Subunit-specific mutational analysis of residue N348 in HIV-1 reverse transcriptase
© Radzio and Sluis-Cremer; licensee BioMed Central Ltd. 2011
Received: 11 April 2011
Accepted: 22 August 2011
Published: 22 August 2011
N348I in HIV-1 reverse transcriptase (RT) confers resistance to zidovudine (AZT) and nevirapine. Biochemical studies demonstrated that N348I indirectly increases AZT resistance by decreasing the frequency of secondary ribonuclease H (RNase H) cleavages that reduce the RNA/DNA duplex length of the template/primer (T/P) and diminish the efficiency of AZT-monophosphate (MP) excision. By contrast, there is some discrepancy in the literature in regard to the mechanisms associated with nevirapine resistance: one study suggested that it is due to decreased inhibitor binding while others suggest that it may be related to the decreased RNase H cleavage phenotype. From a structural perspective, N348 in both subunits of RT resides distal to the enzyme's active sites, to the T/P binding tract and to the nevirapine-binding pocket. As such, the structural mechanisms associated with the resistance phenotypes are not known.
Using a novel modelled structure of RT in complex with an RNA/DNA T/P, we identified a putative interaction between the β14-β15 loop in the p51 subunit of RT and the RNA template. Substitution of the asparagine at codon 348 in the p51 subunit with either isoleucine or leucine abrogated the observed protein-RNA interaction, thus, providing a possible explanation for the decreased RNase H phenotype. By contrast, alanine or glutamine substitutions exerted no effect. To validate this model, we introduced the N348I, N348L, N348A and N348Q mutations into RT and purified enzymes that contained subunit-specific mutations. N348I and N348L significantly decreased the frequency of secondary RNase H cleavages and increased the enzyme's ability to excise AZT-MP. As predicted by the modelling, this phenotype was due to the mutation in the p51 subunit of RT. By contrast, the N348A and N348Q RTs exhibited RNase H cleavage profiles and AZT-MP excision activities similar to the wild-type enzyme. All N348 mutant RTs exhibited decreased nevirapine susceptibility, although the N348I and N348L mutations conferred higher fold resistance values compared to N348A and N348Q. Nevirapine resistance was also largely due to the mutation present in the p51 subunit of RT.
This study demonstrates that N348I-mediated AZT and nevirapine resistance is due to the mutation in the p51 subunit of RT.
HIV-1 reverse transcriptase (RT) is a key target for antiretroviral drug development. To date, 12 RT inhibitors (RTIs) have been approved for the treatment of HIV-1 infection that can be classified into 2 distinct therapeutic groups . These include: (i) the nucleoside/nucleotide RT inhibitors (NRTI) that bind to the DNA polymerase active site of the enzyme and act as competitive inhibitors of DNA polymerization ; and (ii) the nonnucleoside inhibitors (NNRTI) that bind to a non-active site pocket in HIV-1 RT (termed the NNRTI-binding pocket) and act as allosteric inhibitors of DNA polymerization . Although combination therapies that contain two or more RTI have profoundly reduced morbidity and mortality from HIV-1 infection, their long-term efficacy is limited by the selection of drug-resistant variants of HIV-1.
