N348I emerges frequently with failure of first-line antiretroviral therapy (ART) in subtype C human immunodeficiency virus type 1 infection and affects susceptibility to nevirapine, efavirenz, etravirine, and zidovudine. This finding has implications for cross-resistance to subsequent ART regimens in resource-limited settings.
Abstract
Background. It is not known how often mutations in the connection and ribonuclease H domains of reverse transcriptase (RT) emerge with failure of first-line antiretroviral therapy (ART) in subtype C human immunodeficiency virus type 1 (HIV-1) infection and how these mutations affect susceptibility to other antiretrovirals.
Methods. We compared full-length RT sequences in plasma obtained before therapy and at virologic failure of initial ART among 63 participants with subtype C HIV-1 infection enrolled in the Comprehensive International Program of Research on AIDS in South Africa (CIPRA-SA) study. Recombinant viruses containing full-length plasma-derived RT sequences from participants with N348I at virologic failure were assayed for drug susceptibility.
Results. Y181C and M184V mutations in the RT polymerase domain were associated with failure of stavudine-lamivudine-nevirapine (d4T/3TC/NVP; P < .01), and K103N, V106M, and M184V with failure of d4T/3TC/efavirenz (EFV; P < .01). N348I in the RT connection domain emerged in 45% (P = .002) and 12% (P = .06) of participants receiving failing regimens containing NVP or EFV, respectively. Longitudinal analyses revealed that nonnucleoside RT inhibitor resistance mutations in the polymerase domain generally appeared first. N348I emerged at the same time, or after, M184V. N348I in the context of polymerase domain mutations reduced susceptibility to NVP (8.9–13-fold), EFV (4–56-fold), etravirine (ETV; 1.9–4.7-fold) and decreased hypersusceptibility to zidovudine (AZT; 1.4–2.2-fold).
Conclusions. N348I emerges frequently with virologic failure of first-line ART in subtype C HIV-1 infection and reduces susceptibility to NVP, EFV, ETV, and AZT. Additional studies are warranted to characterize the effects of N348I on virologic response to second- and third-line regimens in resource-limited settings where subtype C predominates.
The most widely used first-line antiretroviral therapy (ART) regimens for human immunodeficiency virus type 1 (HIV-1) in resource-limited settings include 2 nucleoside reverse-transcriptase (RT) inhibitors (NRTIs), typically stavudine-lamivudine (d4T/3TC) or zidovudine-3TC (AZT/3TC), combined with either nevirapine (NVP) or efavirenz (EFV), both of which are nonnucleoside RT inhibitors (NNRTIs). First-line ART fails to suppress HIV-1 replication in about 6%–20% of subtype C–infected subjects, and resistance mutations in HIV-1 RT are detected in about 80% of such subjects [1–3].
HIV-1 RT is a 117-kDa heterodimeric enzyme comprised of a 66-kDa (p66) subunit and a p66-derived 51-kDa subunit. The p66 subunit contains the DNA polymerase and ribonuclease H (RNase H) active sites and is comprised of the DNA polymerase (residues 1–319), connection (residues 320–440), and RNase H (residues 441–560) domains [4]. Because NRTIs and NNRTIs bind at or near to the DNA polymerase active site, most genotypic and phenotypic assays of drug resistance typically assess only the polymerase domain of HIV-1 RT. However, there is a growing body of evidence that implicates drug resistance mutations in the connection domain of RT [5–16]. For example, N348I is frequently selected in subtype B HIV-1 and is reported to reduce susceptibility to AZT, d4T, didanosine, NVP, EFV, etravirine (ETV), and delavirdine [5–7, 9]. N348I has also been reported to augment tenofovir and ETV resistance when combined with Y181C or thymidine analog mutations [TAMs] [17, 18].
Most mutations that cause drug resistance are similar across subtypes [19]; however, a few are more common in non-B subtypes. For example, V106M that confers resistance to NNRTI is much more frequent in subtype C HIV-1 than subtype B [20, 21]. Recent studies have assessed the frequency of connection and RNase H domain mutations in non-B subtype HIV-1 [6, 8, 18, 22, 23], but none have compared HIV-1 subtype C RT sequences from the same subject before ART and after virologic failure of a first-line regimen. We therefore compared full-length subtype C RT sequences in plasma samples obtained before initiation of first-line ART and at virologic failure among participants enrolled in the Comprehensive International Program of Research on AIDS in South Africa (CIPRA-SA) “Safeguard the Household” study [24]. In addition to sequence analyses, replication-competent recombinant viruses containing plasma-derived subtype C full-length RT sequences were generated to assess changes in drug susceptibility.
