The K65R Mutation in Human Immunodeficiency Virus
http://www.100md.com
病菌学杂志 2006年第10期
Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
VircoLabs, Durham, North Carolina
ABSTRACT
The K65R mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is selected in vitro by many D-nucleoside analog RT inhibitors (NRTI) but has been rarely detected in treated patients. In recent clinical trials, the K65R mutation has emerged frequently in patients experiencing virologic failure on antiretroviral combinations that do not include 3'-azidothymidine (AZT). The reason for this change is uncertain. To gain insight, we examined trends in the frequency of K65R in a large genotype database, the association of K65R with thymidine analog mutations (TAMs) and other NRTI mutations, and the viral susceptibility profile of HIV-1 with K65R alone and in combination with TAMs. Among >60,000 clinical samples submitted for genotype analysis that contained one or more NRTI resistance mutations, the frequency of K65R increased from 0.4% in 1998 to 3.6% in 2003. Among samples with K65R, a strong negative association was evident with the TAMs M41L, D67N, L210W, T215Y/F, and K219Q/E (P < 0.005) but not with other NRTI mutations, including the Q151M complex. This suggested that K65R and TAMs are antagonistic. To test this possibility, we generated recombinant HIV-1 encoding K65R in two different TAM backgrounds: M41L/L210W/T215Y and D67N/K70R/T215F/K219Q. K65R reduced AZT resistance from >50-fold to <2.5-fold in both backgrounds. In addition, TAMs antagonized the phenotypic effect of K65R, reducing resistance to tenofovir, abacavir, 2',3'-dideoxycytidine, dideoxyinosine, and stavudine. In conclusion, K65R and TAMs exhibit bidirectional phenotypic antagonism. This antagonism likely explains the negative association of these mutations in genotype databases, the rare emergence of K65R with antiretroviral therapies that contain AZT, and its more frequent emergence with combinations that exclude AZT.
INTRODUCTION
The lysine (K)-to-arginine (R) substitution at residue 65 (K65R) in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) results from a single G-to-A transition (AAA to AGA). This mutation was first identified in vitro by serial passage of HIV-1 in peripheral blood mononuclear cell cultures containing the nucleoside analog RT inhibitor (NRTI) 2',3'-dideoxycytidine (ddC) (49) and has subsequently been selected in vitro by many other NRTI (2, 9, 11, 15, 16, 39, 41).
The lysine at codon 65 in RT interacts with the phosphate of the incoming deoxynucleoside triphosphate (dNTP), positioning it properly for incorporation into the nascent DNA chain (19). The K-to-R substitution is believed to alter this positioning, favoring incorporation of the dNTP over NRTI triphosphate and resulting in NRTI resistance (13, 35, 38). To clarify the structural features of NRTI that influence their activity against HIV-1 with K65R, we recently analyzed a diverse panel of NRTI that varied by base component, pseudosugar structure, and enantiomer. The only NRTI that retain activity against HIV-1 with K65R are those having a 3'-azido component in the pseudosugar structure and either a thymine or adenine base (30). Of the eight NRTI currently approved by the U.S. Food and Drug Administration (FDA) for therapy of HIV-1 infection, only 3'-azidothymidine (zidovudine) (AZT) meets these criteria.
Resistance to AZT results from combinations of mutations collectively referred to as thymidine analog mutations (TAMs), which occur most often in two patterns: M41L/L210W/T215Y and D67N/K70R/T215F/K219Q (25, 48). These mutations improve the ATP-catalyzed primer unblocking activity of RT, resulting in the removal of the chain-terminating NRTI and resumption of DNA polymerization (1, 4, 26). This mechanism of resistance is different from that for K65R, which, as noted above, selectively decreases NRTI incorporation (35, 38).
The frequency of K65R emergence in patients treated with NRTI has depended on the antiretroviral treatment regimen. In trials of abacavir monotherapy (CNA2001 and CNA2002), K65R was selected in 13 of 127 (10%) patients (23). The frequency of K65R was lower (3 of 86 [3.5%]) in patients failing therapy with both abacavir and AZT. Of note, two of these three patients developing K65R received 4 weeks of abacavir monotherapy before the addition of AZT. Moreover, K65R was never (CNA2007) or rarely detected in patients treated with abacavir who had baseline virus with TAMs (23, 46). These observations suggested a protective effect of AZT and TAMs against the emergence of K65R, although the mechanism was not defined.
