GT, PRH, NSC, and SHY designed the study. PRH and BW gathered the clinical data, and SHY, DT, JF, MZ, and CWS performed the experiments. GT, SHY, PRH, and VDL performed the statistical analyses. MK performed the structural analyses, and GT, NSC, PRH, SHY, and VDL contributed to writing the paper and analysing the data.
PRH has received honoraria, research and travel grants and served as a speaker from many pharmaceutical companies, and has served as a consultant to HIV drug resistance testing companies including Virco and Monogram Biosciences. SHY, CWS, JF, MZ, DT, VDL, BW, MK, NSC, and GT declare no competing interests.
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.
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 (
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.
Analyzing HIV sequences from a Canadian cohort, Gilda Tachedjian and colleagues identify a common mutation in a little-studied domain of reverse transcriptase that confers resistance to two drug classes.
In the 1980s, infection with the human immunodeficiency virus (HIV), which causes acquired immunodeficiency syndrome (AIDS), was a death sentence. Although the first antiretroviral drugs (compounds that block HIV's life cycle) were developed quickly, single antiretrovirals only transiently suppress HIV infection. HIV rapidly accumulates random changes (mutations) in its genetic material, some of which make it drug resistant. Nowadays, there are many different antiretrovirals. Some inhibit the viral protease, an enzyme used to assemble new viruses. Others block reverse transcriptase (RT), which makes replicates of the genes of the virus. Nucleoside/nucleotide RT inhibitors (NRTIs; for example, zidovudine—also called AZT—and lamivudine) and non-nucleoside RT inhibitors (NNRTIs; for example, nevirapine and efavirenz) interfere with the activity of RT by binding to different sites in its so-called “DNA polymerase domain,” the part of the enzyme that constructs copies of the viral genes. Highly active antiretroviral therapy (HAART), which was introduced in the mid 1990s, combines several antiretrovirals (usually a protease inhibitor and two NRTIs or an NNRTI and two NRTIs) so that the replication of any virus that develops resistance to one drug is inhibited by the other drugs in the mix. When treated with HAART, HIV infection is usually a chronic, stable condition rather than a fatal disease.
Unfortunately, HIV that is resistant to drugs still develops in some patients. To improve the prevention and management of drug resistance, a better understanding of the mutations that cause resistance is needed. Resistance to RT inhibitors usually involves mutations in the DNA polymerase domain that reduce the efficacy of NRTIs (including thymidine analogue mutations—also known as TAMs—and lamivudine-resistance mutations) and NNRTIs. Blood tests that detect these resistance mutations (genotype tests) have been used for several years to guide individualized selection of HIV drugs. Recently, however, mutations outside the DNA polymerase domain have also been implicated in resistance to RT inhibitors. In this study, the researchers have used data and samples collected since the mid 1990s by Canada's British Columbia Centre for Excellence in HIV/AIDS to investigate the clinical relevance of a mutation called N348I. This mutation changes an asparagine (a type of amino acid) to an isoleucine in a region of RT known as the connection domain. The researchers have also investigated how this mutation causes resistance to RT inhibitors in laboratory tests.
The researchers analyzed the first two-thirds of the RT gene in viruses isolated from a large number of the Centre's patients. Virus carrying the N348I mutation was present in less than one in 100 patients whose HIV infection had never been treated, but in more than one in 10 treatment-experienced patients. The mutation appeared early in therapy, often in viruses that had TAMs, a lamivudine-resistance mutation called M184V/I, and/or NNRTI resistance mutations. Patients treated with zidovudine and nevirapine were 2.6 times more likely to have the N348I mutation than patients not treated with these drugs. Furthermore, the appearance of the N348I mutation often coincided with an increase in viral load, although other mutations that appeared at a similar time could have contributed to this increase. When the researchers introduced the N348I mutation into HIV growing in the laboratory, they found that it decreased the susceptibility of the virus to zidovudine and to NNRTIs.