MATERIALS AND METHODS
Study Design and Participant Samples
The CIPRA-SA “Safeguard the Household” study was a randomized study evaluating care given to HIV-1–infected subjects by nurses versus physicians in resource-limited settings (ClinicalTrials.gov No. NCT00255840; see ref [24] for study details). Subjects enrolled were either ART naive or had received single-dose NVP to prevent mother-to-child transmission. ART included d4T/3TC plus either an NNRTI (EFV or NVP) or a protease inhibitor (lopinavir-ritonavir or nelfinavir), chosen by a physician. Participants provided written informed consent, and the study was approved at each site by an institutional review board [24].
Virologic failure for the CIPRA-SA study was define as either a <1.5 log10 decrease in viral load from before therapy to after 12 weeks of treatment (early failure) or confirmed plasma HIV-1 RNA level >1000 copies/mL after 24 weeks (late failure) [24]. For the current analysis, paired pretherapy and virologic failure plasma samples were obtained for full-length RT sequencing from 63 participants who experienced late virologic failure with d4T/3TC/EFV or d4T/3TC/NVP. If a plasma sample from the late virologic failure visit was not available, a sample obtained 12 weeks before or after late virologic failure was substituted. Longitudinal plasma samples were also tested to assess the pattern of emergence of mutations in RT.
Full-length RT Sequencing
Viral RNA was extracted from plasma samples using an automated Roche MagNA Pure LC analyzer and MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche) or ViroSeq HIV-1 Sample Preparation Module (Celera Diagnostics). RNA was converted to complementary DNA using SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity enzyme (Invitrogen) and primer R1 (5′-CCTGACTTTGGGGATTGTAGGGAAT-3′). Full-length RT (codons 1-560) was amplified by nested polymerase chain reaction (PCR) using primers F1 (5′-AGGAAAATGGAAACCAAAAATGATAG-3′) and R1 for first-round PCR and F1 and R2 (5′-CACAGCTAGCTACTATTTCTTTTGC-3′) for second-round PCR. Amplicons were purified with ExoSAP-IT (USB) and bulk sequenced bidirectionally using Big Dye terminator (version 3.1) on an ABI 3730 genetic analyzer (Applied Biosystems). The primers used for sequencing were A (5′-GTAGGACCTACACCTGTCAACAT-3′), B (5′-TCAGGATGGAGTTCATA-3′), C (5′-TATGAACTCCATCCTGA-3′), D (5′-CTGCTCCATCTACATAGAA-3′), E (5′-AGCCACCTGGATTCCTGA-3′), and F (5′-TGCTCTCCAATTGCTGTG-3′). Sequences were assembled and analyzed using SeqScape software, version 2.6 (Applied Biosystems) and compared with consensus subtype C RT (Los Alamos HIV Sequence Database [25]). Sequencing peaks that were >25% of total peak heights were counted as mutant. Alignment using ClustalW2 was performed on pretherapy, longitudinal, and virologic failure sequences to confirm that sequences were from the same participant [26, 27]. All sequences (GenBank accession No. JN601891–JN602028) were subtype C (REGA HIV-1 Subtyping Tool, version 2.0 [28]).
Production of Infectious Virus Containing Participant-Derived RT and Drug Susceptibility Testing
Infectious virus containing full-length participant-derived HIV-1 RT was cloned into pxxLAI 3D, as described elsewhere [29]. Bulk recombinant clones from pretherapy samples and individual clones from failure samples were isolated and DNA sequenced to confirm similarity with the RT sequences from plasma samples. The N348I mutation (codon AAT) was reverted to the wild-type amino acid I348N (codon ATT) in participant-derived recombinant clones from failure samples (QuickChange II XL Site-Directed Mutagenesis Kit). Infectious virus was generated in MT-2 cells, as described elsewhere [30]. The infectivity of virus stocks was determined by a 3-fold end-point dilution in P4/R5 cells (provided by Nathaniel Landau, New York University School of Medicine) [31]. Susceptibility of the recombinant viruses to AZT, d4T, 3TC, EFV, ETV, and NVP (obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health) was determined in P4/R5 cells in a single replication-cycle assay, as described elsewhere [30].
Statistical Analyses
Paired full-length RT sequences from pretherapy and virologic failure samples were analyzed for the emergence of drug resistance mutations in the polymerase domain (International AIDS Society - USA [IAS-USA] 2011 list [32]). The statistical significance of differences in the frequencies of mutations between pretherapy and virologic failure samples was determined using 2-sided exact McNemar's test. Differences were considered statistically significant at P < .01 without correction for multiple comparisons. For drug susceptibility data, the concentrations of drug required to inhibit viral replication by 50% (IC50) from 3–6 independent experiments were log10 transformed and compared for statistically significant differences (P <.05), using the 2-sample Student paired t test.
