INTRODUCTION

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The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) variants is almost always observed during the course of treatment of patients with antiretroviral drugs (3, 10, 14-16, 18, 21, 27; L. T. Bacheler, E. Anton, S. Jeffrey, H. George, G. Hollis, K. Abremski, and the Sustiva Resistance Study Team, Abstr. 2nd Int. Workshop HIV Drug Resistance and Treatment Strategies, abstr. 19, p. 15, 1998). The mutational profile of the resistant viruses generally is characteristic for the particular drug(s) taken. For example, mutations at codons 41, 67, 70, 210, 215, and 219 of reverse transcriptase (RT) typically confer resistance to zidovudine (ZDV) (6, 12, 13, 27). Similarly, mutation M184V in RT has been shown to be specifically associated with high-level (50-fold) phenotypic resistance to lamivudine (3TC) (1, 22, 28). No "specific" mutation(s) associated with moderate levels of phenotypic resistance (4- to <50-fold) to 3TC has been described before. Those mutations that confer moderate (4- to <50-fold) levels of phenotypic resistance to 3TC reported previously always appeared in the context of a constellation of mutations that confer resistance to multiple nucleoside analogues or as a cross-resistance phenomenon that appears with the emergence of resistance to another nucleoside analogue. This has been the case for the nucleoside multidrug resistance complex of mutations Q151M, F77L, F116Y, A62V, and V75I, although the increase in the level of phenotypic resistance to 3TC in viruses that harbor those mutations is slight (9, 20, 24, 25). In the case of the insertion mutations near position 69 of RT, a notable increase in the frequency of 3TC resistance has been reported together with an increased frequency of phenotypic resistance to other nucleosides (2, 17, 29). The K65R mutation appears infrequently during the course of treatment with dideoxyinosine (ddI; didanosine) and dideoxycytosine and confers concomitant low-level 3TC resistance (4, 5). In another study, phenotypic 3TC resistance (from 2.2-fold to 8.6-fold) was observed in the absence of M184V in patient isolates with various levels of phenotypic resistance to ZDV (from 4-fold to 378-fold) (26). However, no specific 3TC resistance-associated mutation was reported in this patient group.

Mutations present simultaneously may act synergistically, causing drug resistance to increase as the number of resistance-associated mutations increases (19). On the other hand, a mutation may reverse the effect of another mutation occurring concurrently in a viral variant. This is the case for the 3TC resistance mutation M184V, which can reverse the effect conferred by the ZDV mutation T215Y (1, 14, 28). Thus, although resistance to a particular antiretroviral drug is conferred by a specific mutation(s) associated with a particular drug, this can also be modulated by other mutations present in the background.

The implementation of high-throughput phenotyping and genotyping assays has allowed us to establish a database containing phenotypic resistance data for and the genotypic sequences of over 6,000 clinical isolates. Correlative data analysis and mutational cluster analysis then enabled us to search for mutational patterns with the accompanying phenotypic resistance profile.

We describe here a novel mutational pattern in HIV-1 RT associated with a moderate level of phenotypic resistance (4- to <50-fold) to 3TC that is distinguishable from the characteristically high levels of phenotypic resistance (50-fold) associated with the M184V mutation and whose expression is modulated by a particular genetic background.

	MATERIALS AND METHODS

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Plasma samples. Plasma samples obtained from HIV-1-infected individuals from routine clinical practices in Europe and the United States were shipped to the laboratory (Virco NV, Virco Ltd.) on dry ice and were stored at 70°C until genotypic and phenotypic analyses. Since the research at this stage is concerned with the discovery and description of the mutational patterns that underlie resistance and with virological and molecular biological aspects of resistance, selection of plasma samples was not necessarily based upon the availability of the therapeutic histories for each subject who provided a sample.