These findings show that the treatment of patients with RT inhibitors can select a drug-resistant HIV variant that has a mutation outside the enzyme's DNA polymerase domain. Because this N348I mutation, which is commonly selected in vivo and has also been seen in other studies, confers resistance to two classes of RT inhibitors and can emerge early during therapy, it could have a large impact on patient responses to antiviral regimens that contain zidovudine and nevirapine. Although these findings do not show that the N348I mutation alone causes treatment failure, they may have implications for genotypic and phenotypic resistance testing, which is often used to guide treatment decisions. At present, genotype tests for resistance to RT inhibitors look for mutations only in the DNA polymerase domain of RT. This study is the first to demonstrate that it might be worth looking for the N348I mutation (and for other mutations outside the DNA polymerase domain) to improve the ability of genotypic and phenotypic resistance tests to predict treatment outcomes.
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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 [
HIV-1 reverse transcriptase (RT) catalyses the conversion of the viral single-stranded genomic RNA into a double-stranded proviral DNA precursor. Due to its essential role in HIV replication, RT is a major target for chemotherapeutic intervention. In this regard, 11 of the 24 anti–HIV-1 inhibitors approved by the US Food and Drug Administration are RT inhibitors (RTIs). These can be divided into two therapeutic classes: (i) the nucleoside/nucleotide RT inhibitors (NRTIs), such as zidovudine (AZT) and lamivudine (3TC), that bind in the active site of the RT and act as competitive chain terminating inhibitors of DNA polymerisation [
HIV-1 RT is an asymmetric dimer that consists of a 66-kDa (p66) subunit and a p66-derived 51-kDa (p51) subunit. The catalytically active p66 subunit comprises the DNA polymerase (residues 1–315), connection (residues 316–437), and ribonuclease H (RNase H; residues 438–560) domains [
Recently, however, a growing body of evidence has emerged that implicates mutations outside of the DNA polymerase domain in RTI resistance. These include the G333D/E polymorphism that facilitates dual AZT/3TC resistance in viruses that contain both thymidine analogue mutations (TAMs) and M184V [
In this study, we evaluated the in vivo relevance and role in drug resistance of a common RT connection domain mutation, N348I, using a variety of complementary approaches. The prevalence of N348I was evaluated in treated versus therapy-naïve individuals; its order of appearance relative to other drug resistance mutations was determined; and its association with virological failure was compared with other TAMs. The impact of N348I on decreased susceptibility to AZT and NNRTIs was determined in cell culture-based assays and further confirmed in biochemical studies with recombinant RT.
Plasma samples and clinical data were obtained from the British Columbia Centre for Excellence in HIV/AIDS (the Centre), which has been previously described in detail [
A detailed description of the collection method and monitoring of data for HIV-1–positive individuals receiving antiretroviral therapy in the Centre was published previously [
Samples were analysed from individuals participating in the HIV/AIDS Drug Treatment Program from 1996 to 2003. The analysis was restricted to patient samples where sequencing was performed up to codon 400 of the RT gene. The latest available “on-therapy” sample was analysed for the treatment-experienced individuals, while a baseline sample, taken prior to therapy, was used for the therapy-naive individuals. The treatment-experienced dataset was randomly divided into two groups: a discovery test set of 500 individuals and an independent validation set of 509 individuals. Samples from 368 therapy naïve individuals were analysed to determine the prevalence of mutations in the absence of antiretroviral treatment.