RESULTS
Of the 812 subjects enrolled in CIPRA-SA, 63 (8%) receiving d4T/3TC/EFV or d4T/3TC/NVP experienced late virologic failure, defined as confirmed plasma HIV-1 RNA levels >1000 copies/mL after 24 weeks [24]. Of these 63 participants, 41 (65%) received d4T/3TC/EFV, and 22 (35%) received d4T/3TC/NVP. Prior exposure to single-dose NVP had occurred in 10 participants (24%) in the d4T/3TC/EFV arm and 7 (32%) in the d4T/3TC/NVP arm.
Emergence of Mutations in the DNA Polymerase Domain of RT
Twenty-nine of 41 participants (71%) who received d4T/3TC/EFV and 16 of 22 (73%) who received d4T/3TC/NVP had NRTI mutations in the polymerase domain of HIV-1 RT at virologic failure, using the IAS-USA 2011 resistance list [32]. Thirty-three participants (80%) in the EFV- and 18 (82%) in the NVP-containing arm had NNRTI resistance mutations. The mutations that emerged most often with failure were K103N, V106M, and M184V for d4T/3TC/EFV and Y181C and M184V for d4T/3TC/NVP (Table 1). These findings are consistent with recently published results from the CIPRA-SA study, which was restricted to analysis of the polymerase domain [33]. Other IAS-USA mutations in the polymerase domain that were more frequent at failure than before therapy, but not significantly associated, were A62V, K65R, D67N, K70R, V75I, and K219E, associated with NRTI resistance, and V90I, A98G, K101E, V106A/I, V108I, V179D, Y188C/H, G190A/S, P225H, and M230L, associated with NNRTI resistance (Table 1).
Table 1.
Resistance Mutations in HIV-1 Reverse Transcriptase Associated With Failure Among Participants Treated With Efavirenz or Nevirapine and Stavudine-Lamivudine.
| Domain | Mutation | Mutations With EFV, % (No.; n = 41) |
Mutations With NVP, % (No.; n = 22) |
||||
|---|---|---|---|---|---|---|---|
| Before Therapy | At Failure | P | Before Therapy | At Failure | P | ||
| Polymerase | A62V | 0.0 (0) | 7.3 (3) | .25 | 0.0 (0) | 0.0 (0) | … |
| K65R | 0.0 (0) | 4.9 (2) | .50 | 0.0 (0) | 0.0 (0) | … | |
| D67N | 0.0 (0) | 2.4 (1) | 1.0 | 0.0 (0) | 0.0 (0) | … | |
| K70R | 0.0 (0) | 2.4 (1) | 1.0 | 0.0 (0) | 0.0 (0) | … | |
| V75I | 0.0 (0) | 2.4 (1) | 1.0 | 0.0 (0) | 4.5 (1) | 1.0 | |
| V90I | 0.0 (0) | 4.9 (2) | .50 | 0.0 (0) | 0.0 (0) | … | |
| A98G | 0.0 (0) | 0.0 (0) | … | 0.0 (0) | 4.5 (1) | 1.0 | |
| K101E | 2.4 (1) | 12.0 (5) | .22 | 0.0 (0) | 4.5 (1) | 1.0 | |
| K103N | 4.9 (2) | 54.0 (22) | <.0001 | 14.0 (3) | 32.0 (7) | .13 | |
| V106A/I | 0.0 (0) | 2.4 (1) | 1.0 | 0.0 (0) | 18.0 (4) | .13 | |
| V106M | 0.0 (0) | 22.0 (9) | .004 | 4.5 (1) | 9.0 (2) | 1.0 | |
| V108I | 0.0 (0) | 2.4 (1) | 1.0 | 0.0 (0) | 9.0 (2) | .50 | |
| V179D | 2.4 (1) | 4.9 (2) | 1.0 | 0.0 (0) | 0.0 (0) | … | |
| Y181C | 2.4 (1) | 0.0 (0) | 1.0 | 0.0 (0) | 41.0 (9) | .004 | |
| M184V | 0.0 (0) | 71.0 (29) | <.0001 | 0.0 (0) | 73.0 (16) | <.0001 | |
| Y188C/H | 0.0 (0) | 7.3 (3) | .25 | 0.0 (0) | 0.0 (0) | … | |
| G190A/S | 4.9 (2) | 12.0 (5) | .37 | 0.0 (0) | 9.0 (2) | .50 | |
| K219E | 0.0 (0) | 2.4 (1) | 1.0 | 0.0 (0) | 0.0 (0) | … | |
| P225H | 0.0 (0) | 12.0 (5) | .06 | 0.0 (0) | 0.0 (0) | … | |
| M230L | 0.0 (0) | 4.9 (2) | .50 | 0.0 (0) | 4.5 (1) | 1.0 | |
| Connection | D324E | 10.0 (4) | 17.0 (7) | .25 | 0.0 (0) | 0.0 (0) | … |
| N348I | 0.0 (0) | 12.0 (5) | .06 | 0.0 (0) | 45.0 (10) | .002 | |
| V365I | 4.9 (2) | 10.0 (4) | .50 | 14.0 (3) | 18.0 (4) | 1.0 | |
| T369I | 0.0 (0) | 7.3 (3) | .25 | 0.0 (0) | 0.0 (0) | … | |
| A371V | 0.0 (0) | 4.9 (2) | .50 | 0.0 (0) | 0.0 (0) | … | |
| M377R | 0.0 (0) | 0.0 (0) | … | 0.0 (0) | 9.0 (2) | .50 | |
| D404N | 0.0 (0) | 7.3 (3) | .25 | 0.0 (0) | 9.0 (2) | .50 | |
| RNase H | I452L | 32.0 (13) | 37.0 (15) | .50 | 45.0 (10) | 27.0 (6) | .13 |
| V467I | 24.0 (10) | 27.0 (11) | 1.0 | 32.0 (7) | 41.0 (9) | .50 | |
| L517I | 12.0 (5) | 22.0 (9) | .13 | 9.0 (2) | 18.0 (4) | .50 | |
Abbreviations: EFV, efavirenz; NVP, nevirapine; RNase H, ribonuclease H.
