The non-nucleoside reverse transcriptase inhibitor efavirenz stimulates replication of human immunodeficiency virus type 1 harboring certain non-nucleoside resistance mutations

Department of Microbiology & Immunology, 601 Elmwood Ave., University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 USA
Virology (Impact Factor: 3.32). 07/2010; 402(2):228-237. DOI: 10.1016/j.virol.2010.03.018


We measured the effects of non-nucleoside reverse transcriptase (RT) inhibitor-resistant mutations K101E+G190S, on replication fitness and EFV-resistance of HIVNL4-3. K101E+G190S reduced fitness in the absence of EFV and increased EFV resistance, compared to either single mutant. Unexpectedly, K101E+G190S also replicated more efficiently in the presence of EFV than in its absence. Addition of the nucleoside resistance mutations L74V or M41L+T215Y to K101E+G190S improved fitness and abolished EFV-dependent stimulation of replication. D10, a clinical RT backbone containing M41L+T215Y and K101E+G190S, also demonstrated EFV-dependent stimulation that was dependent on the presence of K101E. These studies demonstrate that non-nucleoside reverse transcriptase inhibitors can stimulate replication of NNRTI-resistant HIV-1 and that nucleoside-resistant mutants can abolish this stimulation. The ability of EFV to stimulate NNRTI-resistant mutants may contribute to the selection of HIV-1 mutants in vivo. These studies have important implications regarding the treatment of HIV-1 with combination nucleoside and non-nucleoside therapies.

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    • "L74V compensated for the fitness defect of the K101E+G190S double mutation without reducing its level of drug resistance. L74V+ K101E+G190S fitness was increased by 32 % compared to K101E+G190S and by 7 % compared to G190S (Wang et al., 2010b). "
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    ABSTRACT: The fitness of non-nucleoside reverse transcriptase inhibitor (NNRTI) drug resistant reverse transcriptase (RT) mutants of HIV-1 correlates with the amount of RT in the virions and the RNase H activity of the RT. We wanted to understand the mechanism by which secondary NNRTI resistance mutations, L100I and K101E, and the nucleoside resistance mutation, L74V, alter the fitness of K103N and G190S viruses. We measured the amount of RT in virions and the polymerization and RNase H activities of mutant RTs compared to wild type, K103N and G190S. We found that L100I, K101E and L74V did not change the polymerization or RNase H activities of K103N or G190S RTs. However, L100I and K101E reduced the amount of RT in the virions and subsequent addition of L74V restored RT levels back to those of G190S or K103N alone. We conclude that fitness changes caused by L100I, K101E and L74V derive from their effects on RT content.
    Preview · Article · Mar 2013 · Journal of General Virology
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    ABSTRACT: Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are potent and commonly prescribed antiviral agents used in combination therapy (CART) of human immunodeficiency virus type 1 (HIV-1) infection. The development of drug resistance is a major limitation of CART. Reverse transcriptase (RT) genotypes with the NNRTI resistance mutations K101E+G190S are highly resistant to efavirenz (EFV) and can develop during failure of EFV-containing regimens in patients. We have previously shown that virus with K101E+G190S mutations can replicate more efficiently in the presence of EFV than in its absence. In this study, we evaluated the underlying mechanism for drug-dependent stimulation, using a single-cycle cell culture assay in which EFV was added either during the infection or the virus production step. We determined that EFV stimulates K101E+G190S virus during early infection and does not affect late steps of virus replication, such as increasing the amount of active RT incorporated into virions. Additionally, we showed that another NNRTI, nevirapine (NVP), stimulated K101E+G190S virus replication during the early steps of infection similar to EFV, but that the newest NNRTI, etravirine (ETR), did not. We also showed that EFV stimulates K101E+Y188L and K101E+V106I virus, but not K101E+L100I, K101E+K103N, K101E+Y181C, or K101E+G190A virus, suggesting that the stimulation is mutation specific. Real-time PCR of reverse transcription intermediates showed that although the drug did not stimulate minus-strand transfer, it did stimulate minus-strand strong-stop DNA synthesis. Our results indicate that stimulation most likely occurs through a mechanism whereby NNRTIs stimulate priming or elongation of the tRNA.
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    ABSTRACT: An integrated computational and statistical approach was used to determine the association of non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine, efavirenz and etravirine with resistance mutations that cause therapeutic failure and their impact on NNRTI resistance. Mutations detected for nevirapine virological failure with a prevalence greater than 10% in the used patient set were: K103N, Y181C, G190A, and K101E. A support vector regression model, based on matched genotypic/phenotypic data (n=850), showed that among 6365 analyzed mutations, K103N, Y181C and G190A have the first, third, and sixth greatest significance for nevirapine resistance, respectively. The most common indicator of treatment failure for efavirenz was K103N mutation present in 56.7% of the patients where the drug failed, followed by V108I, L100I, and G190A. For efavirenz resistance, K103N, G190, and L100I have the first, fourth, and eighth greatest significance, respectively, as determined in support vector regression model. No positive interactions were observed among nevirapine resistance mutations, while a more complex situation was observed with treatment failure of efavirenz and etravirine, characterized by the accumulation of multiple mutations. Docking simulations and free energy analysis based on docking scores of mutated human immunodeficiency virus (HIV) RT complexes were used to evaluate the influence of selected mutations on drug recognition. Results from support vector regression were confirmed by docking analysis. In particular, for nevirapine and efavirenz, a single mutation K103N was associated with the most unfavorable energetic profile compared to the wild-type sequence. This is in line with recent clinical data reporting that diarylpyrimidine etravirine, a very potent third generation drug effective against a wide range of drug-resistant HIV-1 variants, shows increased affinity towards K103N/S mutants due to its high conformational flexibility.
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