Antimicrobial Agents and Chemotherapy, November 2004, p . 4189-4194, Vol . 48, No . 11

Differential Maintenance of the M184V Substitution in the Reverse Transcriptase of Human Immunodeficiency Virus Type 1 by Various Nucleoside Antiretroviral Agents in Tissue Culture

Marco Petrella, Maureen Oliveira, Daniela Moisi, Mervi Detorio, Bluma G . Brenner, and Mark A . Wainberg^*

McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada

Received 12 December 2003/ Returned for modification 5 February 2004/ Accepted 13 July 2004

A single mutation at position 184 (i.e., M184V) in the HIV-1 RT gene is responsible for high-level resistance (i.e., 500-fold) to 3TC (11, 14, 37), although a more transient substitution, M184I, usually develops first (24, 40) . However, HIV-1 variants harboring M184I are less fit than their M184V counterparts and are therefore rapidly outcompeted by the latter both in vitro and in vivo (1) .

The M184V mutation can also be selected by structurally related compounds, such as (–)-beta-2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC; emtricitabine) (12, 28), as well as by several structurally unrelated NRTIs, such as abacavir (ABC) (18, 30) . Two other NRTIs that less frequently select for M184V are ddI and ddC, and both ddI and ABC retain clinical efficacy against viruses that contain the M184V substitution (13, 16, 60) . This is due, in part at least, to the fact that M184V confers only very low levels of resistance to such drugs as ddI and ABC in tissue culture (i.e., two- to fourfold increases in the 50% inhibitory concentration [IC₅₀]) (34, 39, 54), in contrast to its effect against 3TC . Hence, the potential ddI, ABC, and ddC resistance-conferring effect of M184V may be manifested only in the presence of additional mutations in the RT gene (13, 31) . Although M184V also confers high-level resistance to FTC (41, 46, 53), clinical experience with this compound is limited, and it is not clear whether FTC selects for this mutation as efficiently as 3TC does in patients receiving therapy .

The M184V substitution is also associated with altered RT functions as a possible consequence of the location of M184V within a conserved YMDD motif that constitutes part of the enzymatic active site (21, 25) . These altered functions include decreased RT processivity, reduced nucleoside-dependent primer unblocking, increased polymerase fidelity in biochemical analyses, diminished rates of initiation of reverse transcription (9, 10), hypersensitization to other NRTIs, impaired viral fitness, and a delayed appearance of mutations in the RT gene that are responsible for resistance to thymidine analogue inhibitors such as ZDV and stavudine (d4T) (for reviews, see references 32 and 36) .

For these reasons, some clinicians believe that the M184V mutation should be maintained once it has been initially selected by antiretroviral therapy . However, it is not known whether continuous exposure to therapeutic levels of 3TC is the only means of achieving this goal or if other drugs might also be able to maintain the pressure required to prevent the disappearance of the M184V mutation once it has been selected . Other methods of maintaining M184V could potentially include the use of other NRTIs, combinations of drugs, or even low doses of 3TC . This study was performed to address this subject .

(The research performed by Marco Petrella was in partial fulfillment of the Ph.D . degree, Faculty of Graduate Studies and Research, McGill University, Montreal, Quebec, Canada.)

Clinical isolate 4205 displayed resistance to multiple NRTIs and contained several mutations for resistance to thymidine analogues (i.e., D67N, T215F, and K219Q) as well as M184V . This isolate also harbored mutations in the RT gene at A98G, T69D, V118I, and R211K, in addition to L63P in the protease gene, but did not harbor primary mutations associated with resistance to protease inhibitors . Two other clinical isolates, i.e., 3350 and 4742, a subtype C variant, contained only M184V as a drug resistance-associated mutation . In some experiments, cells were infected with alternating 1:9 or equal (i.e., 5:5) mixtures of the different HIV-1 isolates used . After 7 days, the cells were resuspended in their original culture medium, and 500 µl of supernatant and cell mixture were transferred to a new well with 2 x 10⁶ PBMCs and fresh solutions of the NRTIs . Thereafter, new rounds of infection were initiated by serial passage of the viral cultures at weekly intervals for the duration of the experiments (i.e., 9, 10, or 26 weeks) .

