HIV-1 reverse transcriptase is a key target for the development of antiretroviral drugs. To date, the US Food and Drug Administration have approved 12 reverse transcriptase inhibitors that can be classified into two distinct groups: the nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs) that, once metabolized to their active triphosphate forms, inhibit viral DNA polymerization by acting as chain terminators of nucleic acid synthesis  and the non-NRTIs (NNRTIs) that bind to a hydrophobic pocket in HIV-1 reverse transcriptase and act as allosteric inhibitors of viral reverse transcription .
The USA Panel of the International AIDS Society recommends antiretroviral combination therapies that comprise an NNRTI or protease inhibitor boosted with low-dose ritonavir, each combined with two NRTIs for the treatment of adult HIV infection . The rationale for including two NRTIs stems from studies, which demonstrated synergy between combinations of NRTIs such as zidovudine (ZDV)/didanosine (ddI) or ZDV/lamivudine (3TC) [4–8]. From a clinical perspective, the efficacy of these regimens can also be attributed to a higher genetic barrier to resistance due to the ddI and 3TC resistance mutations (L74V and M184V, respectively) antagonizing the ZDV-associated resistance mutations (e.g. thymidine analogue mutations or TAMs) [9–15]. Similarly, the efficacy of anti-HIV therapies that include both ZDV and an NNRTI such as nevirapine may be explained, in part, by the observed synergistic interactions between these two classes of drugs as well the antagonism between their respective resistance mutations [16–20].
Despite the efficacy of combination antiretroviral therapies (ARTs) containing reverse transcriptase inhibitors, multidrug resistant HIV-1 can still develop, although the current literature suggests that this may require the accumulation of several additional mutations. For example, substitutions at reverse transcriptase codons 44, 118, 207, 208 and 333 have been associated with increased ZDV resistance in viruses that carry both TAMs and M184V [21–23]. Recently, we identified the N348I mutation in HIV-1 reverse transcriptase that confers both ZDV and NNRTI resistance . N348I appears early in therapy and was found to be highly associated with TAMs, M184V/I and the NNRTI resistance mutations K103N, Y181C/I and G190A/S. N348I was also found to be significantly associated with therapies that contained ZDV and nevirapine. Initially, we hypothesized that N348I was selected early in therapy failure because the mutation decreased susceptibility to both ZDV and nevirapine and accordingly provided a simple genetic pathway to resistance that involved only a single-nucleotide change . However, recent investigations into the biochemical mechanisms by which N348I in reverse transcriptase confers ZDV resistance raised additional questions as to whether this mutation may also compensate for the antagonism between TAMs and Y181C (described below).
HIV-1 reverse transcriptase containing TAMs exhibits an increased capacity to unblock ZDV monophosphate (ZDV-MP) terminated primers in the presence of physiological concentrations of ATP by increasing binding affinity of ATP for reverse transcriptase, by increasing the kinetic rate of the ATP-mediated excision reaction or both [26–28]. The Y181C mutation directly antagonizes this ATP-mediated excision activity of reverse transcriptase-containing TAMs [17,18]. By contrast, the N348I mutation in HIV-1 reverse transcriptase indirectly increases ZDV resistance by decreasing the frequency of secondary ribonuclease H (RNase H) cleavages that significantly reduce the RNA/DNA duplex length of the template/primer and diminish the efficiency of ZDV-MP excision [24,29,30]. It is not known, however, whether the Y181C mutation also antagonizes the N348I ZDV resistance phenotype. Accordingly, in this study, our primary goal was to analyze the effects of N348I on the ZDV-MP excision phenotype using recombinant purified reverse transcriptases that contained K70R, Y181C or K70R/Y181C.
