Nucleoside reverse transcriptase inhibitors (NRTIs) contribute importantly to the success of antiretroviral regimens for the treatment of HIV-1 infection. The emergence of drug resistance can limit the long-term success of such regimens, however. Many of the mutations that contribute to NRTI resistance map to the region between amino acids 62 and 78 of HIV-1 reverse transcriptase (RT).1-3 This region, which corresponds to the β3-β4 hairpin loop in the so-called “fingers” subdomain of RT, interacts with the template-primer and the incoming deoxynucleotide triphosphate (dNTP).2,4,5 Mutations at several codons in this region specifically decrease affinity of the enzyme for the nucleoside analogues and may alter processivity of the enzyme.
Although most NRTI resistance mutations result in amino acid substitutions in RT, a number of insertion and deletion mutations have also been described.6 The best characterized of these is a 6-nucleotide insertion between codons 69 and 70.7-9 This insertion usually occurs along with a T69S substitution in viruses carrying thymidine analog resistance mutations (TAMs) and is associated with high-level resistance to most NRTIs. Surveys of large resistance databases and clinic cohorts show a prevalence of the 69 insertion mutation of 1% to 3% among NRTI-experienced patients.10
Deletions in RT are much less common, and are thus less well characterized. Deletions at codon 67 (Δ67) or in the region between codons 67 and 69 have been reported, often in combination with a T69G substitution.11-13 In several cases, the deletion occurred together with Q151M and associated multinucleoside resistance mutations.14 Biochemical characterization showed that RT carrying a Δ67 mutation together with TAMs was able to excise terminal 3′-azido-3′-deoxythymidine monophosphate (AZTMP) at lower adenosine triphosphate (ATP) concentrations than were RTs from other zidovudine (ZDV)-resistant viruses.15 The deletion mutant excised a narrower spectrum of NRTIs than did RT with an insertion in the fingers domain (T69SSG), however.
We identified a deletion at RT codon 70 (Δ70) in a sample from a tenofovir (TFV)- and abacavir (ABC)-treated patient with extensive prior NRTI exposure. The deletion occurred in combination with the L74V mutation encoding didanosine (ddI) resistance and the Q151M multinucleoside resistance mutation. To our knowledge, a total of 6 instances of Δ70 have been reported,16-18 but the virologic characteristics of the Δ70 mutants were not described. We therefore studied the drug susceptibility and fitness of viruses carrying this deletion and associated RT mutations.
METHODS
Cells and Reagents
MT-2 cells were grown in R-10 medium (RPMI 1640 [Cellgro, Herndon, VA] supplemented with 10% fetal calf serum, l-glutamine [2 mM], penicillin [100 U/mL], and streptomycin [100 μg/mL]). HeLa CD4-long terminal repeat (LTR)/β-gal cells, kindly provided by M. Emerman through the AIDS Research and Reference Reagent Program, were propagated in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal calf serum, hygromycin B (0.2 mg/mL), and geneticin (0.2 mg/mL). ABC was provided by GlaxoSmithKline, (Research Triangle Park, NC), ZDV was purchased from Sigma (St. Louis, MO), and 9-[2-(phosphonomethoxy)propyl]adenine [PMPA; TFV] was obtained from Gilead Sciences (Foster City, CA). Human subject aspects of this study were conducted in accordance with relevant federal guidelines. The study was approved by the appropriate institutional review boards, and signed informed consent was obtained.
Cloning and Sequencing of HIV-1 Protease and Reverse Transcriptase
Viral RNA was extracted from patient plasma using the QIAamp Viral RNA Mini kit (Qiagen, Valencia, CA). Population sequencing of HIV-1 protease gene (PR) and the first 242 codons of RT was performed using the TRUGENE HIV-1 Genotyping Kit (Bayer HealthCare, Berkeley, CA). Subsequently, the 1.3-kb pol fragment generated by the TRUGENE reagents was ligated into pGEM-T Easy vector (Promega, Madison, WI) to yield plasmid pGEM-PR-5′RT. In addition, a 1967-base pair (bp) fragment of pol containing the 3′-end of the RT and the 5′-end of integrase (IN) was amplified using the SuperScript 1-step RT polymerase chain reaction (PCR) system (Invitrogen Life Technologies, Carlsbad, CA) with forward primer 5′-CCATACAATACTCCAATATTTGC-3′ (corresponding to nucleotides 2712-2736 of HIV-1NL4-3) and reverse primer 5′-TACTCCTTGACTTTGGGGATTGTAGGG-3′ (corresponding to nucleotides 4652-4679), purified, and ligated into the pGEM-T Easy vector to generate pGEM-3′RT.
