Research Article: N348I in the Connection Domain of HIV-1 Reverse Transcriptase Confers Zidovudine and Nevirapine Resistance

Date Published: December 1, 2007

Publisher: Public Library of Science

Author(s): Soo-Huey Yap, Chih-Wei Sheen, Jonathan Fahey, Mark Zanin, David Tyssen, Viviane Dias Lima, Brian Wynhoven, Michael Kuiper, Nicolas Sluis-Cremer, P. Richard Harrigan, Gilda Tachedjian, Douglas D Richmann

Abstract: BackgroundThe 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 FindingsThe 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 × 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.ConclusionsThis 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.

Partial Text: The advent of highly active antiretroviral therapy has dramatically improved the clinical status of many HIV-infected patients. However, one of the major contributing factors to virological failure during highly active antiretroviral therapy is the selection and evolution of drug-resistant HIV strains [1,2]. Accordingly, a better understanding of drug resistance is needed to effectively prevent and manage resistance.

In this study, we demonstrate a role of the commonly selected N348I mutation in the connection domain of HIV-1 RT in both NRTI (AZT) and NNRTI (NVP and EFV) resistance. N348I is more prevalent in clinical samples from patients treated with RTIs compared with samples from treatment-naïve individuals. In this regard, our analysis ranks N348I as the ninth most prevalent resistance mutation from a total of 39 different RT codons that were evaluated in RTI experienced patients and this mutation was observed more frequently in our cohort than mutations at codons 210, 69, 44, 190, 118, and 74, most of which have been the topic of multiple clinical, genetic, virological, biochemical and structural analyses [59–63]. As expected with HIV, there was a strong codon bias, with most of the mutant N348I (98.6%) encoded by ATT, while more than 97% of WT N348 was encoded by AAT. Furthermore, the N348I mutation is associated with M184V/I, TAMs, K103N and Y181C and is selected by NVP and AZT treatment. The N348I mutation also appears relatively early in virological failure, generally before the appearance of TAMs and usually at the same time as the acquisition of NNRTI resistance mutations. Notably, N348I was the first mutation observed in two patients in our cohort. The early appearance of N348I after initiation of antiretroviral therapy is consistent with it playing a key role in the development of RTI resistance rather than being an accessory mutation that appears after primary mutations [64]. This is supported by our findings that its appearance is associated with an increase in viral load, which is at least as large as that observed for any of the other TAMs.



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