Genotypic analysis. Proviral DNA was extracted and amplified from cultures of the HIV-1 isolates in preparation for genotypic analysis . The TRUGENE HIV-1 genotyping test was used in conjunction with the OpenGene automated DNA sequencing system (Bayer Diagnostics Inc., Toronto, Ontario, Canada) to sequence the RT and protease regions of HIV-1 (45) . The sensitivity of this genotypic assay for the detection of minority viral species in mixtures is reported by the manufacturer to be 20% (OpenGene System User Manual) .

In some experiments, the line probe assay (LiPA; LiPA HIV-1 RT; Innogenetics Inc., Norcross, Ga.) was used as described previously (48, 51) to detect WT and drug resistance-related codons at position 184 in the RT gene . The intensities of the reactive LiPA bands were scored to estimate the percentage of WT or M184V species in mixtures of different viruses whose proportions were predetermined . Previous studies have shown that semiquantitative interpretation of LiPA band signals is possible for various RT codons in order to determine the relative presence of both WT and mutant viruses in clinical samples (56; F . Sheridan, D . Parker, R . Schuurman, et al., Abstr . 12th World AIDS Conf., abstr . 41225, 1998) . The basis for the LiPA technology involves reverse hybridization of a biotin-labeled PCR fragment that encodes the HIV-1 RT or protease gene region of interest to immobilized oligonucleotides that are embedded within nitrocellulose membrane test strips (48, 54) . The presence of WT or drug-resistant variants is determined by using a biotin-streptavidin colorimetric readout in which the relative amount of either species is proportional to the intensities of the bands (7, 43), enabling this test to detect individual genotypes among populations with mixtures of genotypes (7, 17, 43, 48, 55) . LiPA can often reveal the presence of minority species that constitute as little as 1% of the viral population, whereas the cutoff for detection of majority species by DNA sequencing ranges from 10 to 30% (7, 17, 43, 48, 51, 55) .

Phenotypic resistance testing. The drug susceptibility of HIV-1 isolate 4205 was measured by determining the extent to which select NRTIs inhibited replication in tissue culture (45) . Briefly, infected PBMCs were grown in 96-well culture plates in both the absence and the presence of various concentrations of NRTIs . After 7 days, RT assays were performed with culture fluids, in conjunction with Prism analytic software (GraphPad Prism, version 3.03 for Windows; GraphPad Software, San Diego, Calif.), to determine the IC₅₀s (45) . The IC₅₀s obtained for isolates were compared to the IC₅₀s previously determined for WT viruses in order to compute the fold resistance to the NRTIs tested . In instances in which more than one measurement of the IC₅₀ was performed, the results are expressed as the mean IC₅₀ ± the standard deviation (SD) . The antiretroviral drugs used for phenotypic testing included ZDV, 3TC, ABC, ddI, and d4T .

The following NRTIs were kindly provided by the various pharmaceutical companies: ABC, ZDV, and 3TC, GlaxoSmithKline; SPD754 [(–)-2'-deoxy-3'-oxa-4'-thiocytidine], Shire Biochem; ddI and d4T, Bristol-Myers Squibb; and ddC, Roche .

 
The effects of various concentrations of select NRTIs or the absence of drug on maintenance of the M184V substitution in culture was examined with these isolates for 26 weeks . The concentrations of ddI and ABC that were used were one, two, and five times their respective IC₅₀s; and the isolates were assessed for the presence of the M184V mutation in the RT gene after 26 weeks . For clinical isolate 4205, the results in Table 2 show that the absence of drug pressure resulted in reversion to the WT, i.e., M184, during this time, while exposure to ABC at the IC₅₀ (i.e., 0.2 µM) did not provide sufficient drug pressure to fully maintain M184V, with M184 and 184V being detected as a mixture after 26 weeks . However, higher concentrations of ABC (i.e., two and five times the IC₅₀) were able to preserve the M184V mutation over 26 weeks . In this experiment, all concentrations of ddI evaluated (i.e., 2.5, 5.0, and 10.0 µM) were able to maintain M184V over the 26 weeks, and genotypic analysis after that time did not reveal other changes in the RT gene .

 
We also investigated the effects of two concentrations of 3TC on the maintenance of M184V in culture using two different clinical isolates . The data in Table 2 show that M184V was preserved for 26 weeks when either the IC₅₀ of 3TC (0.1 µM) or a subtherapeutic concentration of 3TC (i.e., 0.05 µM) was used .

The effect of antiretroviral pressure on the selection or the preservation of M184V was also investigated in cultures in which infection of PBMCs was initiated with mixtures of WT HXB2D cloned virus and clinical isolate 3350 that contained M184V . The concentrations of the viral inocula were based on the p24 values for viruses that had been grown in PBMCs as described previously (22, 44); and the ratio of viruses used was 1:1, 5:5, or 1:9 .

