Rapid, phenotypic HIV-1 drug sensitivity assay for proteaseand reverse transcriptase inhibitors
Hauke Walter a, Barbara Schmidt a, Klaus Korn a, Anne-Mieke Vandamme b,Thomas Harrer c, Klaus U8 berla a,d,*
a Institut fur Klinische und Molekulare Virologie, Uni6ersitat Erlangen-Nurnberg, Erlangen, Germanyb Rega Institute for Medical Research, Katholieke Uni6ersiteit Leu6en, Leu6en, Belgium
c Institut fur Klinische Immunologie und Rheumatologie, Medizinische Klinik III, Uni6ersitat Erlangen-Nurnberg, Erlangen, Germanyd Institut fur Virologie, Uni6ersitat Leipzig, Liebigstr. 24, D-04103 Leipzig, Germany
Received 13 November 1998; accepted 28 January 1999
Abstract
Background: Development of drug resistance is one of the major reasons for the failure of antiretroviral therapy ofHIV-1 infection. Knowing the drug sensitivity–resistance profile of viruses present in a patient prior to treatment orchange in treatment could help to optimize therapy.
Objective: Development of a rapid standardized phenotypic HIV-1 drug sensitivity assay for protease (PR) andreverse transcriptase (RT) inhibitors.
Design: The PR gene (codons 1–99) and the 5% part of the RT gene (codons 1–300) of HIV-1 is amplified from theplasma of infected individuals by RT-PCR and ligated into a proviral clone of HIV-1 containing a deletion of the PRgene and the 5% part of the RT gene. Bacteria are transformed with the ligation product and plasmid DNA is preparedfrom a library of transformed bacteria.The plasmid DNA is transfected into 293 T cells and recombinant virus isharvested from the supernatant of the transfected cells 2 days after transfection. The sensitivity of the recombinantvirus is determined with the help of a sensitive indicator cell line.
Results: Recombinant viruses were generated with high efficiency. Determination of the drug sensitivity of therecombinant viruses with an indicator cell line was highly reproducible. The recombinant viruses accurately reflectedthe sensitivity–resistance profile of the parental viruses. The phenotypic drug sensitivity determined by this assaycorrelated well with the treatment history of patients.
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1. Introduction
Highly active antiretroviral therapy (HAART)can substantially suppress viral load in manyHIV-infected individuals. Suboptimal suppressionof virus replication can lead to the development ofdrug-resistant viruses and to failure of combina-tion therapy. Therefore, it seems to be importantto make sure that in each patient all compoundsof a treatment regimen are active. Cross-resistanceto previously used drugs or transmission of drug-resistant viruses can reduce the efficacy of com-pounds in pre-treated or naive HIV-1-infectedindividuals, respectively. HIV-1 drug sensitivityassays might help to avoid the inclusion of inac-tive compounds in the combination therapy. Thedrug sensitivity of HIV-1 can either be determinedgenotypically or phenotypically (for review seePerrin and Telenti, 1998). Genotypic drug sensi-tivity assays are based on the determination of thenucleotide sequence of the patient’s virus. Certainpoint mutations in the RT gene and PR geneconfer drug resistance to RT and PR inhibitors,respectively (summarized by Schinazi et al., 1997).These assays only allow the detection of drugresistance, if resistance is due to mutations thathave been identified previously. Mutations confer-ring resistance to a particular drug can also de-pend on the kind and timing of co-administeredantiretroviral drugs (Balzarini et al., 1993). HIV-1sequence diversity and the reversal of drug resis-tance by second site mutations (Tisdale et al.,1993) can render interpretation of the sequencedata difficult. In contrast to genotypic drug sensi-tivity assays, phenotypic assays are more labourintensive. Originally, they were based on measur-ing the replication of virus isolates from patientsin the presence or absence of drugs. Disadvan-tages are the selection of particular virus strainsduring virus isolation and difficulties in standard-ization. The recombinant virus assay described byKellam and Larder (1994), overcomes some ofthese limitations. The RT gene of HIV-1 from theplasma of infected individuals is amplified byRT-PCR. Co-transfection of the PCR productwith a laboratory adapted molecular clone ofHIV-1 containing a deletion in the RT gene into aCD4+ T cell line leads to outgrowth of recombi-
nants containing the RT of the patients’ virus inthe genome of the laboratory adapted virus. Thesensitivity of the recombinants is then determinedin a standardized cell killing assay. A similarapproach was subsequently used for PR inhibitors(Maschera et al., 1995; Boucher et al., 1996) andfor both PR and RT inhibitors simultaneously(Hertogs et al., 1998). However, these assays havesome disadvantages. Due to the low frequency ofrecombination, culture periods of about 10 daysare required for the outgrowth of the recombinantvirus. During this time selection against replica-tion-impaired, drug-resistant viruses (Zhang et al.,1997; Zennou et al., 1998); might occur. In addi-tion, it is very time consuming to control howmany independent recombinants have been gener-ated. This is important to ensure that the recom-binants are representative of the patient’s virusand not just represent one clone. Concerning theread-out system, a disadvantage of the cell killingassay is the strict dependance on the dose ofinfection, requiring the determination of the me-dian tissue culture infectious dose (TCID50) of therecombinants prior to the drug sensitivity assay.We therefore introduced a number of substantialmodifications which made the generation of therecombinant viruses more efficient and improvedthe read-out for the drug sensitivity assay.