The last on-therapy sample from a total of 3,569 patients was analysed for the association of N348I with key drug resistance mutations (
The following baseline predictor variables were investigated: age; gender; CD4 cell count; log10 transformed viral load (pVL); prior exposure to NRTIs, NNRTIs, or protease inhibitors (PIs); physician experience (per 100 patients); an AIDS diagnosis; history of injection drug use; year of first therapy; and adherence. The analysis was restricted to patients who were antiretroviral therapy naïve at baseline. Estimates of adherence to antiretroviral therapy were based on medications actually dispensed, not prescribed. For this study, we limited our measure of adherence to the first year of therapy estimated by dividing the number of months of medications dispensed by the number of months of follow-up. This measure of adherence has been found to be independently associated with HIV-1 viral suppression and survival amongst HIV-1-infected persons enrolled in the HIV/AIDS Drug Treatment Program [
This analysis was performed on a total of 31 individuals who received treatment between June 1996 and February 2006. Neither N348I nor other key drug resistance mutations, as defined by the IAS-USA guidelines [
This analysis was performed on data from 7,074 patients from the BC Centre database between June 1996 and June 2007 where the change in viral load (ΔpVL) was defined as pVL at the time of the first positive test for the mutation subtracted by the pVL immediately before the first positive test for the mutation. The analysis was performed for N348I and for each of the TAMs: M41L, D67N, K70R, L210W, T215Y/F, and K219Q/E. The analysis excluded sequencing data that were not performed up to codon 400 and did not account for other IAS-USA defined RT or PI drug resistance mutations that may have been selected with N348I or TAMs. Mixtures at each codon were considered positive for the mutation. An upper (100,000 copies/ml) and a lower (500 copes/ml) cutoff for viral load were introduced into the analysis to avoid introducing bias due to differences in samples analysed using the original viral load assay (range 500 to 750,000 copies/ml) and the ultrasensitive viral load assay (range 50 to 100,000 copies/ml).
The RT inhibitors AZT, 3TC, NVP, and EFV were obtained from the AIDS Research and Reference Reagent Program, National Institute for Allergy and Infectious Diseases, National Institutes of Health (NIH). 3′-Azido-2′,3′-dideoxythymidine triphosphate (AZT-TP) was purchased from TriLink Biotechnologies. All other nucleotides were purchased from GE Healthcare.
MT-2 cells [
The pDRNLXN construct (a gift from Johnson Mak) was derived from the NL4.3 infectious molecular clone of HIV-1 [
HIV-1 virus stocks were generated by transfecting 239T cells with infectious molecular clones of HIV-1 using the calcium phosphate technique as previously described [
Drug susceptibility assays were performed in either MT-2 cells using cell viability as the readout [
The M41L, L210W, T215Y, and N348I mutations were introduced into WT HIV-1LAI RT [
Heteropolymeric RNA-dependent or DNA-dependent DNA polymerase template/primer (T/P) were prepared as described previously [
A 5′ 32P-labeled 26-nucleotide primer (5′-CCTGTTCGGGCGCCACTGCTAGAGAT-3′) annealed to a 35-nucleotide DNA template (5′-AGAATGGAAAATCTCTAGCAGTGGCGCCCGAACAG-3′) or RNA template (5′-AGAAUGGAAAAUCUCUAGCAGUGGCGCCCGAACAG-3′) was chain-terminated with AZT-monophosphate (AZT-MP), as described previously [
WT and mutant RT RNase H activity was evaluated using the same AZT-MP chain-terminated RNA/DNA T/P substrate described above, except the 5′-end of the RNA was 32P-end-labeled. Assays were carried out using 20 nM T/P, 3 mM ATP, and 10 mM MgCl2 in a buffer containing 50 mM Tris-Cl (pH 8.0) and 50 mM KCl. Reactions were initiated by the addition of 200 nM WT or mutant HIV-1 RT. Aliquots were removed, quenched at varying times, and analysed as described above.