Emergence of Mutations in the Connection and RNase H Domains of RT
The N348I mutation was significantly associated with virologic failure occurring in 45% of participants (10 of 22) receiving d4T/3TC/NVP (P = .002). Of note, after M184V, N348I was the most frequent mutation detected and was observed more often than was Y181C (Table 1). N348I was also detected in 12% of participants (5 of 41) in whom d4T/3TC/EFV therapy failed (P = .06). Of note, the frequency of N348I differed significantly between participants receiving NVP-containing regimens and those receiving EFV-containing regimens (P = .005; Fisher's exact test). Other connection and RNase H domain mutations more frequent at virologic failure compared with pretherapy samples, although not significantly associated (P > .05), were D324E, V365I, T369I, A371V, D404N, I452L, V467I, and L517I with d4T/3TC/EFV and V365I, M377R, D404N, V467I, and L517I with d4T/3TC/NVP (Table 1).
Pattern of Emergence of N348I
To assess the pattern of emergence of N348I in relation to other mutations, we sequenced plasma samples from earlier time points in 12 of the 15 participants in whom N348I was detected at virologic failure (Table 2). NNRTI resistance mutations in the polymerase domain of HIV-1 RT were detected in 11 (92%) of 12 participants at these earlier time points. Of note, 3 participants had NNRTI resistance mutations present before therapy, but only 1 of them had reported prior single-dose NVP exposure. The M184V mutation was present at earlier time points in 6 (50%) of the 12 samples. N348I was also detected in 3 of these 6 samples that contained M184V. Overall, N348I appeared at the same time as M184V in 9 of 12 (75%) participants and after M184V in the other 3 participants.
Table 2.
Pattern of Emergence of Resistance Mutations Among 12 Participants With N348I at Virologic Failure
| Participant | Single-Dose NVP | NNRTI | Before Therapy (Week 0) | Earliest Available Sample (Week)a | Confirmed Virologic Failure (Week)b |
|---|---|---|---|---|---|
| 1 | No | EFV | None | M184V (48) | K103N, M184V, N348I (96) |
| 2 | No | EFV | K103K/N, Y181C/Y, G190A/G | V90I, K103N, M184V (24) | V90I, K101E/K, K103N, M184V, N348I/N (36) |
| 3 | No | EFV | None | K103N (96) | K103N, V108I, M184V, N348I (132) |
| 4 | Yes | EFV | None | K103K/N (48) | K103N, M184V, N348I/N (84) |
| 5 | No | NVP | None | V106M/V, M184V, Y188C, N348I/N (24) | A98G, K101E/K, Y181C, M184V, N348I/N (84) |
| 6 | No | NVP | None | V106A/V, Y188C/Y (96) | Y181C, M184V, N348I (144) |
| 7 | No | NVP | None | V106A, M184V (24) | V106A, M184V, N348I/N (36) |
| 8 | No | NVP | K103N, E138A | K103N, E138A, M184V, N348I (48) | K103N, E138A, M184V, N348I/N (60) |
| 9 | No | NVP | None | K103K/N (4) | K103N, M184V, N348I (24) |
| 10 | Yes | NVP | K103K/N, V106M/V | K103K/N, Y181C/Y (4) | K103K/N, M184V, N348I/N (24) |
| 11 | Yes | NVP | None | K103K/N (12) | K103N, Y181C, M184V, N348I/N (36) |
| 12 | Yes | NVP | None | K103K/N, M184M/V, N348I/N (72) | K103N, M184V, G190A/G, N348I/N (84) |
Abbreviations: EFV, efavirenz; NNRTI, nonnucleoside reverse-transcriptase inhibitors; NVP, nevirapine.
a Sample obtained at earliest available time point after treatment initiation and for which viral RNA was successfully extracted and amplified for sequencing.
b Sample obtained at time of confirmed HIV-1 RNA level >1000 copies/mL after 24 weeks of therapy.