2. Material and methods
2.1. Construction of NL4-3DPRT5 andrecombinant 6irus DNA
The 3% part of the RT gene was amplified withthe primers HDPRT5 (TGCAGGGCCCGG-GTACGTATCTAGAACTGGCAGAAAACAG-GGA) and H4570a (ATGTATTGTTTTTAC-TGGCCAT) from pNL4-3 (Genbank entryM19921). Underlined sequences mark relevant re-striction sites. The PCR product was digestedwith ApaI and MscI and ligated into the ApaI-MscI site of pNL4-3. The resulting plasmid, des-ignated pNL4-3DPRT5, contains a deletion fromnucleotide 2009 to 3446 of pNL4-3. The deletionis flanked 5% by the ApaI restriction site, which ispresent in pNL4-3, and 3% by a XbaI site, whichwas created by mutating the first nucleotide of the
H. Walter et al. / Journal of Clinical Virology 13 (1999) 71–80 73
six base pair recognition site of XbaI. Since themutated nucleotide is cleaved off by Xba1 diges-tion, this point mutation will not be present in therecombinant virus (REC). Using the primersH2001s (TGCAGGGCCCCTAGRAAAARGG-GCTGTT, with R being A or G) and H3454a(AGTGCTAGCTCTGCTTCTTYTGTTAGTG-GTA, with Y being C or T) a fragment compris-ing the region deleted in pNL4-3DPRT5 (PRT5)was amplified by RT-PCR from virus containingsamples. Viral RNA was extracted by the QI-Aamp Viral RNA Kit (Qiagen) and reverse-tran-scribed using Superscript (Gibco BRL).Amplification was performed with the ExpandHigh Fidelity PCR system (BoehringerMannheim) for 30 cycles at 95°C for 20 s, 60°Cfor 30 s, and 72°C for 2 min. During the last 20cycles, the extension time was prolonged by 20 sper cycle. In case of plasma samples, a hemi-nested PCR was performed with primer pairsH2001s/H3532a (TTCTGCTATTAAGTCTTTT-GATGGGTCA) and H2001s/H3454a under theconditions described above. The PCR productwas purified using Gene-Clean (Bio 101) and di-gested with ApaI and NheI. After purificationwith Gene-Clean, the PCR product was ligatedinto ApaI and XbaI digested pNL4-3DPRT5. Ul-tracompetent XL2-Blue cells (Stratagene) weretransformed as described by the manufacturer.Plasmid DNA was prepared from a pool of trans-formed bacteria or single clones using the PlasmidMini Kit (Qiagen).
2.2. Generation of recombinant 6irus stocks
Plasmid DNA (2.5 mg) containing the recombi-nant virus genome or DNA from the ligationreaction was co-transfected with 2.5 mg calf thy-mus carrier DNA (Boehringer Mannheim) into293 T cells using the Superfect transfection kit(Qiagen) as described by the manufacturer. Twodays after transfection, the supernatant of thetransfected cells was cleared by centrifugation at1200 rpm for 10 min and stored in aliquots at−80°C. The TCID50 of the supernatant was de-termined on CEMx174-SIV-SEAP cells (Means etal., 1997) as described (Johnson and Byington,
1990). To determine the complexity of the viruspool, the number of virus producing cells wasdetermined by limiting dilution co-cultures of 293T cells harvested 1 day after transfection withCEMx174-SIV-SEAP.