Materials and methods
The K70R, L74V, Y181C, M184V and N348I mutations were introduced into the wild-type p6HRT-Prot prokaryotic expression vector  by site-directed mutagenesis using the QuikChange mutagenesis kit (Stratagene La Jolla, California, USA). Full-length sequencing of mutant reverse transcriptases was performed to confirm the presence of the desired mutations and to exclude adventitious mutations introduced during mutagenesis. The wild-type, K70R, Y181C, N348I, K70R/Y181C, K70R/Y181C/N348I, K70R/L74V, K70R/L74V/N348I, K70R/M184V, K70R/M184V/N348I HIV-1 reverse transcriptases were purified as described previously [31,32]. The protein concentration of the purified enzymes was determined spectrophotometrically at 280 nm, using an extinction coefficient (ε 280) of 260 450 mol/l/cm, and by Bradford protein assays (Sigma–Aldrich, St. Louis, Missouri, USA). The RNA and DNA-dependent DNA polymerase activities of the purified wild-type and mutant enzymes were essentially identical (data not shown). ZDV triphosphate (ZDV-TP) was purchased from Sierra Bioresearch (Tuscon, Arizona, USA). ATP, deoxyribonucleotide triphosphates (dNTPs) and dideoxy nucleoside triphosphates were purchased from GE Healthcare (Piscataway, New Jersey, USA), and [γ-32P]ATP was acquired from PerkinElmer Life Sciences (Boston, Massachusetts, USA). RNA and DNA oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, Iowa, USA).
Single-round zidovudine monophosphate excision assays
A 26-nucleotide DNA primer (pr26; 5′-CCTGTTCGGGCGCCACTGCTAGAGAT-3′) was 5′-radiolabeled with [γ-32P]ATP and chain terminated with ZDV-MP to generate PZDV as reported previously [20,24,33,34]. PZDV was then annealed to either a 35-nucleotide DNA (TDNA: 5′-AGAATGGAAAATCTCTAGCAGTGGCGCCCGAACAG-3′) or RNA (TRNA: 5′-AGAAUGGAAAAUCUCUAGCAGUGGCGCCCGAACAG-3′) template. ATP-mediated ZDV-MP excision assays were carried out by first incubating 20 nmol/l TRNA/PZDV or TDNA/PZDV with 3 mmol/l ATP, 10 mmol/l MgCl2, 1 μmol/l thymidine triphosphate and 10 μmol/l dideoxycytidine triphosphate in a buffer containing 50 mmol/l Tris–HCl (pH 7.5) and 50 mmol/l KCl. Reactions were initiated by the addition of 200 nmol/l wild-type or mutant reverse transcriptase. Aliquots were removed at defined times, quenched with sample loading buffer (98% deionized formamide, 1 mg/ml each of bromophenol blue and xylene cyanol), denatured at 95°C for 8 min and then product was resolved from substrate by denaturing PAGE and analyzed, as reported previously [20,24,33,34].
Assay for reverse transcriptase ribonuclease H activity
Wild-type and mutant reverse transcriptase RNase H activity was evaluated using the same ZDV-MP chain-terminated RNA/DNA template/primer substrate described above, except the 5′-end of the RNA was 32P-end-labeled. Assays were carried out using 20 nmol/l TRNA/PZDV, 3 mmol/l ATP and 10 mmol/l MgCl2 in a buffer containing 50 mmol/l Tris–HCl (pH 7.5) and 50 mmol/l KCl. Reactions were initiated by the addition of 200 nmol/l wild-type or mutant HIV-1 reverse transcriptase. Aliquots were removed, quenched at varying times and analyzed as described above.
Reverse transcriptase polymerization products formed under continuous DNA polymerization conditions
Heteropolymeric RNA-dependent or DNA-dependent DNA polymerase template/primers were prepared as reported previously [33,34]. DNA polymerization reactions were carried out by incubating 20 nmol/l heteropolymeric template/primer complex with 1 μmol/l concentration of each dNTP, 2 μmol/l of ZDV-TP, 3 mmol/l ATP and 10 mmol/l MgCl2 in buffer containing 50 mmol/l Tris–HCl (pH 7.5) and 50 mmol/l KCl. Reactions were initiated by the addition of 200 nmol/l wild-type or mutant reverse transcriptase. After defined incubation periods, aliquots were removed from the reaction tube and quenched with equal volumes of gel loading dye. Products were separated by denaturing gel electrophoresis and quantified, as described above.
