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

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.

N348I in HIV-1 reverse transcriptase counteracts the synergy between zidovudine and nevirapine.

The efficacy of regimens that include both zidovudine and nevirapine can be explained by the synergistic interactions between these drugs. N348I in HIV-1 reverse transcriptase confers decreased susceptibility to zidovudine and nevirapine. Here, we demonstrate that N348I reverses the synergistic inhibition of HIV-1 by zidovudine and nevirapine. Also, the efficiency of zidovudine-monophosphate excision in the presence of nevirapine is greater for N348I HIV-1 reverse transcriptase compared with the wild-type enzyme. These data help explain the frequent selection of N348I in regimens that contain zidovudine and nevirapine, and suggest that the selection of N348I should be monitored in resource-limited settings where these drugs are routinely used.

N348I in reverse transcriptase provides a genetic pathway for HIV-1 to select thymidine analogue mutations and mutations antagonistic to thymidine analogue mutations.

OBJECTIVE: Several nonnucleoside (e.g. Y181C) and nucleoside (e.g. L74V and M184V) resistance mutations in HIV-1 reverse transcriptase are antagonistic toward thymidine analogue mutations (TAMs) that confer zidovudine (ZDV) resistance. The N348I mutation in the connection domain of reverse transcriptase also confers ZDV resistance; however, the mechanisms involved are different from TAMs. In this study, we examined whether N348I compensates for the antagonism of the TAM K70R by Y181C, L74V and M184V. DESIGN AND METHODS: The ZDV monophosphate and ribonuclease H activities of recombinant-purified HIV-1 reverse transcriptase-containing combinations of K70R, N348I and Y181C, L74V or M184V were assessed using standard biochemical and antiviral assays. RESULTS: As expected, the introduction of the Y181C, L74V or M184V mutations into K70R HIV-1 reverse transcriptase significantly diminished the ATP-mediated ZDV monophosphate excision activity of the enzyme. However, the N348I mutation compensated for this antagonism on RNA/DNA template/primers by significantly decreasing the frequency of secondary ribonuclease H cleavages that reduce the overall efficiency of the excision reaction. CONCLUSION: The acquisition of N348I in HIV-1 reverse transcriptase - which can occur early in therapy, oftentimes before TAMs - may provide a simple genetic pathway that allows the virus to select both TAMs and mutations that are antagonistic toward TAMs.