2.3. Measurement of drug sensiti6ity
To determine virus replication, CEMx174-SIV-SEAP cells (25 000/well) were infected in tripli-cates in 96 well plates in a final volume of 200 ml.If these cells are infected with HIV-1, the viral Tatprotein will transactivate the LTR leading to astrong increase in SEAP activity of the culturesupernatant. Since the titer and the replicationcapacity of the recombinant virus stocks werevariable, the amount of virus present in each virusstock was estimated by infecting CEMx174-SIV-SEAP cells with 1, 5, 10, or 20 ml virus containingsupernatant. The minimal volume of the virusstock resulting in at least 10 000 relative lightunits (RLU) in 20 ml supernatant of CEMx174-SIV-SEAP cells 3 days after infection with thevirus stock was determined as described (Means etal., 1997) using the Phospha-light-kit (Tropix,Bedford, MA, USA). If 20 ml virus containingsupernatant was not sufficient to result in 10 000RLUs, CEMx174 cells were infected with thevirus stock derived from 293 T cells and cultivatedfor a few days to prepare a higher titered virusstock. To determine the actual sensitivity to RTinhibitors, CEMx174-SIV-SEAP cells were in-fected with the 10 000 RLU dose of the recombi-nant virus stock in the presence of 0, 0.01, 0.1, 1,10, and 100 mM of drug and the SEAP activitywas determined 3 days later. For PR inhibitors,the inoculum was reduced by a factor of five andthe SEAP activity was determined 4 days(Saquinavir, Indinavir) or 5 days (Nelfinavir, Ri-tonavir) after infection. For drugs to which onlylow level resistance was observed (Zalcitabine,Didanosine, Stavudine, Saquinavir) intermediatedrug concentrations of 0.03, 0.3, 3 and 30 mMwere also used. As a control, recombinant NL4-3was included in every assay. Abacavir, Zi-dovudine, and Lamivudine were kindly providedby Glaxo Wellcome; Zalcitabine and Saquinavirby Hoffmann la Roche; and Didanosine and
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Stavudine by Bristol-Myers Squibb. Nevirapinewas obtained from Boehringer Ingelheim;Delavirdine from Pharmacia & Upjohn; Indinavirfrom Merck Sharp & Dohme; Ritonavir fromAbbott; and Nelfinavir from AgouronPharmaceuticals.
3. Results
A schematic representation of the assay is givenin Fig. 1. The PR gene (codons 1–99) and the 5%part of the RT gene (codons 1–300) were deletedfrom the proviral DNA of the HIV-1 molecularclone NL4-3. The first 300 amino acids of RTcontain all known drug resistance mutations withthe exception of a mutation at amino acid 333,which was recently found to be involved in resis-tance to Zidovudine in the presence of aLamivudine associated resistance mutation(Kemp et al., 1998). In addition, the deletioncomprises the PR cleavage site between NC/p1and p1/p6, which is sometimes mutated in virusesresistant to PR inhibitors (Zhang et al., 1997;Mammano et al., 1998; Zennou et al., 1998). Thedeletion was introduced in a way that two uniquerestriction sites were generated. Using nested RT-PCR, the PR gene and the relevant part of the RTgene is amplified as one fragment (PRT5) fromthe plasma of HIV-infected patients. The PCR
fragment and the deletion mutant of NL4-3 areligated and transformed into E. coli. PlasmidDNA is prepared from a pool of transformed E.coli clones. The plasmid DNA is transfected into293 T cells. The supernatant of the transfectedcells containing the recombinant viruses is har-vested 2 days after transfection. A sensitive indi-cator cell line (CEMx174-SIV-SEAP, Means etal., 1997) is infected with these supernatants in thepresence of different amounts of the drugs to betested and virus replication is monitored as afunction of drug concentration. The indicatorcells contain the SEAP gene under the control ofthe simian immunodeficiency virus LTR (Meanset al., 1997). If these cells are infected by HIV-1,the viral Tat protein will transactivate the LTRleading to a strong increase in SEAP activity inthe culture supernatant, which can be monitoredeasily.
To determine whether the recombinant viruswould have a similar sensitivity–resistance profileas the virus used as template for the PCR reac-tion, the PRT5 region of two molecular clones ofHIV-1 with defined drug resistance mutations inthe RT (RTMDR1, Larder et al., 1993) or PR(designated PRMDR1; originally described byCondra et al., 1995) region was amplified andligated with NL4-3DPRT5.