More recently, K65R was identified frequently among patients on failing treatment regimens that excluded AZT (18, 24, 28, 34, 40). For example, four of seven (57%) patients in one study and two of six (33%) in another study had early virologic failure with K65R after initiation of dual nucleoside therapy with tenofovir (TNV) and didanosine (ddI) (24, 31) (Table 1). The frequency of K65R was even higher (44 to 92%) among patients experiencing virologic failure on triple nucleoside regimens that excluded AZT (7, 10, 22, 32; J. Jemsek, P. Hutcherson, and E. Harper, Abstr. 11th Conf. Retrovir. Opportunistic Infect. [CROI], abstr. 51, 2004) (summarized in Table 1). The reason for the higher frequency of K65R in these studies was not known.
To investigate this, we examined changes in the prevalence of K65R and TAMs in a large genotype database, analyzed associations of K65R with TAMs from the same database, and determined the resistance profile of K65R in combination with TAMs against the eight FDA-approved NRTI.
MATERIALS AND METHODS
Clinical samples and database queries. To obtain frequency data, a large database of 62,222 genotypes (VircoBVBA, Mechelen, Belgium) was searched for the presence of NRTI mutations defined by the International AIDS Society-USA (IAS-USA) (20). The genotypes analyzed were from samples and sequences submitted between 1998 and 2003 for routine clinical testing. Samples originating from clinical trials were excluded. Patient treatment histories were not available. Genotype analysis of plasma samples was performed by Virco as described previously (17). A mutation was considered to be present in a plasma sample if it was detected in the sequencing reaction either as a pure mutant or as a mixture with another variant at the same position. To examine the associations of K65R with other NRTI mutations, 66,224 genotypes in the database were searched by first selecting the subset of genotypes that had one or more of the IAS-USA-listed mutations associated with resistance to NRTI (20) and then dividing them into subgroups of those with K65R and those without K65R. Chi-square analysis was performed with GraphPad software (GraphPad Software, Inc., San Diego, Calif.). All genotype data for this study were obtained without patient identifiers.
Chemicals. (–)--2',3'-Dideoxy-3'-thiacytidine (lamivudine) (3TC), -l-2',3'-dideoxy-5-fluoro-3'-thiacytidine (emtricitabine) (FTC), and -D-3'-deoxy-2',3'-didehydrothymidine (stavudine) (d4T) were kindly provided by Raymond Schinazi, Emory University. 9-[(R)-2-(Phosphonomethoxy)propyl]adenine (tenofovir; TNV) was provided by Gilead Sciences (Foster City, Calif.). AZT was obtained from TriLink Biotechnologies (San Diego, Calif.) and Sigma Chemical Corporation (St. Louis, Mo.). 2',3'-Dideoxyinosine (ddI; didanosine) and 2',3'-dideoxycytidine (ddC; zalcitabine) were also obtained from Sigma Chemical Corporation (St. Louis, Mo.). (1S,cis)-4-[2-Amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol sulfate (salt) (abacavir) (ABC) (2:1) was obtained from GlaxoSmithKline (Research Triangle Park, N.C.). The compounds were dissolved in dimethyl sulfoxide or sterile water as 10 mM or 30 mM stock solutions and stored at –20°C. Compounds were diluted immediately before use to the desired concentration in Dulbecco's modified Eagle medium, Phenol Red free (Gibco-BRL, Grand Island, N.Y.).
Generation of mutant recombinant HIV-1. Mutations in RT were introduced by site-directed PCR mutagenesis (QuikChange; Stratagene, La Jolla, Calif.). Silent 5' XmaI and 3' XbaI restriction sites in the pxxRT clone (37) facilitated subcloning of the mutated RT fragment into the infectious pxxHIV-1LAI clone. Mutants encoding the following changes in RT were made: K65R alone, M184V alone, K65R/M184V, M41L/L210W/T215Y (TAM41), M41L/L210W/T215Y + K65R (TAM41/65R), D67N/K70R/T215F/K219Q (TAM67), and D67N/K70R/T215F/K219Q + K65R (TAM67/65R). Stock viruses were prepared by electroporating (Gene Pulser; Bio-Rad, Hercules, Calif.) 5 to 10 μg of pxxHIV-1LAI plasmid DNA into 1.3 x 107 MT-2 cells (AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases, National Institutes of Health). Seven days after transfection, cell-free supernatant was harvested and stored at –80°C. The genotype of stock viruses was confirmed by extracting RNA from virions (QIAamp kit; QIAGEN, Valencia, Calif.), treating the extract with DNase I (Roche, Indianapolis, Ind.), amplifying the full-length coding region (amino acids 1 to 560) of RT by RT-PCR, purifying the PCR product (Wizard PCR Purification System; Promega, Madison, Wis.), and sequencing the PCR product with the Big Dye terminator kit, version 3.1, on an ABI 3100 automated DNA sequencer (Applied Biosystems, Foster City, Calif.).
