Characterization of the E138K resistance mutation in HIV-1 reverse transcriptase conferring susceptibility to etravirine in B and non-B HIV-1 subtypes

Abstract

We have selected for resistance to etravirine (ETR) and efavirenz (EFV) in tissue culture using three subtype B, three subtype C, and two CRF02_AG clinical isolates, grown in cord blood mononuclear cells. Genotypic analysis was performed at baseline and at various weeks of selection. Phenotypic resistance in regard to ETR, EFV, and nevirapine (NVP) was evaluated at weeks 25 to 30 for all ETR-selected viruses and in viral clones that contained specific resistance mutations that were inserted by site-directed mutagenesis into pNL-4.3 and AG plasmids. The results show that ETR selected mutations at positions V90I, K101Q, E138K, V179D/E/F, Y181C, V189I, G190E, H221H/Y, and M230L and that E138K was the first of these to emerge in most instances. The time to the emergence of resistance was longer in the case of ETR (18 weeks) compared to EFV (11 weeks), and no differences in the patterns of emergent mutations could be documented between the B and non-B subtypes. Viral clones containing E138K displayed low-level phenotypic resistance to ETR (3.8-fold) and modestly impaired replication capacity (2-fold) compared to wild-type virus. ETR-selected virus showed a high degree of cross-resistance to NVP but not to EFV. We identified K101Q, E138K, V179E, V189I, G190E, and H221Y as mutations not included among the 17 currently recognized resistance-associated mutations for ETR.

FIG. 1.

Baseline polymorphisms of HIV-1, as shown in an amino acid alignment (residues 1 to 240) of RT for all clinical isolates used in the present study. Subtype B: BK132, 5326, and 5331; CRF02_AG, 6383 and 6399; subtype C, Mole 03, BG 05, and 10680. HXB2 was used as a reference sequence. All isolates were sequenced at baseline and passaged simultaneously in the presence or absence of drugs. The sequence of the wild-type RT at baseline was the same as that of isolates passaged without drugs. The dots represent identical positions, while letters indicate variations in amino acids relative to HXB2. Isolate 6383 at baseline harbored the NNRTI resistance mutation V90I that is known to decrease susceptibility to ETR.

FIG. 2.

Comparative phenotypes of recombinant viruses derived from pNL-4.3_E138K and AG_E138K and tested for susceptibilities to ETR, EFV, and NVP in CBMCs. Recombinant clones containing E138K were tested for their susceptibilities to ETR, EFV, and NVP in cell culture assays. The mean EC₅₀s of mutated variants were compared to those of wt pNL-4.3 or AG plasmids. The results shown are means of two or three independent experiments. Bars in the figure represent the mean fold change, while error bars represent ± the standard deviation (SD). Bars with dots, fold change (FC) to ETR; bars with slashes, fold change to EFV; open bars, fold change to NVP.

FIG. 3.

Effect of E138K on viral replication capacity. Viral stocks of both wt and E138K-containing virus were normalized for p24 and used to infect TZM-bl cells. (A and B) The luciferase activity was measured at 48 h postinfection as an indication of viral replication. The relative infectivity of the wt compared to E138K-containing virus is shown on the y axis, while the x axis denotes the input of p24. Statistical analysis using a two-way ANOVA shows that replication capacity was decreased by 2 -and 3-fold compared to the wt for pNL-4.3_E138K and AG_E138K, respectively, after 48 h. A significant difference was observed after a p24 input greater than 10,000 pg/ml. The points denoted by asterisks (***) indicate a significant difference. CBMCs were also infected as described in Materials and Methods, and viral growth was measured by determining the RT activity in culture supernatants at different times (C). Significant differences observed at the peak of infection (days 6 and 8) are indicated by asterisks (***). In the figure, a P value of <0.01 is represented by “**”, while a P value of <0.001 is represented by “***”. Values are means of at least two independent experiments ± the SD. Error bars represent the SD.