Recombinant virus stocks (REC-PR-MDR1,REC-RT-MDR1) were generated by transfectionof the ligation reaction into 293 T cells and prop-agating the recombinant viruses on a CD4
+ cellline. The sensitivity of the recombinant virusesand the parental viruses to RT inhibitors or PRinhibitors was determined by infecting the indica-tor cells in the presence of different concentra-tions of these drugs (Table 1). Both recombinantviruses had similar sensitivity–resistance profilesas the respective parental viruses, demonstratingthat the recombinant viruses accurately reflect thesensitivity of the parental virus to PR and RTinhibitors. The sensitivity of a pair of primaryisolates that were recovered before and after Ri-tonavir treatment (Schmit et al., 1996) was alsodetermined. The recombinant virus of the post-treatment isolate was highly resistant to Ritonavirand resistance to other PR inhibitors was alsoobserved (Table 2). The degree of resistance of the
Fig. 1. Overview of the drug sensitivity assay. RT5: 5% part ofthe RT gene; RT3: 3% part of the RT gene; PRT5: PCRfragment spanning PR and RT5.
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Table 1Comparison of drug sensitivity of parental and recombinant virus
a 50% inhibitory concentration.b HIV-1 clone resistant to multiple RT inhibitors (Larder et al., 1993).c HIV-1 clone resistant to multiple PR inhibitors (Condra et al., 1995).d In repeat experiments the IC50 of Zidovudine for PR-MDR1 was 0.077 and 0.104 mM.
post-treatment recombinant virus was similar topreviously published data for the post-treatmentisolate (Schmit et al., 1996) further supporting theaccuracy of the drug sensitivity assay.
To determine the sensitivity of patients’ viruses,a number of modifications were introduced whichmade the whole assay more sensitive, faster, andless labour intensive. To increase the sensitivity ofthe PCR reaction a hemi-nested PCR was per-formed, which allowed the isolation of the PRT5
fragments from 86% (n=97) of patients with viralloads higher than 500 copies viral RNA/mlplasma (branched DNA assay, Chiron). ThePRT5 fragment could also be amplified from sixout of 22 (27%) patients with viral loads lowerthan 500 copies/ml plasma. The failure to isolate aPCR product from some patients with viral loadsabove 500 copies/ml plasma might therefore bedue to the presence of different viral strains indifferent patients.
Attempts to analyze the drug sensitivity of anumber of patient samples revealed that directtransfection of the ligation reaction into 293 Tcells gave quite variable titers of the recombinantviruses, which often required the amplification ofthe recombinant viruses prior to the drug sensitiv-ity test (data not shown). To increase the viraltiters in the supernatant of the transfected 293 Tcells, E. coli were transformed with the ligationproduct of the PRT5 fragment from a multidrug-resistant patient with the pNL4-3DPRT5. Morethan 1000 ampicillin-resistant colonies were ob-tained per ml ligation reaction. Restriction analy-sis of plasmid DNA isolated from a pool oftransformed colonies revealed that more than 50%of the colonies contained a properely ligated NL4-3DPRT5 with a PRT5 insert. This was confirmed,
Table 2Phenotypic drug sensitivity of a pair of recombinant virusesderived from patient isolates before and after Ritonavir treat-ment
a 50% inhibitory concentration.b Three recombinant virus stocks were prepared from the same plasma sample. Drug sensitivity was determined in independent
assays three times for one recombinant virus stock (1a–c) and once for the other two virus stocks (2 and 3). N.A.: not applicable.
by restriction analyses of plasmid DNA preparedfrom ten randomly selected individual clones oftransformed bacteria. Eight of these ten clonescontained a properly ligated recombinant virus(data not shown). Plasmid DNA from the pool oftransformed clones was then transfected into 293T cells. The virus titer in the supernatant of thetransfected 293 T cells was in the range of 105
median tissue culture infectious doses TCID50/ml,which was high enough for directly performingthe drug sensitivity assay. To ensure that thecomplexity of the plasmid pool would be main-tained in the pool of the recombinant viruses, theminimal number of independently transfected cellswas determined by a limiting dilution co-cultureof the transfected 293 T cells with the indicatorcell line 1 day after transfection. The TCID50/cellvaried from 0.1 to 0.6, corresponding to at least105 independently transfected cells.