To construct a full-length molecular clone of HIV-1 PR and RT from this patient, a 756-bp fragment (fragment “A”) of pGEM-PR-5′RT carrying PR and the first 235 nucleotides of RT was amplified using forward primer 5′p2022 (5′- GGCTGTTGGAAATGCGGAAAGGAAGGACACC-3′) and reverse primer 3′pXbaI2778 (5′-GTTCTCTAGAAATCTACTACTTTCCTCCAAGTACCGTC-3′), into which an XbaI site was introduced (underscored nucleotides). A 1645-bp fragment (fragment “B”) containing nucleotides 222 through 1680 of RT and the 5′-end of IN was amplified from pGEM-3′RT using forward primer 5′pXbaI2778 (5′-GATTTCTAGAGAACTTAATAAGAGAACTCAAGAC-3′), into which an XbaI site was also introduced, and reverse primer 3′p4423 (5′-CAATCTAGTTGCCATATTCCTGG-3′). The purified PCR products were ligated into the pGEM-T Easy vector to generate plasmids pGEM-PR-A and pGEM-B, respectively. Digestion of pGEM-PR-A with XmnI and XbaI yielded a 1762-bp fragment carrying pGEM-T sequences, PR, and the 5′-end of RT; digestion of pGEM-B with the same enzymes yielded a 3654-bp fragment carrying the 3′-end of RT and the 5′-end of IN plus the remainder of pGEM-T. Ligation of these 2 fragments regenerated pGEM-T plus the PR and full-length RT sequences. The XbaI restriction site at the junction of 2 RT segments was deleted by PCR-based site-directed mutagenesis using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), and the final plasmid clones were sequenced using an ABI 377 DNA sequencer (Applied Biosystems, Foster City, CA). The data were imported into DNASTAR (Madison, WI) for further analysis, and the results were compared with the original sequence obtained by bulk sequencing. The consensus RT sequence of the patient virus was deposited in the GenBank data bank under accession number DQ394304.
Site-Directed Mutagenesis
The Δ70, L74V, and Q151M mutations were introduced individually into Hxb2 RT in the pGEM-T Easy vector using the QuickChange site-directed mutagenesis kit (Stratagene). Similarly, Δ70 in RT from the patient isolate was repaired by introduction of the wild-type K70. The presence of the intended alterations and absence of adventitious mutations were confirmed by DNA sequencing.
Generation of Virus Stocks
Infectious recombinant viruses carrying the desired mutations in RT were generated by cotransfecting the PR-RT genes of interest into MT-2 cells together with pHIVΔPR-RT.BstEII, a full-length molecular clone of HIV-1NL4-3 from which PR and the first 306 codons of RT were deleted, as described.19 Initial attempts to generate recombinant viruses using the patient-derived PR-RT clone were unsuccessful, possibly because of the presence of multiple mutations in PR. Therefore, we subcloned the RT-coding sequence from pGEM-PRΔ70RT into pGEM-PR-RT, which carries the Hxb2-PR-RT, to generate pGEM-[PRHxb2-RTPRTΔ70]. Cotransfection of MT-2 cells with the resulting PRHxb2-RTPRTΔ70 chimera plus pHIVΔPR-RT.BstEII produced infectious recombinant viruses. Proviral DNA of infected cells obtained at the end of virus culture was analyzed by automated DNA sequencing to verify the presence of the correct alleles at codons 70, 74, and 151 and to ensure the absence of adventitious mutations. Sequence analysis also verified that recombination between the inserted RT sequences and the NL4-3 sequences of pHIVΔPR-RT.BstEII occurred in the region corresponding to RT codons 532 through 560. No significant differences were found between the sequence of recombinant virus carrying the original patient-derived Δ70RT and the repaired codon 70. Drug susceptibility of recombinant viruses was determined using the PhenoSense HIV Assay (Monogram Biosciences, South San Francisco, CA) and reported as 50% inhibitory concentration (IC50) and as fold change compared with wild type. Because the average coefficient of variance of this clinically validated assay is <2-fold, differences >2-fold were considered significant.20
Growth Competition Assays
Recombinant viruses of interest were mixed together at ratios of 80:20, 50:50, and 20:80, respectively, and inoculated onto 1.5 × 106 MT-2 cells suspended in 300 μL of R-10 medium to yield a multiplicity of infection (m.o.i.) of 0.001. After incubation at 37°C for 2 hours, cells were washed twice with phosphate-buffered saline (PBS), resuspended in 10 mL of R-10 medium at a concentration of 0.15 × 106cells/mL in 25-cm2 tissue culture flasks, and reincubated (day 0). The relative proportions of the 2 competing variants were estimated by quantifying virus-specific sequences in culture supernatants on days 4, 7, 10, and 14 by quantitative real-time RT-PCR with an ABI PRISM 7000 Sequence Detection System (Applied Biosystems) using primers and probes specific for the wild type or deleted sequence at RT codon 70, respectively.