Phenotypic resistance testing. Phenotypic analysis was performed by the recombinant virus assay (11) approach described by Hertogs et al. (7) with the modifications described by Pauwels et al. (Abstr. 2nd Int. Workshop HIV Drug Resistance and Treatment Strategies, Abstr. 51, p. 35, 1998) (Antivirogram; Virco, Mechelen, Belgium). Briefly, protease (PR)- and RT-coding sequences were amplified from patient-derived viral RNA with HIV-1-specific primers. After homologous recombination of amplicons into a proviral clone from which the PR- and RT-coding sequences were deleted, the resulting recombinant viruses were harvested, titrated, and used for testing of in vitro susceptibility to antiretroviral drugs. The results of this analysis are expressed as fold change values, which reflect the fold increase in the mean 50% inhibitory concentration (IC₅₀) (micromolar) of a particular drug when it was tested with patient-derived recombinant virus isolates relative to the mean IC₅₀ (in micromolar) of the same drug obtained when it was tested with a reference wild-type virus isolate (strain IIIB/LAI). The 3TC resistance values generated by the phenotypic assay were grouped into one of three susceptibility categories, susceptible, moderately resistant, and high-level resistant, corresponding to fold changes in the IC₅₀ of between 0 and <4-fold, 4-fold and <50-fold, and 50-fold, respectively.

Sequence analysis and genotypic-phenotypic correlative data analysis. Genotypic analysis was performed by automated population-based full-sequence analysis (ABI). The results of the genotypic analysis are reported as amino acid changes at positions along the RT gene compared to the RT gene sequence of wild-type reference strain (strain HXB2). The database containing the genetic sequences was searched to determine the occurrence and frequencies of occurrence of mutational patterns present in the sequences of the clinical isolates. The corresponding phenotypic resistance profiles of those isolates were retrieved from the phenotypic database for comparison.

Site-directed mutagenesis. Mutations were generated in the RT gene of HXB2, a wild-type laboratory HIV-1 strain, with the QuickChange Site-Directed Mutagenesis Kit (Stratagene; Stratagene Cloning Systems, La Jolla, Calif.).

	RESULTS

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Analysis of the clinical isolates. The phenotypic database was interrogated for isolates susceptible or resistant to ZDV and/or 3TC. The corresponding genotypes were retrieved from the database. These isolates were drawn from a pool of 1,083 clinical samples for which both genotypic and phenotypic resistance data were available. In this group 42.2, 17.5, and 40.3% were found to have phenotypic susceptibility, moderate resistance, and high-level resistance to 3TC, respectively.

Isolates that are phenotypically susceptible (wild type) to both ZDV and 3TC (n = 314). As expected, the frequency of occurrence of any of the mutations listed above was low for the subset of isolates that are phenotypically susceptible to both ZDV and 3TC.

Isolates that are phenotypically susceptible to ZDV but for which the 3TC IC₅₀ is greater than 10-fold (n = 240). Table 1 shows that the frequency of occurrence of 3TC resistance-associated mutation 184V is high among isolates that are phenotypically susceptible to ZDV but for which the 3TC IC₅₀ is greater than 10-fold. The frequencies of occurrence of mutations 44D/A and 118I are low. About 18% of the isolates in this group harbor the major ZDV resistance-associated mutations 41L and 215Y, yet the level of ZDV resistance is low among these isolates, presumably due to the reversal of phenotypic ZDV resistance by the 184V mutation.

Isolates that are phenotypically resistant to ZDV (>10-fold; n = 351). The group of isolates that are phenotypically resistant to ZDV (>10-fold) was divided into the three 3TC resistance categories as detailed above. The mutation frequencies indicate that the rate of occurrence of ZDV resistance-associated mutations 41L and 215Y is high among all three 3TC resistance groups. As expected, the 184V mutation was almost exclusively present in the subset of isolates with high-level phenotypic 3TC resistance (50-fold), whereas the same mutation was low in frequency (4%) in the group of ZDV-resistant isolates with a moderate level of phenotypic 3TC resistance. This mutation was absent from the subset of isolates susceptible to 3TC. Mutations 44D/A and 118I were present in isolates in all 3TC resistance categories, and their incidence was higher among isolates with moderate and high-level phenotypic 3TC resistance than among isolates that were phenotypically susceptible to 3TC.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Frequency and distribution of mutation 65R, the two-amino-acid insertion at position 69, and mutations 151M, 184V, 44D/A, and 118I among the groups susceptible to 3TC and with intermediate and high-level phenotypic resistance to 3TC (n = 1,083).

