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Replication-dependent 65R-->K reversion in human immunodeficiency virus type 1 reverse transcriptase double mutant
Prem L Sharma1, Viktoria Nurpeisov, Kimberly Lee
1Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, GA 30033, USA. plsharm@emory.edu
Virology
|March 31, 2004
Summary
The K65R + L74V mutation in HIV-1 reverse transcriptase is unstable and reverts to K65R during replication. This reversion is linked to viral replication rates, impacting treatment strategies for HIV.
Area of Science:
- Virology
- Molecular Biology
- Drug Resistance
Background:
- Understanding drug resistance mutations in HIV-1 reverse transcriptase (RT) is crucial for developing new therapies.
- Nucleoside reverse transcriptase inhibitor (NRTI)-selected mutations like K65R and L74V are key in HIV-1 drug resistance.
- The combination of K65R and L74V mutations is rarely observed clinically, despite NRTIs selecting for them individually.
Purpose of the Study:
- To investigate the impact of the combined K65R and L74V mutations on HIV-1 replication.
- To analyze the stability and reversion patterns of the K65R + L74V double mutant.
- To understand the relationship between viral replication dynamics and mutation reversion.
Main Methods:
- Site-directed mutagenesis was used to create a double mutant (K65R + L74V) in the HIV-1 pNL4-3 background.
- Replication kinetic assays were performed in PHA-stimulated human peripheral blood mononuclear (PBM) cells and MT-2 cells.
- Viral replication was assessed under varying conditions, including the presence of a novel NRTI (DXG) and suboptimal interleukin-2.
Main Results:
- The K65R + L74V double mutant exhibited instability, with K65R reversion occurring during replication in PBM cells without drug pressure.
- Replication assays in MT-2 cells showed the double mutant was attenuated, with reversion initiation linked to increased RT activity.
- Suppression of viral replication, either by DXG or suboptimal IL-2, minimized or prevented K65R reversion.
Conclusions:
- The reversion of K65R in the K65R + L74V double mutant is dependent on the rate of viral replication.
- These findings suggest a dynamic interplay between viral replication and mutation stability, potentially influencing HIV treatment outcomes in vivo.
- Understanding these mechanisms can inform the design of future antiretroviral therapies and resistance management strategies.