Results and Discussion
Molecular models of wild type (WT) and N348 mutant HIV-1 RT in complex with an RNA/DNA T/P
In the crystal structure of HIV-1 RT in complex with a polypurine tract RNA/DNA hybrid , residue N348 in both subunits is not proximal to the enzyme's active sites, to the RNA/DNA T/P substrate, to residues that comprise the nucleic acid binding tract and to the NNRTI-binding pocket [Figure 1A, B]. Accordingly, the mechanisms by which N348I decreases RT RNase H activity and drug susceptibility cannot be inferred from this structure. It should, however, be noted that although the RNA/DNA duplex extends into the RNase H domain of RT in this structure, it misses the active site by ~ 4 Å. Recently, a crystal structure of the human RNase H1 was solved in complex with an RNA/DNA substrate which extends directly into the enzyme's active site . Because of the similarity between the human RNase H1 and the RNase H domains of HIV-1 RT, the authors were able to model an RNA/DNA duplex into HIV-1 RT that extends from the RNase H active site of the enzyme. It should be noted that due to the orientation and conformation of the bound T/P in this model, HIV-1 RT cannot simultaneously carry-out DNA polymerization and RNase H cleavage. Accordingly, it was proposed that the RNA/DNA T/P substrate would need to toggle between both active sites . A recent study by Beilhartz et al., however, refutes this hypothesis . Nevertheless, in this model, residues Y342, P345 and F346 from the β14-β15 loop of the p51 subunit of HIV-1 RT directly interact with the RNA template backbone [Figure 1C]. The Cβ atom of N348 forms a network of interactions with the Cβ atom and backbone atoms of Y342. [N348 in p66 remains distal to the RNA/DNA substrate in this model (data not shown)]. When the N348I mutation is introduced into the p51 subunit in this structure by molecular modelling (Figure 1D), the position of the β14-β15 loop is shifted such that P345 and F346 no longer contact the RNA template. The repositioning of this loop in the N348I RT is likely due to the bulky side-chain of isoleucine disrupting the network of interactions between this residue and Y342. Similarly, the introduction of leucine (Figure 1F) or glutamic acid (data not shown) at residue 348 in the p51 subunit of RT resulted in a shift of the β14-β15 loop away from the RNA template. By contrast, introduction of alanine (Figure 1E) or glutamine (data not shown) had little impact on the position of this loop. Both these substitutions retain the critical network of interactions between residue 348 and 342. Interestingly, the introduction of an arginine residue appeared to enhance the interactions of the P345 and F346 with the RNA template (data not shown). Taken together, these modelling studies suggest that the N348I mutation in the context of the p51 subunit of HIV-1 RT may decrease the enzyme's RNase H activity via an altered interaction with the RNA template. Importantly, these modelling analyses provided a testable hypothesis.
Subunit-specific mutational analysis of residue N348 in HIV-1 RT
ATP mediated excision of AZT-MP from a chain-terminated T/P by WT and N348 mutant HIV-1 RT
Susceptibility of WT and N348 mutant HIV-1 RT to nevirapine
Susceptibility of WT and N348 mutant HIV-1 to nevirapine
0.52 ± 0.04
1.77 ± 0.50
0.62 ± 0.07
4.60 ± 0.45
0.50 ± 0.11
5.93 ± 0.54
2.96 ± 0.10
1.25 ± 0.13
0.36 ± 0.05
This study demonstrates that N348I-mediated AZT and nevirapine resistance is likely due to the mutation in the p51 subunit of RT. It should be noted, however, that the interpretation of our data is limited by our inability to purify and characterize p66WT/p51N348I HIV-1 RT. The molecular modelling suggests that the N348I mutation abrogates an interaction between the β14-β15 loop in the p51 subunit of RT and the RNA template, which may explain the observed decrease in the secondary or polymerase independent RNase H cleavages. Nevirapine resistance appears complex and may involve both RNase H-dependent and -independent mechanisms.
The co-ordinates for the molecular model of HIV-1 RT in complex with an RNA/DNA duplex that extends into the RNase H active site of the enzyme  were kindly provided by Dr. Marcin Nowotny. To generate the models, we first selected the N348 residue in the p51 subunit and selected all amino acid residues within a 20 Å radius. These residues were then subjected to energy minimization using the Molecular Operating Environment (Chemical Computing Group, Montreal, Quebec, Canada). The rest of RT and the RNA/DNA T/P substrate were frozen and were not subjected to energy minimization. Charges were calculated using the Gasteiger method, and iterative minimizations were carried out using the AMBER 99 forcefield until the energy difference between iterations was less than 0.0001 kcal/mol per Å. These initial minimization experiments had minimal effect on the overall structure of the RT. Next, we introduced the N348I/A/Q/E/R/L mutations into the p51 subunit of HIV-1 RT, and carried out energy minimizations as described above. Modeled RT structures were visualized using MOE or the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR001081).