Analysis of mutant viruses generated by site-directed mutagenesis. In order to investigate the relationship between phenotypic ZDV and/or 3TC resistance and the presence of mutations at positions 41, 215, 184, 44, and 118, different combinations of genotypic profiles were generated by site-directed mutagenesis. Table 3 lists a series of mutants that carried codon changes introduced into the wild-type HXB2 background together with the corresponding fold change in IC₅₀ obtained by a drug susceptibility assay. Three mutants with a change at codon 44, three mutants with a change at codon 118, and three mutants with changes at both codons 44 and 118 were generated. Within each of these three groups two mutants also had changes at positions associated with resistance to ZDV, whereas one mutant had the wild-type sequence at those codons. The drug resistance data for those mutants showed that the presence of mutations at codons 44 and 118, singly or together, could cause moderate phenotypic resistance to 3TC (7- to 32-fold). However, the moderate resistance to 3TC was observed only when mutations at positions 44 and/or 118 were in a ZDV resistance background (41L, 67N, 210W, 215Y). Six mutants with a change at codon 184 were generated, with one of those retaining the wild-type sequences at the other positions, whereas three others carried a series of ZDV resistance-associated mutations and two had changes at codons 44 and 118 as well as a series of ZDV resistance-associated mutations. All six of those mutants had high-level resistance to 3TC (>50-fold) that was distinguishable from the moderate level of phenotypic resistance seen in the mutants with changes at codons 44 and 118. In all of the mutants with the 184V mutation, resistance to 3TC was not related to the presence of the ZDV resistance-associated mutations. All mutants showed the predicted ZDV resistance or susceptibility pattern. The presence of the 184V mutation had a ZDV resensitizing effect on the mutants that carried ZDV resistance-associated mutations. This was not the case for the mutants with changes at codons 44 and 118, as phenotypic ZDV resistance remained unchanged when the mutations were introduced into the ZDV resistance background (Table 3).

Relationship between presence of changes at RT positions 44 or 118 in clinical samples and antiretroviral therapy. As can be deduced from Tables 1 and 2, changes at positions 44 and 118 can occur in viruses with or without the M184V substitution, but their incidence appeared to be the highest in viruses with ZDV resistance. It was therefore of interest to examine the antiretroviral treatments of patients infected with HIV isolates containing 44D or 118I. We identified a subset of 86 samples with isolates with the 44D mutation and 88 samples with isolates with the 118I mutation that originated from patients for whom antiretroviral treatment histories were available. It was not possible to draw conclusions regarding the incidence of changes at position 44 or 118 from this subset according to treatment history, as this was not a randomized study. However, this analysis sheds light on the requirements that lead to mutations at these positions.

	DISCUSSION

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The results presented here indicate that the E44D/A and V118I substitutions in HIV-1 RT confer a moderate level (4- to 50-fold) of phenotypic resistance to 3TC when they occur together with a ZDV resistance background. The presence of the M184V mutation is not required. The results obtained from the site-directed mutagenesis experiments confirmed the phenotypic data for the clinical isolates. This resistance-conferring mutational pattern has not previously been described and was discovered through mutational cluster analysis and correlative data analysis of a database containing phenotypic drug resistance data and the corresponding genetic sequences of these clinical isolates.

The study by Skowron et al. (26) described low-level resistance to 3TC in the absence of the M184V mutation in patient isolates that were also resistant to ZDV. Those investigators noted that the 3TC resistance levels were highly correlated with the ZDV resistance levels. Mutations at positions 41, 67, 70, 215, and 219 were present in the majority of the patient isolates. The investigators reported that patients whose isolates had mutations at codons 70 and 215 experienced a lesser response to treatment with 3TC than patients who did not harbor virus with those mutations, implying that mutations at codons 70 and 215 might interfere with the action of 3TC. In our study, the frequency of the mutation at position 215 (as well as the mutation at position 41) was high in all isolates with intermediate and high-level ZDV resistance, regardless of the level of 3TC resistance. This leads us to conclude that other mutations are responsible for the low and intermediate levels of 3TC resistance that we observed among the isolates in these samples.