Single-replication-cycle drug susceptibility assay. Single-replication-cycle drug susceptibility assays were done as previously described (30). Briefly, threefold serial dilutions of an inhibitor were added in triplicate to P4/R5 cells (provided by Ned Landau, Salk Institute, La Jolla, Calif.), which is a HeLa cell line stably transfected with a Tat-activated -galactosidase gene under the control of an HIV long terminal repeat promoter. Cells were infected with the amount of site-directed mutant that would yield a relative light unit (RLU) value of 100 in the no-drug, virus-infected control wells. A cell lysis buffer and luminescent substrate for -galactosidase (Gal-Screen; Tropix/Applied Biosystems, Foster City, Calif.) were added to each well at 48 h postinfection, and RLU were determined with a luminometer (ThermoLabSystems, Waltham, Mass.). Fold resistance was determined by dividing the concentration of compound required to inhibit virus replication by 50% (IC50) for wild-type HIV-1 by the IC50 for mutant HIV-1. IC50 values from at least three independent experiments were log10 transformed and compared with a two-sample Student t test. P values of less than 0.05 were considered statistically significant.
RESULTS
Changes in the frequency of occurrence of K65R and TAMs. We analyzed a large genotype database to investigate changes over time (1998 to 2003) in the frequency of NRTI resistance mutations. Of the 62,222 genotypes analyzed, the only NRTI mutation that increased in frequency was K65R, rising from 0.4% in 1998 to 3.6% by the beginning of 2003 (Table 2). By contrast, other NRTI mutations, including TAMs (M41L, K70R, L210W, T215F/Y, and K219E/N/Q) and M184I/V, decreased in frequency during the same time period. Of the TAMs, T215Y had the greatest decrease, a 19% change from 1998 to 2003, followed by M41L (17% decrease) and L210W (13% decrease) (Table 2). The frequencies of the 69-insert mutation, Q151M, and Y115F were low and changed little (1.1%) during this time period (Table 2).
Negative association between K65R and TAMs. Because of the divergent trends in the frequency of K65R and TAMs, we queried the database to determine whether a negative association exists between K65R and TAMs. As a control, we also queried the prevalence of K65R with Q151M, in which a positive association has been reported (34, 40, 44, 45; C. Amiel, A. Kara, V. Schneider, G. Pialous, W. Rozenbaum, and J. C. Nicolas, Abstr. 11th Conf. Retrovir. Opportunistic Infect., abstr. 627, 2004). Of the 65,535 genotypes from 1998 to 2003 that had at least one NRTI-associated mutation but not K65R, 32,356 (49.4%) had M41L (Table 3). Of the 689 genotypes that had K65R, only 71 (10.3%) also had M41L. This frequency of both K65R and M41L in the same genotype is significantly lower than expected (P < 0.0001). Significant negative associations were also noted for other TAMs, including D67N, L210W, T215Y, T215F, and K219Q/E (Table 2). Only K70R was not negatively associated with K65R; the subset of genotypes with K70R in the group with K65R was similar to that of the group without K65R (26.7% versus 27.2%; P = 0.775). By contrast, K65R was positively associated with Q151M, as reported previously. Only 2.4% of genotypes without K65R had Q151M, compared to 55% of samples with K65R.
K65R antagonizes resistance of TAMs to AZT. To gain a better understanding of the negative association between K65R and TAMs, we generated site-directed mutants with K65R alone (HIV-165R) and mutants with two different combinations of TAMs: M41L/L210W/T215Y (HIV-1TAM41) and D67N/K70R/T215F/K219Q (HIV-1TAM67) Additionally, we created mutants with K65R in combination with both sets of TAMs: M41L/L210W/T215Y with K65R (HIV-1TAM41/65R) and D67N/K70R/T215F/K219Q with K65R (HIV-1TAM67/65R). Both pathways of TAMs conferred high-level resistance to AZT, with a >50-fold change in IC50 for HIV-1 with the TAM41 combination or the TAM67 combination. The addition of K65R to TAMs significantly decreased this AZT resistance, to 2.2-fold for HIV-1TAM41/65R and to 1.5-fold for HIV-1TAM67/65R (Table 4). This reduction in AZT resistance provides clear evidence of phenotypic antagonism of TAMs by K65R.
