diketo acid which, similar to RAL, belongs to the INSTI group. The resistance profiles observed with this drug followed the same pattern as RAL (data not shown) but the values obtained were in low micromolar range for L,731-988, rather than in the nanomolar range as seen with RAL. Thus DKA, a well-characterized drug and RAL, both of which share the same mechanism of action, probably bind to the same binding site, inhibiting the strand transfer reaction by interfering with the binding of target DNA to IN.Figure 2.

Bottom Line: Importantly, in most cases, an additional mutation at the position G140 is associated with the Q148 pathway.We found that (i) integration is impaired for Q148H when compared with the wild-type, G140S and G140S/Q148H mutants; and (ii) the N155H and G140S mutations confer lower levels of resistance than the Q148H mutation.In conclusion, the Q148H mutation is responsible for resistance to Raltegravir whereas the G140S mutation increases viral fitness in the G140S/Q148H context.

View Article: PubMed Central - PubMed

Affiliation: LBPA, CNRS, Ecole Normale Supérieure de Cachan, 94235 Cachan, France. delelis@lbpa.ens-cachan.fr

ABSTRACT
Raltegravir (MK-0518) is the first integrase (IN) inhibitor to be approved by the US FDA and is currently used in clinical treatment of viruses resistant to other antiretroviral compounds. Virological failure of Raltegravir treatment is associated with mutations in the IN gene following two main distinct genetic pathways involving either the N155 or Q148 residue. Importantly, in most cases, an additional mutation at the position G140 is associated with the Q148 pathway. Here, we investigated the viral DNA kinetics for mutants identified in Raltegravir-resistant patients. We found that (i) integration is impaired for Q148H when compared with the wild-type, G140S and G140S/Q148H mutants; and (ii) the N155H and G140S mutations confer lower levels of resistance than the Q148H mutation. We also characterized the corresponding recombinant INs properties. Enzymatic performances closely parallel ex vivo studies. The Q148H mutation 'freezes' IN into a catalytically inactive state. By contrast, the conformational transition converting the inactive form into an active form is rescued by the G140S/Q148H double mutation. In conclusion, the Q148H mutation is responsible for resistance to Raltegravir whereas the G140S mutation increases viral fitness in the G140S/Q148H context. Altogether, these results account for the predominance of G140S/Q148H mutants in clinical trials using Raltegravir.

Show MeSH
Related in: MedlinePlus