Impact of N348I on NNRTI Susceptibility
To determine the phenotypic effects of N348I in subtype C HIV-1 RT on drug susceptibility, we cloned 12 participant-derived full-length RT genes into our xxLAI 3D vector; 6 from before therapy and 6 from the same participant at virologic failure. Next, using individual clones from participant-derived recombinant viruses at time of failure, paired sets of viruses (with and without N348I) were created by reversion of the N348I mutation. Sequence differences between the cloned full-length RT genes and the consensus subtype C RT gene are listed in Supplementary Tables 1 and 2.
Table 3 shows that N348I in the context of polymerase domain mutations increased the IC50 values for NVP 8.9–13-fold (P ≤ .01) and EFV 4.3–56-fold (P ≤ .05). These increases resulted in >200-fold NVP resistance (P < .01) and 13–9548-fold EFV resistance (P < .01), compared with pretherapy viruses. The impact of N348I on NVP resistance could not be assessed in 2 viruses because the IC50 of the revertant I348N virus was >200 μmol/L, which is the maximal noncytotoxic concentration in the cell line used.
Table 3.
Susceptibility to Nevirapine, Efavirenz, and Etravirine of Participant-Derived Recombinant Viruses With or Without N348I
| Nevirapine |
Efavirenz |
Etravirine |
|||||||
|---|---|---|---|---|---|---|---|---|---|
| Mutations | IC50, Mean ± SD, µmol/L | Failure vs Pretherapya (P) | 348I vs 348Nb (P) | IC50, Mean± SD, nmol/L | Failure vs Pretherapya (P) | 348I vs 348Nb (P) | IC50, Mean ± SD, nmol/L | Failure vs Pretherapya (P) | 348I vs 348Nb (P) |
| Participant 1 | |||||||||
| Pretherapy | 0.032 ± 0.006 | 0.87 ± 0.16 | 1.3 ± 0.2 | ||||||
| K103N/M184V | 16 ± 7.4 | 500 (<.01) | 8.9 (<.01) | 13 ± 6.7 | 15 (.03) | 56 (.01) | 3.4 ± 0.5 | 2.60 (<.01) | 1.9 (.03) |
| K103N/M184V/N348I | 142 ± 44 | 4438 (<.01) | 725 ± 189 | 833 (<.01) | 6.6 ± 2.0 | 5.10 (<.01) | |||
| Participant 6 | |||||||||
| Pretherapy | 0.018 ± 0.002 | 0.46 ± 0.14 | 0.6 ± 0.1 | ||||||
| Y181C/M184V | 98 ± 3.3 | 5444 (<.01) | >2.0 (<.01) | 3.8 ± 1.2 | 8.3 (.02) | 9.6 (<.01) | 6.1 ± 2.5 | 10.0 (.01) | 4.7 (.02) |
| Y181C/M184V/N348I | >200c | >11111 (<.01) | 37 ± 7.7 | 80 (<.01) | 28 ± 3.2 | 47.0 (<.01) | |||
| Participant 7 | |||||||||
| Pretherapy | 0.091 ± 0.019 | 1.28 ± 0.30 | 1.5 ± 0.4 | ||||||
| V106A/M184V | 11 ± 2.1 | 121 (<.01) | 13 (.01) | 2.4 ± 0.4 | 2.0 (<.01) | 6.5 (.01) | 0.53 ± 0.15 | 0.35 (.01) | 3.2 (<.01) |
| V106A/M184V/N348I | 148 ± 45 | 1626 (<.01) | 16 ± 2.6 | 13 (<.01) | 1.7 ± 0.3 | 1.13 (.74) | |||
| Participant 11 | |||||||||
| Pretherapy | 0.149 ± 0.008 | 1.94 ± 0.25 | 1.1 ± 0.1 | ||||||
| K103N/Y181C/M184V | >200c | >1342 (<.01) | … | 22 ± 14 | 11 (<.01) | 5.4 (.05) | 0.93 ± 0.32 | 0.85 (.37) | 2.1 (<.01) |
| K103N/Y181C/M184V/N348I | >200c | >1342 (<.01) | 114 ± 15 | 59 (<.01) | 2.0 ± 0.8 | 1.80 (.04) | |||
| Participant 12 | |||||||||
| Pretherapy | 0.050 ± 0.006 | 0.48 ± 0.12 | 0.5 ± 0.1 | ||||||
| K103N/M184V/G190A | 109 ± 35 | 2180 (<.01) | >1.8 (.06) | 321 ± 251 | 669 (<.01) | 14 (.02) | 0.35 ± 0.19 | 0.70 (.14) | 2.1 (<.01) |
| K103N/M184V/G190A/N348I | >200c | >4000 (<.01) | 4583 ± 2129 | 9548 (<.01) | 0.74 ± 0.26 | 1.48 (.10) | |||
| Participant 13 | |||||||||
| Pretherapy | 1.0 ± 0.4 | 7.87 ± 1.84 | 0.6 ± 0.1 | ||||||
| M184V/G190A | >200c | >200 (<.01) | … | 354 ± 73 | 45 (<.01) | 4.3 (<.01) | 1.1 ± 0.3 | 1.80 (.04) | 1.5 (.02) |
| M184V/G190A/N348I | >200c | >200 (<.01) | 1530 ± 268 | 194 (<.01) | 1.6 ± 0.6 | 2.70 (.02) | |||
Abbreviations: IC50, concentration required to inhibit viral replication by 50%; SD, standard deviation.