Phenotypic drug susceptibility assays
The appropriate drug resistance mutations were introduced by site-directed mutagenesis into the background of wild-type pNL4.3 infectious molecular clone. HIV-1 was recovered by transfection of 293T cells, and drug susceptibility assays were performed in the TZM-bl indicator cell line, as described previously , with the exception that HIV-1 replication was determined by measuring luciferase activity using the Steady-Glo Luciferase Assay System according to manufacturer's instructions (Promega, Madison, Wisconsin, USA). Statistically significant differences in the 50% effective dose (EC50) were determined using the Wilcoxon rank sum test.
Results and discussion
N348I in HIV-1 reverse transcriptase compensates for the antagonism of K70R by Y181C
The Y181C mutation in reverse transcriptase increases HIV-1 sensitivity to ZDV, even when multiple TAMs are coselected on the same genome . Previous biochemical studies suggested that this phenotype was due to the Y181C mutation decreasing the ZDV-MP excision activity of both wild-type and D67N/K70R/T215F/K219Q HIV-1 reverse transcriptase by directly impacting ATP binding, the rate of ZDV-MP excision or both [18,35]. Because the mechanism by which N348I in HIV-1 reverse transcriptase confers ZDV resistance is different from that conferred by TAMs (described in Introduction) [24,29,30], we hypothesized that Y181C might not antagonize the excision activity of N348I HIV-1 reverse transcriptase and, accordingly, that N348I may compensate for the Y181C-mediated antagonism of TAMs. To investigate this hypothesis, we examined the ability of recombinant HIV-1 reverse transcriptase containing combinations of Y181C, K70R and N348I to excise ZDV-MP using established biochemical assay systems. In this study, we focused on a single TAM (i.e. K70R) because we were interested to determine how N348I influenced the interplay between TAMs and Y181C at the onset of resistance development. In this regard, the K70R mutation is one of the first TAMs to appear under ZDV drug pressure [36,37]. Furthermore, K70R in HIV-1 reverse transcriptase is associated with a strong ATP-mediated excision phenotype in vitro .
We first examined the ability of wild-type, K70R, Y181C, K70R/Y181C and K70R/Y181C/N348I HIV-1 reverse transcriptase to excise ZDV-MP and rescue DNA synthesis from chain-terminated DNA/DNA and RNA/DNA template/primers (Fig. 1). As described previously [18,35], Y181C HIV-1 reverse transcriptase unblocked ZDV-MP chain-terminated primers inefficiently on both DNA/DNA and RNA/DNA template/primers (Fig. 1b and c). As expected, Y181C also antagonized the ability of K70R reverse transcriptase to excise ZDV-MP and recover DNA synthesis, although this defect was more pronounced on an RNA/DNA template/primer than on a DNA/DNA template/primer (Fig. 1b and c). Introduction of the N348I mutation into reverse transcriptase containing K70R/Y181C reverse transcriptase increased the enzymes excision activity on the RNA/DNA template/primer but not on the DNA/DNA template/primer (Fig. 1b and c).