Statistical analyses of the prevalence of mutations from codons 240 to 400 in drug-naïve compared to drug-treated individuals and the association of N348I with key drug resistance mutations were performed using the Chi-square test. In the descriptive analysis used to determine which antiretroviral drug treatment was associated with the emergence of N348I, categorical variables were compared using the Chi-square or Fisher Exact test, and continuous variables were compared using the Wilcoxon rank-sum test. In the case of multiple comparisons, the Bonferroni and/or the Benjamini and Hochberg methods were used [
The prevalence of mutations at HIV RT codons 1–400 observed by population sequencing analysis of plasma-derived HIV from individuals with known treatment histories (
Prevalence of RT Mutations in Treated and Untreated Patients
To determine whether N348I was associated with the presence of other IAS-USA-defined RTI resistance mutations, we analysed the last on-therapy sample from 161 patients with N348I compared with 3,408 without N348I. N348I was highly associated with several key drug resistance mutations, including M184V/I (
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
The association of N348I with several baseline predictor variables was analysed. Univariate analyses demonstrated a significant association with male gender (odds ratio [OR] 2.23, 95% confidence interval [CI] 1.21–4.13), increased adherence (OR 1.13, 95% CI 1.05–1.22), and increased physician's experience (OR 1.24, 95% CI 1.03–1.48), AZT treatment (OR 1.60, 95% CI 1.07–2.40), and NVP treatment (OR 1.53, 95% CI 1.02–2.29). The association of predictor variables with N348I was analysed by multivariate logistic regression analysis. These data demonstrate that treatment with AZT compared with treatment that did not consist of either AZT or NVP was associated with an increased risk for N348I, while there was no increased risk for N348I with NVP treatment compared with treatment with neither AZT nor NVP (
Association of N348I with Specific Drug Therapy
In order to determine whether N348I appears early in drug therapy and its pattern of emergence with respect to key drug resistance mutations, we performed an analysis on samples from a subset of patients in the Centre's database who had neither N348I nor other IAS-USA-resistance mutations present at baseline and for whom complete treatment history and viral data were known. Of the 31 patients fitting these criteria, we observed that N348I tended to appear in plasma virus relatively early after initiation of antiretroviral therapy. Moreover, the appearance of N348I was observed during virological failure (viral loads 283–591,000 copies/ml, median 3,600). In general, N348I appeared at the same time as M184V/I and before the appearance of TAMs (
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.
To determine the potential in vivo significance of N348I, we examined whether the appearance of N348I was associated with an increase in viral load and compared this with the changes in viral load associated with each of the other TAMs (
Appearance of N348I and TAMs Is Associated with an Increase in Viral Load
The impact of N348I on AZT susceptibility was examined by introducing this mutation by site-directed mutagenesis into the molecular clones HXB-2 (HX/348) and NL4.3 (NL/348) in order to evaluate the effect of N348I in two genetically distinct WT backbones. Phenotypic susceptibility assays were performed in MT-2 cells for the HXB-2 strain (HX) or in the TZM-bl indicator cell line for NL4.3 strain (NL). In comparison with the WT HIV-1, both the HX/348 (
Effect of N348I on AZT and 3TC Resistance
Although N348I did not appear to confer 3TC resistance by itself, when combined with M41L and T215Y (NL/2AZT + 348), it conferred a 3.3-fold and 1.8-fold decrease in 3TC susceptibility compared to the WT (
M184V suppresses phenotypic resistance conferred by TAMs, although the effect decreases with an increase in the number of TAMs [
Since N348I is also highly associated with key mutations that confer NNRTI resistance (
Effect of N348I on NNRTI Resistance
Drug Susceptibility of Recombinant RT to AZT-TP and NNRTIs
NRTI-associated resistance mutations can be broadly categorised into two groups depending on their phenotypic mechanism of resistance [
Our data show that each of the four recombinant enzymes (WT, N348I, 3AZT, and 3AZT + N348I) was equally sensitive to inhibition by AZT-TP (
The experimental conditions for this experiment are described in
(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.