a Mean fold change in IC50 of recombinant virus with participant-derived reverse-transcriptase at virologic failure versus before therapy (pretherapy).
b Mean fold change in IC50 of recombinant virus with 348I vs 348N.
c Nevirapine cytotoxicity was observed at >200 μmol/L.
Table 3 also shows that 2 of 6 viruses with N348I and K103N/M184V or Y181C/M184V had 5.1- and 47-fold resistance to ETV (P < .01), respectively, compared with pretherapy viruses. This corresponded to 1.9- and 4.7-fold increases in ETV resistance, respectively, compared with virus without N348I (P < .05). Of the other 4 viruses, 1 remained susceptible and the other 3 had low-level resistance to ETV (1.5–2.7-fold resistance) compared with pretherapy viruses. Reversion of the N348I mutation in all 4 of these viruses increased ETV sensitivity by 1.5–3.2-fold.
Impact of N348I on NRTI Susceptibility
Recombinant viruses, with and without N348I, were also tested for susceptibility to AZT, d4T ,and 3TC (Table 4). All viruses were >910-fold resistant to 3TC compared with pretherapy recombinant viruses as a result of the M184V mutation (data not shown). The N348I mutation decreased AZT susceptibility by 2.2–2.3-fold in 2 of 6 viruses and decreased AZT hypersusceptibility (compared with pretherapy viruses) by 1.4–2.2-fold in the other 4 viruses. By contrast, N348I had had little or no effect on d4T susceptibility.
Table 4.
Susceptibility to Zidovudine and Stavudine of Participant-Derived Recombinant Viruses With or Without N348I
| Mutations | Zidovudine |
Stavudine |
||||
|---|---|---|---|---|---|---|
| IC50, Mean ± SD, µmol/L | Failure vs Pretherapya (P) | 348I vs 348Nb (P) | IC50, Mean ± SD, µmol/L | Failure vs Pretherapya (P) | 348I vs 348Nb (P) | |
| Participant 1 | ||||||
| Pretherapy | 0.50 ± 0.21 | 10.0 ± 3.50 | ||||
| K103N/M184V | 0.31 ± 0.21 | 0.62 (.24) | 1.4 (.02) | 4.25 ± 0.59 | 0.43 (.03) | 1.5 (.16) |
| K103N/M184V/N348I | 0.42 ± 0.26 | 0.84 (.93) | 6.25 ± 1.53 | 0.63 (.22) | ||
| Participant 6 | ||||||
| Pretherapy | 0.47 ± 0.16 | 7.70 ± 1.30 | ||||
| Y181C/M184V | 0.20 ± 0.04 | 0.43 (<.01) | 1.8 (.04) | 4.40 ± 1.22 | 0.57 (.10) | 1.7 (.03) |
| Y181C/M184V/N348I | 0.36 ± 0.06 | 0.77 (.17) | 7.44 ± 1.14 | 0.97 (.68) | ||
| Participant 7 | ||||||
| Pretherapy | 0.25 ± 0.09 | 6.00 ± 1.30 | ||||
| V106A/M184V | 0.22 ± 0.09 | 0.88 (.93) | 2.2 (<.01) | 5.02 ± 0.64 | 0.84 (.92) | 1.2 (.07) |
| V106A/M184V/N348I | 0.49 ± 0.21 | 1.96 (.04) | 5.83 ± 0.69 | 0.97 (.11) | ||
| Participant 11 | ||||||
| Pretherapy | 0.39 ± 0.17 | 9.90 ± 1.70 | ||||
| K103N/Y181C/M184V | 0.10 ± 0.04 | 0.26 (<.01) | 2.2 (<.01) | 4.69 ± 0.96 | 0.47 (.03) | 1.0 (.81) |
| K103N/Y181C/M184V/N348I | 0.22 ± 0.08 | 0.56 (.05) | 4.44 ± 0.49 | 0.45 (.02) | ||
| Participant 12 | ||||||
| Pretherapy | 0.22 ± 0.03 | 5.70 ± 0.80 | ||||
| K103N/M184V/G190A | 0.17 ± 0.07 | 0.77 (.26) | 2.3 (<.01) | 4.21 ± 0.27 | 0.74 (.14) | 1.4 (.19) |
| K103N/M184V/G190A/N348I | 0.40 ± 0.19 | 1.80 (.12) | 5.87 ± 1.46 | 1.03 (.29) | ||
| Participant 13 | ||||||
| Pretherapy | 0.26 ± 0.08 | 5.10 ± 1.00 | ||||
| M184V/G190A | 0.19 ± 0.03 | 0.73 (.37) | 1.6 (.18) | 4.92 ± 2.19 | 0.96 (.97) | 0.94 (.92) |
| M184V/G190A/N348I | 0.31 ± 0.19 | 1.19 (.71) | 4.60 ± 1.36 | 0.90 (.92) | ||
Abbreviations: IC50, concentration required to inhibit viral replication by 50%; SD, standard deviation.
