N348I in the connection domain of HIV-1 reverse transcriptase confers zidovudine and nevirapine resistance

Abstract

Background: The catalytically active 66-kDa subunit of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) consists of DNA polymerase, connection, and ribonuclease H (RNase H) domains. Almost all known RT inhibitor resistance mutations identified to date map to the polymerase domain of the enzyme. However, the connection and RNase H domains are not routinely analysed in clinical samples and none of the genotyping assays available for patient management sequence the entire RT coding region. The British Columbia Centre for Excellence in HIV/AIDS (the Centre) genotypes clinical isolates up to codon 400 in RT, and our retrospective statistical analyses of the Centre's database have identified an N348I mutation in the RT connection domain in treatment-experienced individuals. The objective of this multidisciplinary study was to establish the in vivo relevance of this mutation and its role in drug resistance.

Methods and findings: The prevalence of N348I in clinical isolates, the time taken for it to emerge under selective drug pressure, and its association with changes in viral load, specific drug treatment, and known drug resistance mutations was analysed from genotypes, viral loads, and treatment histories from the Centre's database. N348I increased in prevalence from below 1% in 368 treatment-naïve individuals to 12.1% in 1,009 treatment-experienced patients (p = 7.7 x 10(-12)). N348I appeared early in therapy and was highly associated with thymidine analogue mutations (TAMs) M41L and T215Y/F (p < 0.001), the lamivudine resistance mutations M184V/I (p < 0.001), and non-nucleoside RTI (NNRTI) resistance mutations K103N and Y181C/I (p < 0.001). The association with TAMs and NNRTI resistance mutations was consistent with the selection of N348I in patients treated with regimens that included both zidovudine and nevirapine (odds ratio 2.62, 95% confidence interval 1.43-4.81). The appearance of N348I was associated with a significant increase in viral load (p < 0.001), which was as large as the viral load increases observed for any of the TAMs. However, this analysis did not account for the simultaneous selection of other RT or protease inhibitor resistance mutations on viral load. To delineate the role of this mutation in RT inhibitor resistance, N348I was introduced into HIV-1 molecular clones containing different genetic backbones. N348I decreased zidovudine susceptibility 2- to 4-fold in the context of wild-type HIV-1 or when combined with TAMs. N348I also decreased susceptibility to nevirapine (7.4-fold) and efavirenz (2.5-fold) and significantly potentiated resistance to these drugs when combined with K103N. Biochemical analyses of recombinant RT containing N348I provide supporting evidence for the role of this mutation in zidovudine and NNRTI resistance and give some insight into the molecular mechanism of resistance.

Conclusions: This study provides the first in vivo evidence that treatment with RT inhibitors can select a mutation (i.e., N348I) outside the polymerase domain of the HIV-1 RT that confers dual-class resistance. Its emergence, which can happen early during therapy, may significantly impact on a patient's response to antiretroviral therapies containing zidovudine and nevirapine. This study also provides compelling evidence for investigating the role of other mutations in the connection and RNase H domains in virological failure.

Figure 1. Association of N348I with Key Drug Resistance Mutations

The prevalence of key drug resistance mutations without N348I (red bars) compared to their prevalence with N348I (blue bars). The last on therapy sample from a total of 3,569 patients was analysed where 3,408 and 161 samples did not or did contain N348I, respectively. Statistically significant differences between the two groups were determined by Chi-square analysis. The asterisk above the bars denotes p < 0.001, except for L210 where p = 0.002.

Figure 2. Pattern of Emergence of N348I Early in Drug Therapy Failure

The pattern of emergence of N348I relative to key drug resistance mutations in the RT is shown with respect to days post therapy. These patients had neither N348I nor key drug resistance mutations present at baseline. Mixtures of WT and mutants were considered mutant in this analysis. The presence of the indicated key mutations was included from data available from each time point up to the appearance of N348I. If subsequent time points also contained N348I, then the key mutations present at this time point was also included in the analysis.

Figure 3. Steady-State DNA Synthesis by WT and Mutant HIV-1 RT in the Presence of AZT-TP and 3 mM ATP

The experimental conditions for this experiment are described in Methods. (A) DNA synthesis was evaluated on a DNA/DNA T/P. (B) DNA synthesis was evaluated on an RNA/DNA T/P. For both (A) and (B), lanes demarcated as 1–4 included control reactions for the WT, N348I, 3AZT, and 3AZT + N348I RTs where all substituents were added except ATP. The primer, final product, and AZT-MP chain-termination sites are indicated.

Figure 4. Isotherms for ATP-Mediated Phosphorolytic Excision of AZT-MP by WT, 3AZT, N348I, and 3AZT + N348I

HIV-1 RT on DNA/DNA T/P (A) and RNA/DNA T/P (B). Data are the mean of three or four independent experiments. Autoradiogram of RNase H products generated during ATP-mediated excision assays on the RNA/DNA T/P (C). Experiments were carried out as described in Methods.

Figure 5. Position of N348 in the HIV-1 RT Structure

Structural model of HIV-1 demonstrating the position of N348 in the p66 (blue) and p51 RT (pink) subunits relative to the polymerase active site (gold spheres), the NNRTI-binding pocket (green) and the dimer interface (gold). N348 in p66 (red sphere) is located in the connection domain and is in close proximity to the hinge region of the p66 thumb (turquoise). In contrast, N348 in p51 is located far from the dimer interface. The RT coordinates were derived from 1RTD [78], with C280 reverted to WT S280 and with missing residues of p51 added (amino acids 218 to 230 and 430 to 440), while the DNA/DNA duplex was converted to an RNA/DNA duplex. The image was prepared using POVRAY (Persistence of Vision Pty, Ltd, Williamstown, Victoria, Australia) and GNU Image Manipulation Program (GIMP) (http://www.gimp.org/).