To further determine the role of N348I in the AZT excision phenotype, we next evaluated the ability of the WT or mutant RT to excise AZT-MP from the 3' terminus of the primer and to rescue DNA synthesis, as described previously [
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
To further characterise this phenotype, we also assessed the RNase H cleavage events that occurred during the AZT-MP excision reactions described in the latter experiment. This analysis showed that the N348I mutation, alone and in combination with 3AZT, significantly decreased RNase H cleavage activity; and in particular, decreased formation of a secondary cleavage product that reduced the RNA template to 17 nucleotides (the corresponding RNA/DNA duplex length is reduced to ten nucleotides) (
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 [
The high prevalence of N348I is not unique to the Centre's database as it was also identified in a separate analysis of mutations beyond RT codon 240 in a large US clinical database (Quest Diagnostics) [
To delineate the role of N348I in RTI resistance, this mutation was introduced into molecular HIV-1 clones with defined genetic backbones. In this regard, N348I, alone or in combination with TAMs, conferred 2- to 4-fold AZT resistance. The level of AZT resistance conferred by N348I alone is comparable to the levels of resistance conferred by other individual TAMs. For example, the M41L mutation has been reported to confer between 1.4- to 4-fold AZT resistance, the K70R mutation up to 8-fold resistance, and T215Y up to 16-fold resistance [
N348I was also significantly associated with M184V/I in our cohort. N348I conferred a small increase in 3TC resistance in the context of TAMs but did not counteract the previously observed antagonism between M184V and TAMs. This is in contrast to another connection domain mutation, G333D/E, which is associated with dual resistance to AZT and 3TC [
Apart from Q145M, which has been reported to confer broad resistance to several NRTIs and NNRTIs [
Biochemical analyses designed to elucidate the mechanism by which N348I confers AZT resistance indicate that this mutation, consistent with other TAMs, acts by an excision rather than discrimination phenotype. However, an increase in AZT excision was only observed with a RNA/DNA T/P and not with a DNA/DNA T/P. Recently, it was suggested that mutations in the RT that decrease RNase H activity will enhance the AZT excision phenotype by slowing down the rate at which the RNA template strand is degraded [
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 [
One of the major limitations of this study is that while the data demonstrate that the appearance of N348I is associated with an increase in viral load, which is at least as large as the recognised TAMs, it does not account for the simultaneous appearance of other key PI or RT drug resistance mutations that could also contribute to the observed increase in viral load. Nevertheless, this caveat also applies to our analysis of the individual TAMs. Even with this large study, the number of permutations of mutations is too large to be able to exclude the contribution of other mutations, particularly as these patients are on combination therapy. Nevertheless, this study represents, to our knowledge, the most thorough evaluation of the in vivo relevance of a drug resistance mutation that can contribute to HIV drug resistance, but is not presently included in most genotypic and phenotypic resistance assays.
In conclusion, we have demonstrated that a mutation in the connection domain of the HIV-1 RT, N348I, is prevalent in drug-treated individuals, appears relatively early in drug therapy, and confers decreased susceptibility to AZT and NNRTIs. The mechanism of AZT resistance can be ascribed to increased nucleotide excision, as observed from an RNA/DNA T/P, and is likely to be manifested through decreased RNase H activity conferred by N348I. Decreased susceptibility to NNRTIs can be demonstrated at the enzyme level. Taken together, our data underscore the important role of N348I in conferring drug resistance to AZT and NNRTIs.
The GenBank (
We thank the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute for Allergy and Infectious Diseases, NIH, for the supply of efavirenz, nevirapine, lamivudine and zidovudine, and TZM-bl cells. We thank Johnson Mak for providing the pDRNLXN clone and Jenny Lewis and Margaret Hellard for advice on the statistical analyses performed in this study.
lamivudine
Akaike Information Criterion
zidovudine
AZT-monophosphate
AZT-triphosphate
confidence interval
efavirenz
human immunodeficiency virus type 1
International AIDS Society-USA
non-nucleoside reverse transcriptase inhibitor
nucleoside/nucleotide reverse transcriptase inhibitor
nevirapine
odds ratio
protease inhibitor
log10-transformed viral load
ribonuclease H
reverse transcriptase
RT inhibitor
thymidine analogue mutation
template/primer
wild-type