AZT- TP was purchased from Sierra Bioresearch (Tuscon, AZ). Nevirapine was obtained from the NIH AIDS Research and Reference Reagent Program. ATP, dNTPs, and ddNTPs were purchased from GE Healthcare (Piscataway, NJ), and [γ-32P]ATP was acquired from PerkinElmer Life Sciences (Boston, MA). RNA and DNA oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA).
Site-directed mutagenesis and protein expression
The N348A, N348E, N348L, N348Q and N348R mutations were introduced into the wild-type (WT) p6HRT-Prot prokaryotic expression vector  by site-directed mutagenesis using the QuikChange mutagenesis kit (Stratagene La Jolla, CA). Full-length sequencing of mutant RTs was performed to confirm the presence of the desired mutations and to exclude adventitious mutations introduced during mutagenesis. The mutant HIV-1 RTs were purified as described previously . For subunit selective mutagenesis, the p66 and p51 RT genes were cloned into the pET-DUET vector (Novagen-EMD Biosciences Inc., San Diego, California). The p66 subunit was expressed as an N-terminal hexahistidine fusion protein whereas p51 was expressed as an N-terminal FLAG fusion protein. The p66 and p51 subunits of RT were also cloned into the pBAD/His B (Invitrogen) and pT7-FLAG (Sigma) expression vectors to generate His-p66 and FLAG-p51, respectively. Enzymes were expressed and purified as described previously using a double-tag strategy . The protein concentration of the purified enzymes was determined spectrophotometrically at 280 nm using an extinction coefficient (ε280) of 260450 M-1 cm-1, and by Bradford protein assays (Sigma-Aldrich, St. Louis, MO).
AZT-MP excision assays
A 26-nucleotide DNA primer (pr26; 5'-CCTGTTCGGGCGCCACTGCTAGAGAT-3') was 5'-radiolabeled with [γ-32P]ATP and chain-terminated with AZT-MP to generate PAZT as reported previously [6, 7, 16, 17]. PAZT was then annealed to a 35-nucleotide RNA template (TRNA: 5'-AGAAUGGAAAAUCUCUAGCAGUGGCGCCCG AACAG-3'). ATP-mediated AZT-MP excision assays were carried out by first incubating 20 nM TRNA/PAZT with 3 mM ATP, 10 mM MgCl2, 1 μM dTTP and 10 μM ddCTP in a buffer containing 50 mM Tris-HCl (pH 7.5) and 50 mM KCl. Reactions were initiated by the addition of 200 nM WT or mutant RT. 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 polyacylamide gel electrophoresis and analyzed, as reported previously [6, 7, 16, 17].
Assay for RT RNase H activity
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-labelled. Assays were carried out using 20 nM TRNA/PAZT, 3 mM ATP and 10 mM MgCl2 in a buffer containing 50 mM Tris-HCl (pH 7.5) 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 analyzed as described above.
Inhibition of WT and N348 mutant HIV-1 RT by nevirapine
Fixed time point assays using a heteropolymeric T/P substrate were used to determine HIV-1 RT DNA polymerase activity, as reported previously . The sequence of the DNA primer and RNA template were 5'-TCGGGCGCCACTGCTAGAGA-3' and 5'-UCAGACCCUUUUAGUCAGAAUGGAAAGUCUCUAGCAGUGGCGCCCGAACAGGGACA-3', respectively. The primer was synthesized with a biotin label on their 5'-terminus. DNA polymerase reactions using heteropolymeric T/P (600 nM) were carried out in 50 mM Tris-HCl pH 7.5 (37°C), 50 mM KCl, 10 mM MgClB2B containing 600 nM T/P, 10 μM of each [P3PH]dNTP, and variable concentrations of nevirapine (0-20 μM). Reactions were initiated by the addition of 25 nM of RT, incubated for 20 min at 37°C and then quenched with 0.5 M EDTA. Streptavidin Scintillation Proximity Assay beads (GE Healthcare, Piscataway, NJ) were then added to each reaction, and the extent of radionucleotide incorporation was determined by scintillation spectrometry using a 1450 Microbeta Liquid Scintillation Counter (Perkin Elmer, Waltham, MA).
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 (PART) grant (T32 AI065380).
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