The cluster analysis of the approximately 1,000 genotypically and phenotypically characterized clinical isolates and the results from the site-directed mutagenesis experiment confirm that mutations at codons 44 and 118 are indeed associated with moderate levels of phenotypic resistance to 3TC when they are present with ZDV resistance-associated mutations. The analysis of the clinical samples from patients for whom therapeutic histories were available and for which prior ZDV exposure was shown to be extensive confirmed the results obtained from our large clinical data set in that mutations 44D/A and 118I appeared in the context of ZDV mutations. The appearance of a mutation at position 118 in a ZDV resistance background has been documented previously (23). In a study of 39 patients who received ZDV and ddI combination therapy for 2 years, isolates from 5 patients were reported to have a mutation at position 118 at the end of the 2-year period. All five patients had received ZDV prior to the start of the combination therapy. The isolates from the five patients all had mutations at positions 41 and 215. Interestingly, isolates from one of the patients in this group of five patients harbored the mutation at position 118 at the start of combination therapy and also had a mutation at position 215 at the start of combination therapy.

Mutations 44D/A and 118I in the context of ZDV mutations are both capable of independently generating resistance to 3TC. The site-directed mutagenesis experiments did not indicate the existence of synergistic effects between the two mutations with respect to their phenotypic effect on 3TC resistance.

The 3TC resistance mutation 184V appears independently of a ZDV resistance background and clearly appears to be due to 3TC selection pressure, whereas the accumulation of 44D/A and 118I appears to be driven by ZDV and, fortuitously, confers resistance to 3TC. The mutational patterns of the mutants with site-directed mutations and the clinical isolates showed that the 44D/A and 118I mutations can occur with or without the 184V mutation. This indicates that 44D/A and 118I are not necessarily alternative mutations to 184V but suggests that 184V is a more dominant mutation with respect to 3TC resistance. We are not able to differentiate the effects of 44D/A and 118I from the effect of 184V in the isolates in which they occur together. Thus, we do not know whether 44D/A and 118I contribute to phenotypic resistance when 184V is simultaneously present.

The data presented here thus point to the existence of one possible genetic pathway for intermediate phenotypic 3TC resistance in clinical isolates of HIV-1. The data in Table 1 show that not all recombinant clinical isolates with an intermediate level of phenotypic resistance to 3TC and with high-level resistance to ZDV possess mutations at positions 44 and 118. This implies that other polymorphisms with an effect on phenotypic 3TC resistance may exist. These polymorphisms will need to be identified through further study.

The clinical relevance of the accumulation of 44D/A and 118I in a ZDV resistance background is under investigation by means of a longitudinal follow-up study. Further studies are also required to establish whether the 3TC resistance-associated changes that occur in the absence of 3TC treatment may provide an explanation for the possible delayed appearance of 184V during subsequent 3TC treatment (data not shown).

It is of interest to examine the positions 44 and 118 in the three-dimensional structures of the RT enzyme. The recently described structure of the catalytic complex of RT with a deoxynucleoside triphosphate template and primer sheds new insight on the possible way that nucleoside analog mutations confer resistance (8). Residue 215 is located near the incoming nucleotide-binding site. Of interest, residue 118 is located close to residue 215 in the three-dimensional structure of the catalytic complex. Furthermore, residue 44 is located in close proximity to residue 41. It is conceivable that changes at positions 215 and 41 of RT facilitate the accumulation of changes at positions 118 and 44 because of three-dimensional structural constraints. However, the mutations at positions 44 and 118 do not affect phenotypic ZDV resistance. Whether the changes at positions 118 and 44 influence the replicative fitness of ZDV-resistant viruses remains to be determined.

Our analysis shows that the pathway toward resistance to a particular drug may critically depend on the sequence in which drugs are administered in the course of therapy. As new drugs are approved for treatment, physicians will not only need to continue to evaluate the effects of the drugs in the context of the concurrently administered drugs but will also need to consider previously taken drugs. This not only is because of the possibility of cross-resistance but is also because of phenomena such as the one discussed here. This is also true in situations in which recycling of drugs is being considered. The results presented in this report indicate that the significant differences in phenotype which will probably be reflected in the response to treatment may not be readily predicted by the genotype. This points to the importance of combining the knowledge about both the phenotype and the genotype when therapeutic decisions are made.