Mutations in the connection domain of HIV-1 reverse transcriptase increase 3'-azido-3'-deoxythymidine resistance

Abstract

We previously proposed that a balance between nucleotide excision and template RNA degradation plays an important role in nucleoside reverse transcriptase inhibitor (NRTI) resistance. To explore the predictions of this concept, we analyzed the role of patient-derived C-terminal domains of HIV-1 reverse transcriptase (RT) in NRTI resistance. We found that when the polymerase domain contained previously described thymidine analog resistance mutations, mutations in the connection domain increased resistance to 3'-azido-3'-deoxythymidine (AZT) from 11-fold to as much as 536-fold over wild-type RT. Mutational analysis showed that amino acid substitutions E312Q, G335C/D, N348I, A360I/V, V365I, and A376S were associated strongly with the observed increase in AZT resistance; several of these mutations also decreased RT template switching, suggesting that they alter the predicted balance between nucleotide excision and template RNA degradation. These results indicate that mutations in the C-terminal domain of RT significantly enhance clinical NRTI resistance and should be considered in genotypic and phenotypic drug resistance studies.

Fig. 1.

Drug resistance and replicative capacity associated with the C-terminal RT domains derived from treatment-experienced and treatment-naïve patients. (a) Schematic representation of the HIV-1 vector pHL[WT] used for antiretroviral drug resistance testing. Patient RT subdomains (gray boxes) were subcloned either in the context of a wild-type pol domain (−TAMs) or a TAMs (D67N, K70R, T215Y, and K219Q)-containing pol domain (+TAMs). LTR, long-terminal repeat; gag, gag gene; PR, protease gene; IN, integrase gene; luc, luciferase gene. The letters above the vector designate restriction sites: M, MscI; E, Eco47III; S, SpeI; C, ClaI. (b) AZT and d4T IC₅₀ values for treatment-experienced and treatment-naïve patient C-terminal RT domains in the context of a wild-type pol domain. (c) AZT and d4T IC₅₀ values for treatment-experienced and treatment-naïve patient C-terminal RT domains in the context of a TAMs-containing pol domain. (d) EFV IC₅₀ values for treatment-experienced patient C-terminal RT domains in the context of a TAMs-containing pol domain. In b–d, the fold changes in IC₅₀ values vs. wild-type virus (WT) are shown above each bar. (e) Replicative capacity of virus containing treatment-experienced patient C-terminal RT domains in the context of a TAMs-containing pol domain. Statistically significant differences (∗) in IC₅₀ values or replicative capacities were measured vs. WT (b, dashed reference line) or vs. TAMs control (c–e, dashed reference line). Error bars represent SEM from 3 to 10 replicates per experiment.

Fig. 2.

The effects of the pol, cn, and rh domains on AZT resistance levels. (a) The resistance levels to AZT for viruses containing the patient-derived C-terminal domains, rh domains, or cn domains in conjunction with TAMs. The left side schematically shows RT divided into pol, cn, and rh domains. Gray boxes indicate patient-derived amino acid sequences; white boxes indicate wild-type amino acid sequences. The amino acids representing the cluster of TAMs are shown in the white box corresponding to pol. The numbers in the table represent the mean of fold differences in resistance to AZT over WT for the corresponding combinations of domains from three or more experiments. (b) AZT resistance level for the combination of patients' connection domains with different clusters of AZT resistance mutations in pol. AZT resistance is shown as the fold difference in AZT IC₅₀ over WT (mean of three or more experiments ± SEM). Black bars correspond to the combination of TAMs with WT cn domain; white and gray bars correspond to the combinations of TAMs with T-3 and T-4 cn domains, respectively. The first group of TAMs is D67N, K70R, T215Y, and K219Q; the second group (TAM₁) is M41L, L210W, and T215Y; the third group (TAM₂) is D67N, K70R, T215F, and K219Q. (c) AZT resistance levels for the patient's entire RT, patient's RT with the rh domain replaced by WT, and the combination of the patient's pol domain with WT C-terminal RT domain. All designations are the same as for a. The following mutations were present in the pol domain from each patient: T-3 = V60I, K64H, D67N, T69N, K70R, V106I, K122E, I135T, Y188L, T215F, D218E, K219Q, K233Q, and L228H; T-4 = M41L, K43E, E44A, D67N, L100I, K102R, K103N, V118I, K122E, D123N, T139K, D177N, M184V, G196E, L210W, R211T/A, T215Y, and K219N; T-6 = V35L, M41L, E44D, D67N, L74V, R83K, L100I, K103N, K122P, A158S, Q174K, D177E, M184V, E194D, and T215Y; and T-8 = M41L, L74I, K103N, V108I, V118I, L210W, and T215Y.

Fig. 3.

Identification of mutations that enhance AZT resistance in the C-terminal domain of RT from treatment-experienced patients. Each line named T-3, T-4, T-6, T-8, and T-10 in the top portion indicates the patient's cn domain amino acids (297–423) that are different from wild-type pNL4–3. The underlined amino acids were present in one or more cn domains from treatment-naïve patients; amino acids indicated in bold were reverted to the wild type in the mutational analysis. The results of mutational analysis are shown below, and each group is labeled with the patient number (T-3 to T-10). Only the amino acids targeted for mutational analysis are shown. The top line in each group represents the control that contains all mutations in this patients' cn domain. The absence of amino acids in subsequent lines indicates reversion to the WT amino acid. The bars next to each line correspond to the AZT resistance level from three or more experiments ± SEM; the number is the fold difference in AZT IC₅₀ over WT (1-fold).

Fig. 4.

Effect of RT amino acid sequence on template-switching frequency. (a) Simplified structure of HIV-1-GFFP provirus with two overlapping fragments of the gfp gene (gray box). HIV-1-GFFP provirus mobilized with a helper construct undergoes one cycle of replication; during reverse transcription, template-switching events within homologous repeats functionally reconstitute the GFP gene. Quantitation of the GFP-positive infected cells by flow cytometry provides a measure of the frequency of RT template switching in a single replication cycle. (b) The left side schematically shows RT divided into pol, cn, and rh domains. Gray boxes represent patient-derived amino acid sequences; white boxes represent WT amino acid sequences. White stripes in the gray cn domain boxes represent a reversion of the patient-derived mutant amino acid back to WT. The bars on the right represent the template-switching frequency (% GFP reconstitution) as measured in the direct-repeat-deletion assay (mean of two or more experiments ± SEM.).