Primers and TaqMan minor groove-binding (MGB) probes specific for RT that was wild type or deleted at codon 70 spanned the region from nucleotides 2698 or 2701 through 2806 (Hxb2 sequence [available at: http://hiv-web.lanl.gov]). All MGB probes for the wild type (K70) were labeled with the reporter dye FAM (6-carboxyfluorescein) at their 5′ ends, whereas MGB probes for the Δ70 variants were labeled with VIC (6-carboxyrhodamine 6G, succinimidyl ester) at their 5′ ends; all probes included a nonfluorescent quencher at their 3′ ends (Table 1). Thermal cycling parameters were as follows: 50°C for 30 minutes and then 95°C for 10 minutes and 40 cycles (95°C for 15 seconds, 60°C for 1 min). To verify that the proportion of wild-type and Δ70 RT could be determined accurately, purified DNA from pHIVK70-RTHxb2 and pHIVΔ70-RTHxb2 as well as from pHIVK70-PRT and pHIV-Δ70-PRT was combined at defined ratios of 0:100, 1:99, 5:95, 20:80, 50:50, 80:20, and 90:10. The samples were analyzed by real-time PCR, and the proportions of wild-type and Δ70 RT were determined by quantifying the number of RNA molecules of each competing virus. Standard curves were obtained using serial dilutions of plasmid DNA carrying wild-type and Δ70 RT sequences. The measured and nominal proportions were highly correlated (R2 > 0.99). Quantitative real-time RT-PCR was performed in triplicate on each sample. Data shown represent the means (±SD) of 3 independent growth competition experiments.
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TABLE 1: Primers and Probes for Detection of Wild-Type and Codon 70-Deleted RT Sequences*
Estimation of Viral Fitness
The relative fitness (w) of each virus was obtained from the average of the results of 3 independent growth competition assays (inoculated at ratios of 80:20, 50:50, and 20:80).21 For each pair of recombinant viruses tested, the final ratio of the 2 viruses was determined by quantitative real-time PCR at day 10 as described previously. The fitness difference (WD) was estimated by the ratio of the relative fitness values (WD = WM/WL), where WM is the more fit virus and WL is the less fit virus in the growth competition assays.21
Infectivity Assays and Fitness Profiles
Viral infectivity was determined on MAGI cells, obtained from the National Institutes of Health AIDS Research and Reference Reagent Program, by a modification of the method of Mammano et al,22 as described.19 Infectivity was determined from mean β-galactosidase production in triplicate wells and expressed as a percentage of wild-type infectivity. To determine the replicative advantage of mutants carrying Δ70 as a function of drug (ABC, TFV, and ZDV) concentration, we calculated the infectivity ratios (in terms of relative β-galactosidase production) of the following virus pairs at drug concentrations ranging from 0 to 10 μM: Δ70 to wild type, Δ70/L74V to L74V, Δ70/Q151M to Q151M, and patient RT (Δ70-PRT) to the repaired deletion (K70-PRT). At least 3 independent experiments were performed for each mutant; the values plotted represent the mean infectivity ratios ±95% confidence intervals (CIs) obtained at each drug concentration.
