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Characterization of human immunodeficiency virus type 1 mutants with decreased sensitivity to proteinase inhibitor Ro
H Jacobsen1, K Yasargil, D L Winslow
1Hoffmann-LaRoche AG, Pharmaceutical Research New Technologies/Biology, Basel, Switzerland.
Abstract:
A human immunodeficiency virus type 1 (HIV-1) variant with highly reduced susceptibility to Ro 31-8959, an inhibitor of the viral proteinase, has been selected by repeated passage of wild-type virus in CEM cells in the presence of increasing concentrations of the inhibitor. Peptide sequences of the proteinase of selected virus were obtained from proviral DNA. Sequence comparison to wild-type (wt) proteinase demonstrated two amino acid substitutions in the resistant virus, a Gly to Val exchange at position 48 and a Leu to Met exchange at position 90. Furthermore, sequences of intermediate passage virus suggest contributions from positions 12, 36, 57, and 63 in early steps of resistance development. The selected virus showed a ca. 40-fold increase in 50% inhibitory concentration of Ro 31-8959. Growth kinetics of resistant virus were comparable to wild-type virus and the resistant genotype proved to be stable in the absence of inhibitor. Directed mutagenesis of the HIV-1 HXB2 proteinase at positions 48 and 90 suggested that each mutation alone led to a moderate decrease in sensitivity of the recombinant virus to proteinase inhibitor. However, a recombinant virus carrying both mutations in the proteinase gene showed a significant reduction in its sensitivity to Ro 31-8959 thus proving the importance of these exchanges for the resistance phenotype.
Insights
Researchers developed a human immunodeficiency virus type 1 (HIV-1) variant resistant to Ro 31-8959, a proteinase inhibitor. This resistance is linked to specific amino acid substitutions in the viral proteinase, impacting drug efficacy.
Area of Science:
- Virology
- Drug Resistance Studies
- Molecular Biology
Background:
- Human immunodeficiency virus type 1 (HIV-1) proteinase is a key target for antiviral therapies.
- Development of drug resistance is a major challenge in HIV-1 treatment.
- Ro 31-8959 is an inhibitor designed to target the HIV-1 viral proteinase.
Purpose of the Study:
- To investigate the mechanisms of resistance to the HIV-1 proteinase inhibitor Ro 31-8959.
- To identify specific mutations in the viral proteinase conferring reduced susceptibility to the inhibitor.
- To assess the stability and growth characteristics of the resistant HIV-1 variant.
Main Methods:
- Selection of a resistant HIV-1 variant through repeated passage in the presence of increasing Ro 31-8959 concentrations.
- Peptide sequencing of the viral proteinase from proviral DNA of selected and intermediate passage viruses.
- Directed mutagenesis to introduce specific amino acid substitutions into the HIV-1 HXB2 proteinase gene.
- Assessment of viral susceptibility to Ro 31-8959 using 50% inhibitory concentration (IC50) assays.
- Comparison of growth kinetics between wild-type and resistant HIV-1 strains.
Main Results:
- A HIV-1 variant with approximately 40-fold reduced susceptibility to Ro 31-8959 was successfully selected.
- Two primary amino acid substitutions, Gly48Val and Leu90Met, were identified in the proteinase of the resistant virus.
- Intermediate passage viruses suggested early contributions to resistance from mutations at positions 12, 36, 57, and 63.
- Recombinant viruses with individual mutations showed moderate resistance, while the double mutant exhibited significant resistance to Ro 31-8959.
- The resistant HIV-1 genotype was stable in the absence of the inhibitor, with growth kinetics comparable to wild-type virus.
Conclusions:
- The amino acid substitutions at positions 48 and 90 in the HIV-1 proteinase are critical for the development of high-level resistance to Ro 31-8959.
- These mutations significantly reduce the sensitivity of the virus to the proteinase inhibitor.
- Understanding these resistance mechanisms is crucial for the development of more effective HIV-1 therapies and strategies to overcome drug resistance.