The data in Table 3 show that ddC at a concentration of 0.05 µM, i.e., the IC₅₀, was not able to select for M184V in cultures of WT HXB2D but show that this concentration of drug was able to maintain M184V over 9 weeks in all of the cultures with dual infections with HXB2D and clinical isolate 3350 . Control cultures containing only isolate 3350 reverted or deselected to either a mixed 184M-184V genotype or the WT during this period in the absence of drug pressure .

 
In 1:9 mixtures of WT HXB2D and isolate 3350, 0.05 µM ddC was more effective than 0.006 µM ddC at maintaining M184V over 9 weeks . In the latter case, mixtures of both WT viruses and viruses with the M184V mutation were found . Reversion to M184 with 0.006 µM ddC was also observed in cultures containing equal mixtures of both viruses . In contrast, at 0.05 µM either ddC or 3TC provided adequate pressure to maintain M184V . In cultures infected with 9:1 mixtures of WT virus and isolate 3350, M184V was effectively selected and/or maintained with 0.05 µM ddC, indicating the importance of the concentration of ddC used .

The effectiveness of ABC and ddI for the selection and/or maintenance of M184V were also evaluated by using different mixtures of infecting viruses in tissue culture . The results in Table 4 show that treatment with 3.0 µM ddI for 10 weeks was unable to select for the M184V mutation in the HXB2D WT strain . In contrast, 3.0 µM SPD754 (60% 184V) and 1.28 µM ABC (50% 184V) were only partially selective for this mutation over this time . However, selection of M184V did occur with 1.28 µM 3TC . In the absence of drug pressure, reversion to WT M184 was much more efficient with the M184V clone (isolate 3350) than with the multinucleoside-resistant HIV-1 variant (isolate 4205) . In the latter case, LiPA testing revealed that the frequency of M184V codons was 70% after 10 weeks without treatment with antiretroviral drugs .

 
When M184V-containing species were already present in the infecting viral population, all of the NRTIs used were capable of preserving the M184V mutation in various mixtures of HXB2D and isolate 4205 . However, in mixtures of HXB2D and isolate 3350, 3.0 µM ddI was not able to maintain the presence of M184V to the same extent as 1.28 µM either ABC or 3TC after 10 weeks of tissue culture in the presence of these drugs . In this study, the use of 3.0 µM SPD754 also provided adequate drug pressure to preserve the M184V mutation in all mixtures of HXB2D and isolate 3350 .

M184V is known to impart a fitness disadvantage to HIV-1 . In the absence of selection pressure for this mutation, the replication capacity of viruses harboring M184V may be reduced by 3 to 10% (8, 38, 49) to as much as 48 to 57% in comparison to that of WT HIV-1 (M . D . Miller, K . L . White, C . J . Petropoulos, et al., Abstr . 10th Conf . Retrovir . Opportunistic Infect., abstr . 616, 2003), depending on the viral strain and the laboratory methods used . In addition, infection with viruses containing M184V often results in lower levels of viremia in plasma as a result of their diminished replication competence (5) . The rapid disappearance of M184V has been documented in treatment-experienced patients in whom selection pressure for this mutation was removed (62); and this has been shown to be frequently associated with a sharp rebound of plasma viremia, as was the case following the removal of 3TC from a triple-drug induction regimen (6) and, more recently, in therapy-experienced patients undergoing partial treatment interruption in which NRTIs were discontinued from a regimen that initially contained both these drugs and protease inhibitors (S . G . Deeks, J . N . Martin, R . Hoh, et al., Abstr . 10th Conf . Retrovir . Opportunistic Infect., abstr . 640, 2003) . Moreover, virological and immunological discordance in which CD4 cell counts are stable or increased, despite persistent plasma viremia (i.e., HIV-1 RNA levels, >10,000 copies/ml), has been reported in a subset of heavily pretreated patients who were infected with isolates with the M184V substitution and who experienced virological failure during antiretroviral therapy (35) .