TAMs antagonize K65R resistance to ABC, TNV, and ddC. To analyze the effect of TAMs on the phenotype of K65R, we evaluated the in vitro susceptibilities of wild-type HIV-1, HIV-165R, HIV-1TAM41, HIV-1TAM67, HIV-1TAM41/65R, and HIV-1TAM67/65R to several FDA-approved NRTI, including zalcitabine (ddC), didanosine (ddI), stavudine (d4T), ABC, and TNV (Table 5). HIV-1TAM41 had wild-type susceptibility to d4T, ddC, ddI, ABC, and TNV (0.8- to 1.2-fold change; P > 0.05). HIV-1TAM67 exhibited decreased susceptibility to d4T and ABC (2.3- and 2.0-fold, respectively; P < 0.01) but not to other NRTI. As expected (30), HIV-165R exhibited reduced susceptibility to all five NRTI, ranging from 2.7-fold to 5.0-fold (Table 5). The most striking antagonism of K65R by TAMs was observed for ABC, ddC, and TNV. The addition of the TAM41 combination significantly reduced resistance of HIV-1 with K65R to ABC, from 4.2-fold to 2.6-fold (P < 0.005), and the addition of the TAM67 combination significantly reduced resistance to ABC from 4.2-fold to 2.4-fold (P < 0.005). Similarly, both combinations of TAMs reduced resistance of HIV-1 with K65R to ddC from 5.0-fold to 2.2-fold for HIV-1TAM41/65R (P = 0.04) and from 5.0-fold to 2.1-fold for HIV-1TAM67/65R (P = 0.04). Only the TAM67 combination significantly diminished resistance of K65R to TNV (2.8-fold to 1.0-fold; P < 0.005); TNV susceptibility was unchanged by the TAM41 combination (2.4-fold for HIV-1TAM41/65R compared to 2.8-fold for HIV-165R) (Table 5).
For the other two NRTI tested, trends for antagonism of K65R by TAMs were observed. Resistance of K65R to ddI (2.7-fold) was reduced slightly more by the TAM67 combination (to 1.8-fold) than by the TAM41 combination (to 2.1-fold), but these differences from K65R without TAMs were not significant (P > 0.05). Similarly, resistance of HIV-165R to d4T (3.8-fold) was diminished by both sets of TAMs (HIV-1TAM41/65R, 1.5-fold; HIV-1TAM67/65R, 2.1-fold), but these differences were also not significant (P > 0.05) (Table 5).
TAMs do not antagonize K65R resistance to 3TC or FTC. We also tested 3TC and FTC to determine whether TAMs antagonized resistance of K65R to these compounds. HIV-165R was highly resistant to both 3TC (58-fold) and FTC (21-fold). The addition of the TAM41 combination to K65R did not alter susceptibility to 3TC (58-fold versus 65-fold; P = 0.90) and marginally increased resistance to FTC (from 21-fold to 57-fold; P = 0.04). The addition of TAM67 to K65R did not significantly increase resistance to 3TC (from 58-fold to 79-fold; P = 0.58) or to FTC (from 21-fold to 44-fold; P = 0.16) (Table 6).
K65R enhances resistance of M184V to ABC, ddI, and TNV. Finally, to assess whether the observed antagonism is specific to K65R and TAMs, recombinant mutants with both M184V and K65R (HIV-165R/184V) were generated to examine their combined effects (Table 7). K65R significantly increased resistance of HIV-1 with M184V alone (HIV-1184V) to ddC (from 1.8-fold to 4.9-fold), ddI (from 1.5-fold to 3.4-fold), and ABC (from 2.7-fold to 11-fold) (P < 0.001). Of note, HIV-165R reversed hypersusceptibility of HIV-1184V to TNV (0.5-fold), restoring HIV-165R/184V to wild-type susceptibility (1.1-fold) (P < 0.001). There was no discernible effect of the double mutant K65R/M184V on susceptibility to the other NRTI, including 3TC, FTC, AZT, and d4T. Resistance to 3TC and FTC was maximal at the highest drug concentration tested (90 μM) for HIV-1 with M184V alone or with K65R and M184V.