Our group  as well as Ehteshami et al.  and Delviks-Frankenberry et al.  have demonstrated that the N348I mutation in HIV-1 reverse transcriptase increases ZDV resistance by decreasing the frequency of secondary RNase H cleavages that significantly reduce the RNA/DNA duplex length of the template/primer and diminish the efficiency of ZDV-MP excision. In this regard, we previously delineated the relationship between ZDV-MP excision efficiency and RNase H activity on the RNA/DNA template/primer substrate used in these experiments [20,33]. These studies showed that the primary polymerase-dependent RNase H cleavage of reverse transcriptase does not impact the enzyme's ZDV-MP excision efficiency, but polymerase-independent RNase H cleavages that reduce the RNA/DNA duplex length to less than 12 nucleotides abolish ZDV-MP excision activity [20,33]. In light of these data, we next evaluated the RNase H activity of wild-type and mutant reverse transcriptase that occurred during the ATP-mediated excision reactions described in Fig. 1. As reported previously , N348I significantly reduced the frequency of a polymerase-independent cleavage event that decreases the RNA/DNA duplex to 10 nucleotides (Fig. 2b). In comparison with the wild-type enzyme, the K70R and Y181C mutations had minimal impact on the RNase H activity of reverse transcriptase (Fig. 2). Introduction of the N348I mutation into K70R/Y181C reverse transcriptase, however, significantly reduced the frequency of this polymerase-independent cleavage event (Fig. 2), a finding that is consistent with the notion that N348I in HIV-1 reverse transcriptase impacts the efficiency of the ZDV-MP excision reaction by RNase H-dependent mechanism.
In the experiments described above, we evaluated the ZDV-MP excision and RNase H cleavage activities of the wild-type and mutant enzymes on a defined (in terms of sequence and length) RNA/DNA template/primer. Because both excision and RNase H activities of reverse transcriptase are likely affected by nucleic acid sequence and length, we next evaluated the ability of wild-type and mutant enzymes to synthesize DNA in the presence of ZDV-TP and ATP, using a long heteropolymeric RNA template, corresponding to the HIV-1 sequence used for (−) strong stop DNA synthesis, primed with a DNA oligonucleotide. The 173-nucleotide incorporation events needed to produce full-length DNA product in this assay system allow for multiple ZDV-TP incorporation and ZDV-MP excision events during the formation of full-length final product [33,34]. In the presence of 3 mmol/l ATP, the N348I and K70R enzymes were significantly more efficient than wild-type enzyme in synthesizing full-length DNA product (Fig. 3). By contrast, both the Y181C and K70R/Y181C enzymes were less efficient in generating full-length DNA product. Consistent with the data in Fig. 1, the N348I mutation partially compensated for the antagonism between K70R and Y181C; more final DNA synthesis product was evident for the K70R/Y181C/N348I reverse transcriptase compared with the wild-type, Y181C and K70R/Y181C reverse transcriptases (Fig. 3).
We also assessed the susceptibility of HIV-1 containing K70R, K70R/Y181C or K70R/Y181C/N348I to ZDV in a TZM-bl indicator cell line. HIV-1 containing K70R did not confer significant resistance to ZDV in these assays. The EC50 value for inhibition of replication of K70R HIV-1 by ZDV (0.27 ± 0.04 μmol/l, n = 6) was increased only 1.2-fold relative to the wild-type virus (EC50 = 0.23 ± 0.04 μmol/l, n = 6). Accordingly, the assay window was not significantly large enough to reproducibly measure ZDV resistance, antagonism of K70R by Y181C, and the subsequent recovery of the ZDV resistance phenotype by introduction of the N348I mutation. However, in viruses that contained both K70R and T215Y (EC50 = 0.94 ± 0.13 μmol/l, n = 5, 4.1-fold ZDV resistance, P = 0.009), the introduction of the N348I mutation clearly compensated for the antagonism of the ZDV-resistance phenotype by Y181C; the EC50 value for inhibition of replication of K70R/T215Y/Y181C/N348I HIV-1 by ZDV (1.3 ± 0.5 μmol/l, n = 4) was increased 2.8-fold relative to the K70R/T215Y/Y181C virus (EC50 = 0.46 ± 0.14 μmol/l, n = 5, P = 0.032).
Taken together, these data show that the Y181C mutation does not antagonize the ability of N348I to excise ZDV-MP via an RNase H-dependent mechanism in HIV-1 reverse transcriptase containing K70R. Accordingly, N348I in HIV-1 reverse transcriptase compensates for the antagonism between TAMs and Y181C. N348I in HIV-1 reverse transcriptase also compensates for the antagonism of K70R by L74V and M181V.
