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Effect of translocation defective reverse transcriptase inhibitors on the activity of N348I, a connection subdomain drug resistant HIV-1 reverse transcriptase mutant.

ABSTRACT: 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a highly potent inhibitor of HIV-1 reverse transcriptase (RT). We have previously shown that its exceptional antiviral activity stems from a unique mechanism of action that is based primarily on blocking translocation of RT; therefore we named EFdA a Translocation Defective RT Inhibitor (TDRTI). The N348I mutation at the connection subdomain (CS) of HIV-1 RT confers clinically significant resistance to both nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). In this study we tested EFdA-triphosphate (TP) together with a related compound, ENdA-TP (4'-ethynyl-2-amino-2'-deoxdyadenosine triphosphate) against HIV-1 RTs that carry clinically relevant drug resistance mutations: N348I, D67N/K70R/L210Q/T215F, D67N/K70R/L210Q/T215F/N348I, and A62V/V5I/F77L/F116Y/Q151M. We demonstrate that these enzymes remain susceptible to TDRTIs. Similar to WT RT, the N348I RT is inhibited by EFdA mainly at the point of incorporation through decreased translocation. In addition, the N348I substitution decreases the RNase H cleavage of DNA terminated with EFdA-MP (T/P(EFdA-MP)). Moreover, N348I RT unblocks EFdA-terminated primers with similar efficiency as the WT enzyme, and further enhances EFdA unblocking in the background of AZT-resistance mutations. This study provides biochemical insights into the mechanism of inhibition of N348I RT by TDRTIs and highlights the excellent efficacy of this class of inhibitors against WT and drug-resistant HIV-1 RTs.

SUBMITTER: Michailidis E

PROVIDER: S-EPMC3551986 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Effect of translocation defective reverse transcriptase inhibitors on the activity of N348I, a connection subdomain drug resistant HIV-1 reverse transcriptase mutant.

Michailidis E E Singh K K Ryan E M EM Hachiya A A Ong Y T YT Kirby K A KA Marchand B B Kodama E N EN Mitsuya H H Parniak M A MA Sarafianos S G SG

Cellular and molecular biology (Noisy-le-Grand, France) 20121222 1

4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a highly potent inhibitor of HIV-1 reverse transcriptase (RT). We have previously shown that its exceptional antiviral activity stems from a unique mechanism of action that is based primarily on blocking translocation of RT; therefore we named EFdA a Translocation Defective RT Inhibitor (TDRTI). The N348I mutation at the connection subdomain (CS) of HIV-1 RT confers clinically significant resistance to both nucleoside (NRTIs) and non-nucleoside RT ...[more]

PMID: 23273211

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Project description: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 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.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.

Dataset Information

Effect of translocation defective reverse transcriptase inhibitors on the activity of N348I, a connection subdomain drug resistant HIV-1 reverse transcriptase mutant.

Publications

Effect of translocation defective reverse transcriptase inhibitors on the activity of N348I, a connection subdomain drug resistant HIV-1 reverse transcriptase mutant.

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