Using these experimental conditions, the repro-ducibility of the drug sensitivity assay was testedwith a recombinant virus derived from the plasmasample of a multidrug-resistant patient. The IC50
values and fold resistance values of three indepen-dent assays performed with the same virus stockon 3 different days differed by less than a factorof three for most drugs tested (Table 3, stock1a–c). A larger variation was only observed forStavudine which differed by a factor of 4.8. To
further test the interassay variability, two newrecombinant virus stocks were prepared from thesame plasma sample 4 and 6 months later andtested in independent assays (Table 3, stock 2 and3). The IC50 values and fold resistance values ofthese new stocks were in same range as observedfor the first stock. In one of the five stavudinesensitivity tests the IC50 value and the fold resis-tance value was 6.4 fold lower than the mean ofthe determinations. For all other tested drugs,single values of all five determinations varied byless than a factor of three from the mean of thesefive determinations (Table 3), indicating that phe-notypic drug sensitivity can be reproduciblydetermined.
A comparison of the results of the phenotypicdrug sensitivity assay with the treatment historyof three patients and the nucleotide sequences ofthe viruses present in the plasma of these patientsis shown in Table 4. In the untreated patient(patient 1), full sensitivity to all drugs tested isrevealed by phenotypic and genotypic drug sensi-tivity analyses.
Patient 2 was treated with Didanosine,Stavudine, Lamivudine, Loviride and Saquinavirat the time of analysis. A viral load of 53 000HIV-1 RNA copies/ml plasma indicated ineffi-cient therapy. Phenotypic drug resistance was de-tected against all used nucleoside analogue RT
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Table 4Comparison of phenotypic drug sensitivity with genotype and treatment history
63P, 71V, 82A51 Wt 51Nelfinavir 36I no 19 63P, 77I 6/97-51Ritonavir Wt 51 20R, 36I no 96 54V, 71V, 82A No
a Ratio of IC of patient-derived recombinant virus and NL4-3.b Codons associated with drug resistance were taken from Schinazi et al., (1997). Wt: wild type codon at all positions associated with resistance to indicated drug;
numbers indicate the codon mutated in RT (for RT inhibitors) or PR (for PR inhibitors) and are followed by the one letter abbreviation of the deduced amino acidat this position.
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inhibitors and against Nevirapine (the sensitivityto Loviride was not determined phenotypically,but cross-resistance to Nevirapine is frequentlyobserved). Sequencing of the RT gene of thepatient’s virus also revealed drug resistance muta-tions against all used RT inhibitors except forStavudine. Phenotypic drug sensitivity assay indi-cated full sensitivity to all PR inhibitors testedincluding Nelfinavir. Genotypic analysis revealedthree drug resistance associated mutations in thePR gene. After Saquinavir was replaced by Nelfi-navir, the viral load in this patient dropped to 920copies/ml plasma within 2 months. The lack oftherapy response to Saquinavir in the absence ofresistance may be due to the low bioavailability ofthe Saquinavir formulation used at that time (IN-VIRASE). However, even after change to Nelfi-navir the low viral load level could be maintainedfor only another 2 months, since only one of thefive compounds used was still active.
Patient 3 had also been administered varioustreatment regimens. He harboured viruses resis-tant to all nucleoside analogue RT inhibitors andPR inhibitors tested in phenotypic and genotypicdrug sensitivity assays. In agreement with theobserved multidrug resistance, changing the treat-ment regimen from Zidovudine, Lamivudine,Saquinavir, and Nelfinavir to Didanosine,Stavudine and Nelfinavir did not reduce the viralload levels by more than a factor of three. Fullsensitivity to non-nucleoside RT inhibitors, a classof compounds not used in this patient before,suggests that inclusion of these compounds intothe treatment regimen might have been beneficial.
