A novel molecular mechanism of dual resistance to nucleoside and nonnucleoside reverse transcriptase inhibitors

Abstract

Recently, mutations in the connection subdomain (CN) and RNase H domain of HIV-1 reverse transcriptase (RT) were observed to exhibit dual resistance to nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs). To elucidate the mechanism by which CN and RH mutations confer resistance to NNRTIs, we hypothesized that these mutations reduce RNase H cleavage and provide more time for the NNRTI to dissociate from the RT, resulting in the resumption of DNA synthesis and enhanced NNRTI resistance. We observed that the effect of the reduction in RNase H cleavage on NNRTI resistance is dependent upon the affinity of each NNRTI to the RT and further influenced by the presence of NNRTI-binding pocket (BP) mutants. D549N, Q475A, and Y501A mutants, which reduce RNase H cleavage, enhance resistance to nevirapine (NVP) and delavirdine (DLV), but not to efavirenz (EFV) and etravirine (ETR), consistent with their increase in affinity for RT. Combining the D549N mutant with NNRTI BP mutants further increases NNRTI resistance from 3- to 30-fold, supporting the role of NNRTI-RT affinity in our NNRTI resistance model. We also demonstrated that CNs from treatment-experienced patients, previously reported to enhance NRTI resistance, also reduce RNase H cleavage and enhance NNRTI resistance in the context of the patient RT pol domain or a wild-type pol domain. Together, these results confirm key predictions of our NNRTI resistance model and provide support for a unifying mechanism by which CN and RH mutations can exhibit dual NRTI and NNRTI resistance.

FIG. 1.

Effect of CN mutations from patients' RTs on NNRTI resistance. Fold changes in the resistance level to NVP, DLV, EFV, and ETR are shown as vertical bars (mean of two or more independent experiments ± standard error) for viruses containing wild-type pol and CN from treatment-experienced patients' RT T-3, T-4, T-6, T-8, and T-10 (A) or CN from treatment-naïve patients' RT N-16, N-18, N-19, N-22, and N-24 (B). An asterisk within the bar indicates a statistically significant increase in resistance versus wild-type (WT) control (t test, P < 0.05).

FIG. 2.

Effect of CN mutations from treatment-experienced patients' RTs on NNRTI resistance. (A to D) IC₅₀ determinations for the viruses containing CN mutations from treatment-experienced patients' RTs in the context of their own pol to NVP (A), DLV (B), EFV (C), ETR (D). White bars represent the average IC₅₀ for the wild-type RT control; gray bars represent RTs containing pol domains originated from the patients' virus; black bars represent both pol and CN originated from the patients' virus. Numbers in front of black bars represent fold changes, and asterisks within the black bars indicate a statistically significant difference in IC₅₀ within each pair of RTs connected by a bracket (t test, P < 0.05). Numbers within bars represent the maximum drug concentration used for the testing that was not sufficient to inhibit viral replication. (E and F) IC₅₀ determinations for the viruses containing specific CN mutation(s) from treatment-experienced patients' RTs to NVP (E) and EFV (F). Numbers in front of gray bars represent fold changes, and asterisks within the gray bars indicate a statistically significant difference in IC₅₀ versus the black bar for each group connected by a bracket (t test, P < 0.05). Direction of arrow located near the number indicates a decrease or increase in resistance caused by mutation. An additional asterisk within the bar indicates a statistically significant change in resistance for the double mutant versus the single mutant within the group (t test, P < 0.05). Schematics of the corresponding RT structures are depicted on the left-hand side of panels A, B, C, D (top part), and E and F (bottom part). Groups of constructs are connected by a bracket. White boxes in schematics represent the wild-type sequences, and gray boxes represent the patients' RT sequences; white strips within the gray box indicate the reversion of the mutant amino acid to the wild type; gray strips within the white box indicate a replacement of the wild-type amino acid to the mutant amino acid. Specific mutations are shown near each construct where applicable, designating either the reversion of this mutation to the wild-type amino acid (e.g., ×360I) or the reversion of the wild-type amino-acid to the mutant amino acid (e.g., +348I). Linear regression analysis of resistance changes to AZT versus NVP (G), and to AZT versus EFV (H) caused by CN mutations from treatment-experienced patients. Resistance data for NVP and EFV are collected from Fig. 1A and panels A, C, E, and F of this figure and, for AZT, from reports by Delviks-Frankenberry et al. (12) and Nikolenko et al. (34). The correlation coefficients (R) were determined using SigmaPlot 8.0 software. Fold resistance was calculated as a ratio between IC₅₀s for virus containing specific mutation(s) and correspondent control virus without this mutation(s).

FIG. 3.

A model for NNRTI-mediated abrogation of HIV-1 replication. DNA primer strand and RNA template strand (stretches of black and white circles, respectively), RT (large gray oval), and NNRTI (dark gray cylinder) are shown schematically. The thick arrow reflects sequential events during HIV-1 replication in the presence of NNRTI. Once initiated, polymerization continues until NNRTI binds to RT-template-primer complex forming a PI complex. The left panel represents events for wild-type RT leading to NNRTI-sensitive phenotype; the middle and right panels represent events for RT with reduced affinity to NNRTI and RNase H-defective RT, which lead to NNRTI-resistant phenotype. The sequential events in the reverse transcription during NNRTI exposure include RNase H cleavage, dissociation of NNRTI, and either formation of a PI complex for NNRTI-susceptible phenotype or continuation of polymerization for NNRTI-resistant phenotype.

FIG. 4.

Effect of reduced RNase H activity on the resistance level to NNRTIs NVP (A), DLV (B), EFV (C), and ETR (D). Vertical bars represent IC₅₀s (nM) for each drug; gray bars correspond to the IC₅₀s by viruses bearing the wild-type RNase H domain; black bars represent viruses with the D549N mutation. Fold changes in IC₅₀s versus wild-type control are indicated above each bar. Error bars represent the standard error for the results from 2 to 11 experiments. (E) Summary table for the K_d values for the NNRTI-RT complex from the work by Xia et al. (58) and IC₅₀s for WT viruses. (F) Linear regression analysis of fold NNRTI resistance associated with NNRTI BP mutations in the presence (x axis) or absence of the D549N substitution (y axis). Fold resistance was calculated as a ratio between IC₅₀s for virus containing specific mutation(s) versus WT control using resistance data from panels A, B, C, and D. The correlation coefficient (R) was determined using SigmaPlot 8.0 software.

FIG. 5.

RNase H cleavage of NNRTI-resistant mutants. (A) RNase H primary cleavages for D549N, Q475A, and Y501A mutants. The assay is schematically depicted above the gel autoradiogram; horizontal arrows show the size of the RNA product. The average percentages of cleaved product (proportion of 15- and 14-nt products) are shown for the representative gel from two independent experiments. (B) RNase H primary cleavages for the patients' RT pol alone and in combination with patient's CN. Fold reduction in RNase H cleavages is calculated for each pair of RTs (connected by a bracket) and presented below as an average of the results from five independent experiments. (C) RNase H cleavages by mutant RTs containing NNRTI BP resistant mutations. The percentages of cleaved product (proportion of 15- and 14-nt products) are shown for the representative gel. Fold reduction in cleavages is calculated versus WT control. Average fold reduction was calculated from the results for five independent experiments.