a Mean fold change in IC50 of recombinant virus with participant-derived reverse-transcriptase at virologic failure vs before therapy (pretherapy).
b Mean fold change in IC50 of recombinant virus with 348I vs 348N.
DISCUSSION
The current study is the first to compare full-length HIV-1 subtype C RT sequences in paired plasma samples from before initiation of ART and at virologic failure. Comparisons of sequences before ART and at virologic failure are essential to control for baseline polymorphisms and establish that mutations arose with antiretroviral selection. Our analysis revealed that the connection domain mutation N348I was the second most frequent mutation, after M184V, to emerge with failure of d4T/3TC/NVP. The N348I mutation also emerged with failure of d4T/3TC/EFV, but less commonly. Longitudinal sequence analyses revealed that polymerase domain mutations emerged before N348I in 9 of 12 participants, with NNRTI resistance mutations preceding N348I in 6 of 9. In 9 of 12 participants, N348I also emerged at the same time as M184V. In the other 3 participants, M184V appeared first. This finding is largely consistent with that of previous studies showing that N348I emerged at the same time as M184V [5] and is associated with polymerase domain mutations [10, 11].
Several other mutations in the connection and RNase H domain of HIV-1 RT were more frequent at virologic failure than before therapy, but were not significantly associated with failure and thus have uncertain relevance. Although our study is the largest comparison of HIV-1 subtype C full-length RT sequences before therapy and at virologic failure, the sample size (n = 63 pairs) provides limited power to detect mutations that emerge at low prevalence (<6%). As such, our study cannot exclude the infrequent emergence of previously reported or novel mutations in the connection or RNase H domains of RT with failure of first-line ART (see [34–36] for recent reviews).
Our study is also the first to compare the drug susceptibility of recombinant viruses containing full-length plasma-derived subtype C RT from both pretherapy and virologic failure time points, with and without reversion of the N348I mutation. Phenotypic analyses confirmed that the N348I mutation in the context of polymerase domain mutations decreased EFV (4–56-fold) and NVP (2–13-fold) susceptibility in subtype C RT [5, 7, 9]. These phenotypic effects of N348I probably explain, at least in part, the emergence of N348I with failure of NVP- or EFV-containing regimens. Our study extends these findings to ETV, a potent NNRTI with activity in patients with NNRTI-resistant virus [37]. Two of the 6 recombinant viruses in our study had >3-fold increases in ETV IC50 compared with the pretherapy recombinant virus. Because an increase in ETV IC50 of ≥3-fold is associated with reduced treatment response to ETV [38], our findings have important potential implications for virologic response to ETV-containing regimens. Similarly, Gupta et al showed that for subtype B HIV-1, the N348I mutation, in combination with other NNRTI resistance mutations, increased ETV resistance >3-fold when compared with a NL4-3 reference strain [18].
In prior studies, N348I was found to be associated with TAMs, the 3TC resistance mutation M184V/I, and the NNRTI resistance mutations K103N and Y181C/I [5, 10, 11, 18]. The association of N348I with TAMs, M184V, and NNRTI resistance mutations is consistent with failure of regimens that include AZT, 3TC and NVP [10, 11]. Recently, Radzio et al showed that N348I decreases the phenotypic antagonism of TAMs by Y181C and M184V and hypothesized that N348I permits the coselection of TAMs and mutations antagonistic to TAMs (eg Y181C and M184V) on the same viral genome [39]. In the current study, participants received d4T and 3TC, and d4T is known to select for TAMs in subtype C–infected patients in South Africa with failure of first-line d4T/3TC-containing ART [1]. The absence of TAMs in the current study is likely due to the sensitive definition of virologic failure (plasma HIV-1 RNA level >1000 copies/mL), which limited the opportunity for multiple mutations to accumulate. The emergence of N348I in our study might have facilitated the accumulation of TAMs had failing therapy been continued. In support of this concept, N348I decreased AZT susceptibility or reduced AZT hypersusceptibility of viruses that contained M184V and/or Y181C, although its effects on d4T susceptibility were inconsistent. Additional longitudinal analyses would be needed to support the association of N348I with the subsequent emergence of TAMs in subtype C HIV-1. Of note, von Wyl et al also recently suggested a compensatory interaction between N348I and M184V, and further demonstrated that N348I partly restores the reduced RT processivity of the M184V mutant [16], which may help explain the frequent selection of N348I in our study.
