Analysis of HIV cross-resistance to protease inhibitors using a rapid single-cycle recombinant virus assay for patients failing on combination therapies

Race, Esther; Dam, Elizabeth M.; Obry, Véronique; Paulous, Sylvie; Clavel, François

doi:10.1097/00002030-199910220-00008

Cited by 121 publications

(86 citation statements)

References 33 publications

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“…3 Cross-resistance within each of the three classes of approved antiretroviral drugs is extensive and often limits the treatment options for patients who are receiving their third or fourth regimen. [4][5][6] New classes of drugs directed at targets other than the reverse transcriptase or protease would be of great benefit.…”

Section: Resultsmentioning

confidence: 99%

Efficacy of Enfuvirtide in Patients Infected with Drug-Resistant HIV-1 in Europe and Australia

et al. 2003

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Section: Resultsmentioning

confidence: 99%

Efficacy of Enfuvirtide in Patients Infected with Drug-Resistant HIV-1 in Europe and Australia

et al. 2003

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“…Phenotyping was performed as previously described using the Phenoscript single-cycle recombinant virus assay (7,42). This assay is based on reverse transcription and PCR amplification of HIV-1 viral RNA, recombination into an HIV-1 molecular clone, and evaluation of PI susceptibility in a single cycle of infection in producer cells treated with different concentrations of PI.…”

Section: Methodsmentioning

confidence: 99%

Molecular Characterization of Clinical Isolates of Human Immunodeficiency Virus Resistant to the Protease Inhibitor Darunavir

Šašková

Kožíšek

Řezáčová

et al. 2009

J Virol

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Darunavir is the most recently approved human immunodeficiency virus (HIV) protease (PR) inhibitor (PI)and is active against many HIV type 1 PR variants resistant to earlier-generation PIs. Darunavir shows a high genetic barrier to resistance development, and virus strains with lower sensitivity to darunavir have a higher number of PI resistance-associated mutations than viruses resistant to other PIs. In this work, we have enzymologically and structurally characterized a number of highly mutated clinically derived PRs with high levels of phenotypic resistance to darunavir. With 18 to 21 amino acid residue changes, the PR variants studied in this work are the most highly mutated HIV PR species ever studied by means of enzyme kinetics and X-ray crystallography. The recombinant proteins showed major defects in substrate binding, while the substrate turnover was less affected. Remarkably, the overall catalytic efficiency of the recombinant PRs (5% that of the wild-type enzyme) is still sufficient to support polyprotein processing and particle maturation in the corresponding viruses. The X-ray structures of drug-resistant PRs complexed with darunavir suggest that the impaired inhibitor binding could be explained by change in the PR-inhibitor hydrogen bond pattern in the P2 binding pocket due to a substantial shift of the aminophenyl moiety of the inhibitor. Recombinant virus phenotypic characterization, enzyme kinetics, and X-ray structural analysis thus help to explain darunavir resistance development in HIV-positive patients.

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“…To evaluate the effect of hydroxyurea pretreatment of target cells on the replication of viruses carrying resistance mutations in RT, a modification of the previously described recombi-VOL. 79,2005 EFFECT OF RT MUTATIONS ON VIRAL DNA SYNTHESIS 813 nant-virus assay was used (29,38,43). The plasmid used in these studies (pSRT) was derived from pNL4-3 and contains deletions in the regions coding for RT (nucleotides 2618 to 3886) and envelope (nucleotides 6343 to 7611).…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

Quantification of the Effects on Viral DNA Synthesis of Reverse Transcriptase Mutations Conferring Human Immunodeficiency Virus Type 1 Resistance to Nucleoside Analogues

Bouchonnet

Dam²,

Mammano

et al. 2005

J Virol

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Human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) resistance mutations reduce the susceptibility of the virus to nucleoside analogues but may also impair viral DNA synthesis. To further characterize the effect of nucleoside analogue resistance mutations on the efficiency and kinetics of HIV-1 DNA synthesis and to evaluate the impact of the depletion of deoxynucleoside triphosphates (dNTP) on this process, DNA synthesis was evaluated by allowing DNA synthesis to proceed with natural HIV-1 templates and primers, either within permeabilized viral particles or in newly infected cells, and quantifying the products by real-time PCR. Three recombinant viruses derived from three pNL4-3 molecular clones expressing mutations associated with resistance to zidovudine: a clone expressing RT mutation M184V, a clone expressing mutations M41L plus T215Y (M41L؉T215Y), and clinical isolate BV34 (carrying seven resistance mutations). Following infection of P4 cells, the BV34 mutant, but not viruses expressing the M184V mutation or M41L؉T215Y, exhibited a defect in DNA synthesis. Importantly, however, for mutants carrying the M184V mutation or M41L؉T215Y mutations, a defect could be detected by using target cells in which dATP pools had been reduced by pretreatment with hydroxyurea. Based on these observations, we developed a recombinant-virus assay to assess the effects of hydroxyurea pretreatment on infectivity of viruses carrying plasma-derived RT sequences from patients with nucleoside resistance. Using this assay, we found that many, but not all, viruses carrying RT resistance mutations display an increased sensitivity to hydroxyurea, suggesting that the impact of RT resistance mutations on viral replication may be more profound in cell populations characterized by smaller dNTP pools.

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Analysis of HIV cross-resistance to protease inhibitors using a rapid single-cycle recombinant virus assay for patients failing on combination therapies

Abstract: These results suggest that, although the total number of protease mutations correlates with the degree of cross-resistance, the specific mechanisms accounting for primary resistance and for cross-resistance may be different.

Cited by 121 publications

References 33 publications

Efficacy of Enfuvirtide in Patients Infected with Drug-Resistant HIV-1 in Europe and Australia

Efficacy of Enfuvirtide in Patients Infected with Drug-Resistant HIV-1 in Europe and Australia

Molecular Characterization of Clinical Isolates of Human Immunodeficiency Virus Resistant to the Protease Inhibitor Darunavir

Quantification of the Effects on Viral DNA Synthesis of Reverse Transcriptase Mutations Conferring Human Immunodeficiency Virus Type 1 Resistance to Nucleoside Analogues

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