RESULTS
Identification of Deletion at Reverse Transcriptase Codon 70
An HIV-1 variant with a deletion at RT codon 70 (Δ70-PRT) was identified in a sample from a patient evaluated for entry into AIDS Clinical Trials Group protocol A5118, a pilot study to evaluate the safety and antiviral activity of amdoxovir (diaminopurine dioxolane [DAPD]) in antiretroviral-experienced patients with virologic failure.23 The patient had received extensive prior treatment with a variety of antiretroviral drugs over an 8-year period, including ZDV, ABC, TFV, ddI, lamivudine (3TC), stavudine (d4T), zalcitabine, nevirapine, efavirenz, indinavir, ritonavir, amprenavir, and ritonavir-boosted lopinavir alone or in various combinations. The regimen at the time of evaluation included ABC, TFV, and ritonavir-boosted lopinavir. The plasma HIV-1 RNA level was 4.01 log10 copies/mL (HIV-1 Monitor Assay; Roche Molecular Systems, Pleasanton, CA), and the CD4 count was 105 cells/μL. A genotypic resistance test revealed an in-frame 3-nucleotide deletion at codon 70. Additional RT substitutions at positions possibly associated with drug resistance included S68G, L74V, A98S, Y115F, Q151M, Y181C, G190A, Q207A, and R211K. Deletion of codon 70 was confirmed by sequence analysis of full-length molecularly cloned RT from plasma virus. Phenotypic resistance testing using the Phenosense HIV Drug Resistance Assay (Monogram Biosciences) revealed resistance to all NRTIs, with the exception of TFV (Table 2). High-level resistance to all nonnucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors was also noted (data not shown).
TABLE 2: Susceptibility of Recombinant HIV-1 Isolates to NRTIs
Drug Susceptibility of Site-Directed Mutants
Table 2 shows the NRTI susceptibility of HIV-1 recombinants expressing control and mutant RTs. Presence of the Δ70 mutation increased the IC50 for 3TC and emtricitabine (FTC) by 2.8- and 4.4-fold, respectively, compared with the isogenic wild-type control but had minimal effect on susceptibility to ABC, ddI, d4T, or TFV. By contrast, the IC50 for ZDV decreased approximately 5-fold. Introduction of Δ70 into a Q151M mutant resulted in 2.6- and 4.1-fold increases in IC50 for 3TC and FTC, respectively, as compared to the Q151M mutant with a wild-type codon 70 but had little effect on susceptibility to other NRTIs. By contrast, deletion of codon 70 in an L74V background resulted in a 2.8- to 4.6-fold increase in IC50 for ABC, ddI, FTC, d4T, and ZDV but had less than a 2-fold effect on susceptibility to 3TC and TFV. Recombinant virus expressing the patient-derived RT (Δ70-PRT) showed the highest IC50s to all NRTIs. Repair of the deletion by reintroduction of the wild-type lysine at position 70 (K70-PRT) reduced IC50s for the NRTIs by 1.7- to 3.1-fold (see Table 2).
Effect of Deletion at Codon 70 on Fitness of Wild-Type and Nucleoside Reverse Transcriptase Inhibitor-Resistant Mutants of HIV-1
The relative replicative fitness of recombinant viruses carrying various combinations of RT mutations was tested in a series of pairwise growth competitions assays in the absence of drug. The recombinant carrying the patient-derived RT (Δ70-PRT) was substantially more fit than a recombinant in which the deletion had been repaired (K70-PRT) (Fig. 1A). Deletion of codon 70 also resulted in increased fitness of the wild-type, L74V, and Q151M mutants (see Figs. 1B-D). Relative fitness values for both recombinant viruses in each growth competition experiment were calculated as described21 and used to determine the fitness difference (WD) for each pair of recombinant viruses tested (Table 3). There was a 10.7-fold fitness difference between the Δ70-PRT and K70-PRT recombinants and a 5.3-fold fitness difference between the Δ70 and wild-type HXb2 recombinants. Smaller fold differences in fitness were observed for the other pairs tested (see Table 3).
TABLE 3: Relative Fitness Values of HIV-1 RT Codon 70 Deletion Mutants From Growth Competition Assays
FIGURE 1: Growth competition assays between recombinant viruses carrying Δ70-PRT (closed circles) and K70-PRT (open circles) (A), Δ70/L74V (closed circles) and L74V (open circles) (B), Δ70/Q151M (closed circles) and Q151M (open circles) (C), and Δ70 (closed circles) and wild type (WT; open circles) (D) in the absence of drug. The recombinants were inoculated at a ratio of 50:50; the day 0 values show the actual measured amount of each viral species in the inoculum. The data shown are means ± SD of 3 independent experiments.