Our study did not discern whether the frequent emergence of N348I was due to the ART regimen or the HIV-1 subtype. The N348I substitution is the result of a 1-nucleotide transversion (AAT → ATT) in both subtype B and C viruses, implying that the emergence of N348I may be influenced more by regimen than subtype. Indeed, in the current study, N348I emerged more often with NVP- than EFV-containing ART. Moreover, our group recently reported that mutations in the connection and RNase H domains of RT were not associated with virologic failure of 2 NRTI plus EFV in the AIDS Clinical Trials Group study A5142 [40]. However, A5142 had a stricter definition of virologic failure than the current study, and tenofovir, AZT, or d4T were used with 3TC. Additional studies are needed to assess the influence of subtype and regimen on the selection of N348I, as well as the phenotypic and fitness changes that drive selection.
In summary, the N348I mutation emerged frequently with virologic failure of NVP-containing first-line ART in subjects infected with HIV-1 subtype C and also with EFV-containing therapy, although less often. Phenotypic analyses revealed that N348I in subtype C RT decreases susceptibility to NVP, EFV, ETV, and AZT or reduces hypersusceptibility to AZT when in the context of polymerase domain mutations. The frequent emergence and phenotypic effects of N348I warrant further efforts to characterize its effects on virologic response to second- and third-line regimens in resource-limited settings where subtype C predominates. Such studies will address whether inclusion of codon 348 in resistance genotypes used for clinical management could improve the selection of more effective second- and third-line ART regimens and justify the additional cost.
Supplementary Data
Supplementary materials are available at Clinical Infectious Diseases online (http://cid.oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.
Notes
Acknowledgments. We thank Christina Lalama and Michael Hughes (Harvard School of Public Health, Boston, Massachusetts) for providing SAS programs and guidance for statistical analysis, the study sites and their personnel, and the participants for their participation in the study.
Author contributions. J. H. B., C. L. W., W. S. S., N. S. C., and J. W. M. designed the study. J. H. B. and D. L. K. performed all of the experiments. K. A. S. and J. H. B. performed statistical analysis. I. S., R. W., and J. A. M. provided samples and data from the CIPRA-SA study protocol. J. H. B., N. S. C., and J. W. M. wrote the manuscript, and all authors provided critical input.
CIPRA-SA Project 1 Study Team. The following investigators participated in CIPRA-SA: (Sharlaa Badal-Faesen, Mildred Botile, Nastassja Choonilal, Francesca Conradie, Jennipher Gelant, Janet Grab, Veronica Graham, Najma Hafejee, Lynda Hamber, Sindesh Harduth, Johean Hendricks, Colleen Herman, Mellissa Hero, Prudence Ive, Richard Kaplan, Nicola Killa, Daniella Klemp, Faisel Laher, Thandi Mabiletsa, Zanele Madlala, Ntombekaya Mafukuzela, Bontle Mahlatsi, Helgard Marias, Nomakhaya Mfundisi, Buang Motloba, Cindy Moyo, Mcebisi Mtshizana, Lundi Ncana, Kevin Newell, Catherine Orell, Sean Palmer, Deborah Pearce, Mary-Ann Potts, Daphne Radebe, Anne Reyneke, Anna Segeneco, Jennifer Sekgale, Jan Steyn, Pinky Thebe, Handre Truter, Diederik van Niekerk, Frieda Verheye-Dua, Karlien Voges, Helen Woolgar, Jennifer Zeinecker), the Endpoint Review Committee (Gary Maartens, David Spencer; Charles van der Horst).
Financial support. This study was supported by the National Institute Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH; grants R01 AI081571 and R01 AI081571-02S1), the Pitt AIDS Research Training Program (NIAID grant 5T32 AI065380-04), the University of Pittsburgh Clinical Translational Science Institute (NAIAD grant T32-5TL1 RR024155-04), and the National Cancer Institute (SAIC contract 25XS119). The CIPRA-SA trial was funded by the Division of AIDS of the NIAID, NIH (grant 1U19AI53217-01).
Potential conflicts of interest. J. W. M is a consultant to Gilead Sciences, Merck, and RFS Pharmaceuticals and owns share options in RFS Pharmaceuticals. All other authors report no potential conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
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