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. 2006 Sep 15;353(1):184-92.
doi: 10.1016/j.virol.2006.05.021. Epub 2006 Jun 21.

Relative Replication Fitness of Efavirenz-Resistant Mutants of HIV-1: Correlation With Frequency During Clinical Therapy and Evidence of Compensation for the Reduced Fitness of K103N + L100I by the Nucleoside Resistance Mutation L74V

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Free PMC article

Relative Replication Fitness of Efavirenz-Resistant Mutants of HIV-1: Correlation With Frequency During Clinical Therapy and Evidence of Compensation for the Reduced Fitness of K103N + L100I by the Nucleoside Resistance Mutation L74V

Christine E Koval et al. Virology. .
Free PMC article

Abstract

Efavirenz resistance during HIV-1 treatment failure is usually associated with the reverse transcriptase mutation K103N. L100I, V108I, or P225H can emerge after K103N and increase its level of efavirenz resistance. K103N + L100I is the most drug-resistant of the double mutants but is the least common clinically. We hypothesized that differences in replication efficiency, or fitness, influence the relative frequencies of these secondary efavirenz resistance mutations in clinical isolates. We measured fitness of each secondary mutant introduced into HIV(NL4-3), alone and in combination with K103N, using growth competition assays in H9 cells. In the absence of efavirenz, the fitness of V108I was indistinguishable from wild type. K103N, L100I, and P225H were minimally, but consistently, less fit than wild type. K103N + L100I had a greater reduction in fitness and was less fit than K103N + V108I and K103N + P225H. The fitness defect of K103N + L100I relative to K103N was completely compensated for by the addition of the nucleoside resistance mutation L74V. In the presence of efavirenz, L100I was less fit than K103N, and K103N + L100I was more fit than K103N + V108I. Our studies suggest the primary driving force behind the selection of secondary efavirenz resistance mutations is the acquisition of higher levels of drug resistance, but the specific secondary mutations to emerge are those with the least cost in terms of replication efficiency. In addition, nucleoside and NNRTI resistance mutations can interact to affect HIV replication efficiency; these interactions may influence which mutations emerge during treatment failure. These studies have important implications for the design of more durable NNRTI-nucleoside combination regimens.

Figures

Fig. 1
Fig. 1
Correlation between quantification of mutant prevalence by direct sequencing of PCR product versus clonal analysis. Results from five independent growth competition assays using the L100I, K103N, V108I, P225H, or P236L mutants were pooled for use in this analysis. The same PCR amplified product was either directly sequenced or cloned into a bacterial plasmid and transformed into E. coli, with subsequent sequence analysis of DNA isolated from each clone. Twenty-four consecutive, unselected clonal sequences were utilized for each determination. The results of one assay were not known when analyzing the results of the other assay.
Fig. 2
Fig. 2
Growth competition assays between efavirenz-resistant and wild-type NL4-3, inoculated at a ratio of 1:1, based on p24 antigen content. The reference strain in each case is wild-type NL4-3. (A) K103N. (B) V108I. (C) P225H. (D) L100I. (E) P236L.
Fig. 3
Fig. 3
Growth competition assays between K103N and L100I, in the absence and presence of efavirenz. The L100I and K103N variants were inoculated at a ratio of 1:1, based on p24 antigen content. Solid lines, proportion of K103N, measured by direct sequence analysis at codon 103. Dotted lines, proportion of L100I, inferred from sequence analysis at codon 103. The same results were obtained when the prevalence of L100I was measured directly by sequence analysis at codon 100 (data not shown). ♦, No efavirenz;▪, 10 nM efavirenz;▲, 100 nM efavirenz; •, 1 µM efavirenz.
Fig. 4
Fig. 4
Growth competition assay between K103N + L100I and K103N + V108I in the absence and presence of efavirenz. Mutant viruses were inoculated at a ratio of 1:1, based on p24 antigen content. Results represent the average ± standard error of the mean of at least three independent experiments. Solid lines, prevalence of K103N + V108I, based on sequence analysis at codon 108. Dotted lines, inferred prevalence of K103N + L100I, based on sequence analysis at codon 108. Sequence analysis at codon 100 gave the same results (data not shown). ◊, No efavirenz; Δ, 100 nM efavirenz; ○, 1 µM efavirenz, ×, 10 µM efavirenz.
Fig. 5
Fig. 5
Growth competition assay between K103N + L100I and K103N + L100I + L74V (inoculated at a 75:25 ratio, respectively, based on p24 antigen content) in the absence of efavirenz. Results represent the average ± standard error of the mean of at least three independent experiments.

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