Effect of Deletion at Codon 70 in Hxb2 and Patient-Derived Reverse Transcriptase on Relative Viral Infectivity
To determine the relative replication advantage of wild-type and codon 70-deleted viruses in the presence of drug, we determined the fitness profiles for each of the mutants over a range of ABC and TFV concentrations. A recombinant virus expressing the patient-derived RT (Δ70-PRT) showed an increasing advantage over virus with the repaired deletion (K70-PRT) in the presence of ABC, peaking at 5 μM (Fig. 2). Similarly, the Δ70/L74V Hxb2 mutant showed a marked advantage at ABC concentrations ranging from 1 to 5 μM. Only a modest replication advantage was observed for the Δ70 mutant compared with wild type or between the Δ70/Q151M and 151M mutants. By contrast, the Δ70 mutation did not confer a substantial replication advantage in the presence of TFV (see Fig. 2B).
FIGURE 2: Fitness profiles of HIV-1 recombinants carrying RT mutations. The infectivity ratios of viruses that were wild type (WT) or deleted at codon 70 in different genetic backgrounds (measured as relative β-galactosidase activity) were determined in the presence of 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, and 10.0 μM of ABC (A) or TDF (B). Ratios shown include Δ70-PRT to K70-PRT (•), Δ70 to wild type (WT; ○), Δ70/L74V to L74V (▾), and Δ70/Q151M to Q151M (▿). The profiles shown correspond to the average values ±95% CIs of at least 3 independent experiments. The dashed line indicates a ratio of 1.0.
DISCUSSION
Although insertions and deletions are relatively common in the HIV-1 envelope, they are found less often in the enzymes necessary for viral replication. In one report, deletions occurred in <0.1% of RT gene sequences analyzed from nearly 8400 patients;14 those deletions all mapped to codons 67 through 69. A previous report of a deletion at RT codon 70 noted an association with the Q151M mutation but provided no phenotypic data about that virus.17 A second report of a codon 70 deletion again noted an association with Q151M and found resistance to all NRTIs by drug susceptibility testing.16 A third report noted the deletion of codon 70 in 3 (0.06%) of 5157 samples genotyped from June 1998 to December 1999.18 Virus from 1 of the 3 samples also carried the Q151M mutation, but no data on phenotype or treatment history are provided. We identified a deletion at RT codon 70 in HIV-1 from a patient with a history of extensive NRTI treatment. We performed a systematic analysis of the effect of the codon 70 deletion on the susceptibility and fitness of HIV-1 recombinants carrying patient-derived RT and in site-directed mutants carrying various combinations of antiretroviral drug resistance mutations identified in the patient virus.
As in most of the previously reported cases, the Δ70 mutant that we identified carried numerous other RT inhibitor resistance mutations, including the multinucleoside resistance mutation Q151M. Likewise, deletions at codon 67, which are more common than codon 70 deletions, usually occur together with Q151M.12,14,16 The Q151M mutation typically is associated with mutations at codons 62, 75, 77 in the β3-β4 region and at codon 116.24,25 The presence of these additional mutations affects the magnitude and breadth of drug resistance and improves the replicative fitness of Q151M mutants.26
Given the association of deletions at codons 67 and 70 with the Q151M mutation, it is possible that these deletions subsume some of the compensatory role of secondary mutations in the β3-β4 fingers subdomain. To test this hypothesis, we constructed a series of recombinant viruses by site-directed mutagenesis to determine the effect of Δ70 on the fitness and relative infectivity of mutants carrying 151M in an Hxb2 backbone. Although the Δ70/Q151M mutant showed 2- to 4-fold higher resistance to 3TC and FTC, respectively, susceptibility to other NRTIs and to TFV was unchanged. In addition, the codon 70 deletion had only a small effect on the relative fitness of the Q151M mutant in the absence of drug (1.4-fold) and produced minimal increases in relative infectivity in the presence of ABC or TDF. Thus, we are unable to explain the association between this deletion and Q151M on the basis of our results.