Previous studies [9–15] have demonstrated that the NRTI discrimination mutations L74V and M184V antagonize the ZDV-MP excision phenotype of reverse transcriptase containing TAMs. To determine whether L74V or M184V-mediated antagonism of TAMs could also be rescued by the N348I mutation, we compared the ZDV-MP excision and RNase H activities of K70R/L74V HIV-1 reverse transcriptase with K70R/L74V/N348I HIV-1 reverse transcriptase and K70R/M184V HIV-1 reverse transcriptase with K70R/M184V/N348I HIV-1 reverse transcriptase (Figs. 4 and 5). Consistent with previously published data [12–15], the introduction of either the L74V or the M184V mutations into HIV-1 reverse transcriptase containing K70R dramatically decreased the ATP-mediated ZDV-MP excision activity of HIV-1 reverse transcriptase (Fig. 4). However, the N348I mutation completely alleviated the antagonism of K70R by L74V (Fig. 4b) and partially compensated for the antagonism of K70R by M184V (Fig. 4a). As expected, N348I decreased the formation of the polymerase-independent RNase H cleavage product that reduced the RNA/DNA duplex to 10 nucleotides in length for both the K70R/M184V (Fig. 5a) and K70R/L74V (Fig. 5b) reverse transcriptases.
In the RNase H activity experiments described above, we only assessed the activity of enzymes that contained both K70R and a mutation antagonistic to K70R (i.e. Y181C, M184V or L74V). In Fig. 5(c), we showed that the formation of the polymerase-independent RNase H cleavage product that reduced the RNA/DNA duplex to 10 nucleotides by N348I reverse transcriptase relative to the wild-type reverse transcriptase is not significantly impacted by the introduction of the Y181C, L74V or M184V mutations.
Taken together, these findings demonstrate that neither L74V nor M184V antagonizes the ability of N348I in HIV-1 reverse transcriptase to excise ZDV-MP via an RNase H-dependent mechanism. Therefore, N348I in HIV-1 reverse transcriptase can also compensate for the antagonism of TAMs by L74V and M184V.
This study suggests that the acquisition of N348I in HIV-1 reverse transcriptase, which can occur early during therapy oftentimes before TAMs , may provide a simple genetic pathway that allows the virus to select both TAMs and mutations that are antagonistic to TAMs (e.g. L74V, Y181C and M184V). This finding is consistent with recent studies that show a strong association between N348I with TAMs, M184V/I and Y181C  or that N348I is frequently observed in ZDV and/or ddI-containing therapies . In fact, in the Stanford University HIV Database, N348I is frequently observed in viruses from patients failing combination ARTs that contained an NNRTI and either ZDV/3TC (frequency of N348I = 22.2%) or ZDV/ddI (frequency of N348I = 9.5%). Finally, this study further highlights the complex but potentially important role of mutations in the C-terminal domains of HIV-1 reverse transcriptase in drug resistance.
This study was supported by a grant (#R01 AI081571) from the National Institute of Health Allergy and Infectious Diseases, National Institutes of Health to N.S.-C. J.R. was supported by a fellowship from the Pitt AIDS Research Training grant (#T32 AI065380). G.T. was supported by the National Health and Medical Research Council of Australia Senior Research Fellowship #543105 and NHMRC project grant #433903. S.H.Y. was supported by the Monash University Postgraduate Award.
N.S-C and G. T. designed the study. J.R and S.H.Y. performed all of the experiments. All authors contributed to data analysis. J.R., G.T. and N.S.-C. wrote the manuscript.
There are no conflicts of interest
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Keywords:© 2010 Lippincott Williams & Wilkins, Inc.
antagonism; HIV-1; N348I; resistance; reverse transcriptase; zidovudine