4. Discussion
A number of modifications were introduced inthe recombinant virus assay for the determinationof HIV-1 drug sensitivity as described by Kellamand Larder (1994), which overcome some of itslimitations. By ligating the patient-derived PCRfragment with a deletion mutant of a molecularclone of HIV-1, a larger number of independentrecombinants can be obtained than by ho-mologous recombination. A plasmid library ofthese recombinant viruses was first amplified in
bacteria and plasmid DNA prepared from a poolof transformed bacteria was then transfected into293 T cells. A library of recombinant viruses washarvested from the supernatant of transfectedcells 2 days after transfection. Since no virusreplication can occur in 293 T cells due to the lackof the CD4 receptor, there should be no selectivepressure acting on the library of recombinantviruses. This might be beneficial since there isevidence that drug-resistant viruses emerging inpatients can have reduced replication properties(Zhang et al., 1997; Zennou et al., 1998). In caseof viruses resistant to PR inhibitors, mutationswere detected primarily in the C-terminal PRcleavage sites of gag, which counteract the replica-tion defect (Zhang et al., 1997; Mammano et al.,1998; Zennou et al., 1998). Since the patient-derived PRT5 fragment spans the PR cleavagesites between NC/p1 and p1/p6, most of the C-terminal mutations are also part of the recombi-nant virus. In contrast to the previously describedrecombinant virus assays, the complexity of theplasmid library can be easily determined by plat-ing out an aliquot of the transformed bacteria andcounting the number of transformed colonies.This ensures that the recombinant viruses trulyrepresent the viruses in the plasma sample. Theonly bottleneck in the complexity of the library ofthe recombinant virus seems to be the number oftemplates in the PCR reaction, particularly inpatients with low viral loads. To increase theefficiency of the PCR reaction, we only amplifiedthe 5% part of RT and not the entire RT gene sinceamplification of longer PCR products results in aloss of sensitivity. However, one has to bear inmind that there is one mutation outside the PRT5fragment which has recently been shown to beinvolved in resistance to Zidovudine in the pres-ence of a resistance mutation to Lamivudine(Kemp et al., 1998). Therefore, resistance to Zi-dovudine can not be excluded if the recombinantvirus is resistant to Lamivudine and sensitive toZidovudine. We also changed the read-out systemfor determination of the drug sensitivity. Due tothe higher sensitivity of the indicator cell lineused, results are obtained 3–5 days after infectionrather than 6 days after infection. In addition, thecell killing assays used previously have a narrow
H. Walter et al. / Journal of Clinical Virology 13 (1999) 71–80 79
linear range, requiring the careful titration of therecombinant virus stocks prior to the drug sensi-tivity assay, while in the assay described here a 3day infection period of the indicator cells with therecombinant virus stocks is used to determine theinfectious dose.
The assay was validated by a number of inde-pendent approaches. Using multidrug-resistantmolecular clones, the recombinant and theparental viruses were shown to have very similardrug sensitivity profiles. The level of resistance ofrecombinant viruses derived from a pair of patientisolates recovered before and after PR inhibitortreatment was comparable with previously pub-lished resistance levels (Schmit et al., 1996). Theassay was reproducible and IC50 values differedby less than a factor of two from the mean formost drugs tested. If required, occasional largervariations as observed for AZT (Table 1, PR-MDR1) or Stavudine (Table 3, 1b) can be over-come by including intermediate drugconcentrations in the drug sensitivity assay or byrepeat experiments.
The phenotypic drug sensitivity was in agree-ment with the reported treatment history. Pheno-typic and genotypic drug sensitivity assays gavesimilar results in most cases analyzed so far. How-ever, the genotype did not always correlate withphenotypic drug sensitivity, particularly in case ofPR inhibitors (data not shown). A comparison ofphenotypic drug sensitivity, genotype, and theoutcome of treatment in a larger number of pa-tients is needed, in order to determine the relativeimportance of phenotypic and genotypic drug sen-sitivity testing. It is important to note, that bothapproaches only assess the sensitivity of the dom-inant virus variant. Since drug-resistant virusespresent as a minority species might lead to rapidtherapy failure, the clinical impact of drug sensi-tivity assays remains to be determined. The phe-notypic drug sensitivity assay described is nowroutinely used to monitor drug sensitivity andmore than 60 samples have been analyzed so far.Clinical applications for routine phenotypic drugresistance testing are the initiation of antiretrovi-ral therapy and any change in the treatment regi-men to ensure that all compounds of thecombination therapy are active against the virusof each HIV-infected individual.
Acknowledgements
The authors thank B. Moschik and C. Paatz forexcellent technical assistance and E. De Clercqand B. Fleckenstein for helpful discussion andcontinuous support. The indicator cell line waskindly provided by R.Means and R.C. Desrosiers.The authors thank M. Helm, J.-C. Schmit, and B.Clotet, for providing clinical samples and infor-mation on treatment history. The followingreagent was obtained through the AIDS Researchand Reference Reagent Program, Division ofAIDS, NIAID, NIH: HIV-1 L10R/ M46I/ L63P/V82T/ I84V from E. Emini and HIV-1RTMDR1
from B. Larder.
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