The codon 70 deletion did have significant effects on NRTI resistance in viruses expressing the patient-derived RT. Susceptibility to ABC, 3TC, FTC, d4T, and ZDV was reduced an additional 2- to 3-fold in recombinants expressing RT derived from the patient's virus (Δ70-PRT) as compared to an isogenic recombinant virus carrying the wild-type amino acid at position 70 (K70-PRT). Similar effects were observed when Δ70 was combined with L74V in Hxb2 RT recombinants. It thus seems that the codon 70 deletion enhances NRTI resistance to purine and pyrimidine analogues and contributes to multinucleoside resistance in the appropriate viral genetic background.
Recombinant viruses that expressed the patient virus-derived RT showed substantially greater relative fitness (nearly 11-fold) than an isogenic recombinant with the repaired deletion (K70-PRT). The effect on relative infectivity was greatest in the presence of ABC. A similar effect of Δ70 on relative infectivity in the presence of ABC was observed in site-directed mutants of Hxb2 carrying a 74V mutation. It is noteworthy that 74V is selected by ABC in vitro and in vivo and confers 3- to 4-fold resistance to the drug.27,28 Curiously, site-directed mutants with a deletion of codon 70 in an otherwise wild-type Hxb2 RT showed more than 5-fold greater relative fitness compared with wild type. Because wild-type virus generally is assumed to be the most fit, this finding is surprising. If confirmed in other viral backbones, this observation would suggest that other evolutionary pressures not present in vitro (eg, immune responses) might select against emergence of the codon 70 deletion under most circumstances.
The β3-β4 hairpin loop in the fingers subdomain of p66 is involved in the interaction between the template-primer and the incoming dNTP2,4 and affects positioning of the nucleic acid at the polymerase active site.15 Deletion of codon 67 in this region has local and long-range effects on RT enzyme structure. Local changes in the β3-β4 region are thought to alter the orientation of the primer-template complex, resulting in increased nucleotide selectivity.2 Structural changes in the fingers subdomain might trigger additional changes in the palm subdomain, particularly movement of helices C and E.14 Additional modeling studies are needed to determine whether deletion of the lysine at position 70 results in similar structural changes and whether those changes explain the observed phenotype of this mutant.
A limitation of this study is that we did not investigate the effect of each of the other resistance-associated mutations present in the original isolate (S68G, A98S, F115Y, Y181C, G190A, Q207A, and R211K) on the properties of the codon 70 deletion. It is possible that the presence of some or all of these mutations and other differences in the RT backbone might explain differences in the effect of the codon 70 deletion on the resistance and fitness profiles of the original isolate as compared to viral recombinants expressing Hxb2 RT with individual RT mutations. We chose to focus on the 74V and 151M mutations because of their significant role in NRTI resistance. The S68G mutation has been associated with the K65R mutation and was present together with a deletion at codon 69 but was not present in the RT of the other reported codon 70 deletions.16-18,29 The S68G mutation also occurs frequently in association with Q151M and improves the fitness of variants carrying a Q151L mutation, a likely intermediate in the acquisition of Q151M.30 The A98S, Y181C, and G190A mutations are all associated with NNRTI resistance but, likewise, have no specific association with mutations at codon 70. The F115Y mutation contributes to ABC resistance but has less impact on ABC susceptibility and response to treatment than the L74V mutation.27 Substitutions at codons 207 and 211, which may be present as polymorphisms, contribute modestly to ZDV resistance in the presence of TAMs but have not been reported to play a role in NRTI resistance when present together with the Q151M mutation.31
In summary, a codon 70 deletion was identified in HIV-1 RT from a patient with an extensive history of NRTI exposure. As is the case with 3 of 6 previously reported deletions at this position as well as the deletion at codon 67, this deletion occurred together with the Q151M multinucleoside resistance mutation. In the context of other NRTI and NNRTI resistance mutations present in the patient virus, the codon 70 deletion increased the level of NRTI resistance and relative viral fitness of the mutant virus. It should be interesting to compare the effects of Δ70 on biochemical and kinetic properties of RT with those of other insertion/deletion mutations in the β3-β4 fingers subdomain.
ACKNOWLEDGMENTS
The authors thank Janet Steele and Lindsay Ware for administrative assistance and Kelly Hartman for expert technical assistance.
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