However, the selection or maintenance of M184V by NRTIs other than 3TC has not been extensively investigated . By using a multinucleoside-resistant viral isolate that harbored M184V, in addition to several other mutations for resistance to nucleoside analogues, both ABC (0.4 and 1.0 µM) and ddI (2.5, 5.0, and 10.0 µM) effectively maintained M184V over a period of 26 weeks . Similarly, treatment of an M184V-containing clinical isolate with ddC (0.05 or 0.006 µM) for 9 weeks also maintained M184V in vitro . Exposure to 0.05 µM ddC for 9 weeks did not select for M184V with HIV-1 HXB2D, a laboratory WT clone .

LiPA was used instead of DNA sequencing for some experiments (Table 4) in order to detect the proportions of WT and M184V variants in primary cell cultures infected with different mixtures of these viruses . In our study, HIV-1 genotyping by LiPA revealed a considerably lower proportion of M184V-substituted codons in the 9:1 (0% 184V), 5:5 (60% 184V), and 1:9 (70% 184V) mixtures of HXB2D and isolate 3350 that were maintained with 30.0 µM ddI for 10 weeks than in cell cultures exposed to 12.8 µM either ABC or 3TC for the same length of time . Although the relative frequency of mutants with M184V maintained by ddI in the viral mixtures was observed to increase contemporaneously with the dilution of WT species, our data suggest that ddI may not be able to provide selection pressure to the same degrees as ABC and 3TC to preserve M184V, especially when the latter constitutes a minority species in the viral population .

Genotypic analysis indicated that isolate 4205 contained V118I . This mutation, when it is present either alone or together with E44A/D, can confer moderate phenotypic resistance to 3TC (3, 20, 42, 50) . In addition, data from other studies (B . G . Brenner, M . Oliveira, V . Micheli, et al., Abstr . XII Int . HIV Drug Resist . Workshop, abstr . 139, 2003) suggest that V118I may also mediate a negative compensatory effect in regard to M184V that could possibly improve the replicative fitness of multidrug-resistant HIV-1 variants that also harbor the M184V substitution in the RT gene . It is possible that V118I may have imparted a competitive advantage to isolate 4205 that allowed this virus to replicate to high levels and persist for 10 weeks in mixed viral populations that also contained WT species .

The M184V substitution in the RT gene confers low-level cross-resistance to structurally unrelated NRTIs, such as ABC, ddC, and ddI, in tissue culture (13, 31, 29) . However, the presence of M184V, which was detected in 60% of 3TC-experienced patients with virological failure, did not compromise the virological outcomes during subsequent treatment with new regimens that included d4T plus ddI (59) . Furthermore, M184V was detected in only 27% of these patients, whereas it was detected in 100% of the patients who received an alternate regimen that included d4T plus 3TC (59) . Similar results were reported in another study (10), in which virological responses during 8 weeks of treatment with ddI or ddI-hydroxyurea were not adversely affected by the presence of M184V . Thus, clinical studies suggest that the presence of the M184V mutation does not appear to adversely affect the virological response to ddI-containing regimens . Moreover, our LiPA results with ddI are consistent with the clinical data that show that treatment with this drug is relatively ineffective at maintaining M184V in clinical samples (52, 59) .

One important limitation of this work is that we have not carried out these studies using isogenically matched WT and M184V-containing clinical variants . Consequently, the results obtained could conceivably have been affected by differences in alleles in a variety of viral genes that might have affected viral fitness or the ability of other mutations to compensate for the effects of M184V . The construction of several pairs of matched clinical isolates containing multiple resistance-associated mutations is in progress . The fact that a variety of both clinical and laboratory-generated viruses have been used in this study and the fact that consistent results have been obtained with each type make it unlikely that the results that will ultimately be obtained with isogenic pairs of viruses will be different from those described here .

We have demonstrated that a variety of drugs, including ABC, the novel nucleoside analogue SPD754, and a subtherapeutic concentration of 3TC (i.e., 0.05 µM), can be used in tissue culture to maintain the M184V substitution in RT . These findings may have important clinical implications, insofar as the presence of this mutation has been shown in certain circumstances to be associated with improved therapeutic outcomes . It should be noted that a recent clinical study in which 3TC was either maintained or discontinued in a second-line regimen in which patients otherwise received three effective new drugs did not show virological benefit for the maintenance of M184V (U . Dragsted, Z . Fox, L . Mathiesen, et al., Abstr . 11th Conf . Retrovir . Opportunistic Infect., abstr . 549, 2004) . However, this study was not sufficiently powered to reveal small differences between the arms in regard to the levels of plasma viremia, since all of the patients had received new active drugs . Prospective clinical trials are still warranted to investigate whether the maintenance of M184V can confer clinical benefit .

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