DISCUSSION
This is the first study to demonstrate bidirectional phenotypic antagonism between K65R and TAMs. Two independent lines of evidence support the existence and relevance of this antagonism: (i) clinical and epidemiological observations of increasing K65R frequency and negative association with TAMs, and (ii) a systematic analysis of mutants containing K65R with two different clinically relevant combinations of TAMs against all eight FDA-approved NRTI.
From our database analyses, we observed that the frequency of K65R increased while that of all individual TAMs decreased over the same time period. We had first noticed this increase in 2003 (U. M. Parikh, D. L. Koontz, J. L. Hammond, L. T. Bacheler, R. F. Schinazi, P. R. Meyer, and J. W. Mellors, Abstr. XII Int. HIV Drug Resist. Workshop, abstr. 136, 2003). Since then, other studies have observed increases in the frequency of K65R (5). For instance, Valer and colleagues found that the prevalence of K65R increased from 0.6% in 1999 to 11.5% in 2004 in their database of 1,846 HIV-infected patients in Spain (40). In a retrospective analysis, Winston and colleagues noticed a significant increase in K65R, from 1.7% in 2000 to 4% in 2002, from 997 regimen-failing patients from Chelsea and Westminster Hospital, United Kingdom (45). Kagan and colleagues also noticed a significant increase in K65R, from 0.94% to 4.6% (P < 0.0001) from 1999 to 2003 in the Quest Diagnostics clinical database (21). The reason for the increasing frequency of K65R is uncertain, but antagonism between K65R and TAMs is likely to be involved. It is possible that the increasing use of TNV (which selects K65R) after its approval in 2001, without concomitant use of AZT, contributed to the rise in K65R as well as to the decrease in TAMs. In our study, however, patient treatment information was not available to directly test this hypothesis.
Using a large sample size (66,224 genotypes), we found that K65R shows a strong negative association with specific TAMs, including M41L, D67N, L210W, T215F/Y, and K219Q/E. TAMs facilitate the excision reaction by increasing binding of ATP through substitutions at 210 and 215 (6, 48). M41L and D67N may enhance the effect of T215F/Y by altering the interaction of the 3'-azido group of AZT with the dNTP binding pocket, favoring excision (33). In the database, we found that K70R was not among the TAMs negatively correlated with K65R. The crystal structure of RT predicts that, like K65R, K70R may interact with the phosphate of the incoming dNTP (19). In addition, K70R, in the absence of other TAMs, may have minor discrimination activity against AZT and ddI (36). Thus, in the absence of other TAMs, K70R and K65R may not be antagonistic. The combinations of NRTI used and their order of use, which was likely to be variable among patients with samples in the database, may also have contributed to the associations or lack of associations observed between K65R and specific TAMs.
We further investigated these two observations—the divergent prevalence trends of K65R and TAMs and the negative association of K65R with TAMs—by analyzing the phenotypic effects of both K65R and TAMs on HIV-1 susceptibility to the eight FDA-approved NRTI. We found that K65R significantly diminishes AZT resistance (from >50-fold to <2.5-fold) in the context of two clinically relevant combinations of TAMs (M41L/L210W/T215Y and D67N/K70R/T215F/K219Q) (Table 4). Reversal of resistance to AZT by K65R was first reported by Bazmi and colleagues in a different, clinically uncommon TAM background (2). Our study extends the finding that K65R antagonizes TAMs. M184V, which also has an antagonistic effect on TAMs, has been shown to diminish the primer-unblocking activity of TAMs. M184V may alter the position of the primer terminus, causing a reduction in the efficiency of the excision reaction (3, 12). Biochemical studies have also shown that RT with K65R excises NRTI less efficiently than wild-type RT (43) and that K65R diminishes the excision activity of TAMs (27; U. M. Parikh, D. L. Koontz, N. Sluis-Cremer, J. L. Hammond, L. Bacheler, R. F. Schinazi, and J. W. Mellors, Abstr. 11th Conf. Retrovir. Opportunistic Infect., abstr. 54, 2004). The mechanism for antagonism of TAMs by K65R may be different from that of M184V. Preliminary modeling of RT suggests that K65R alters the alignment of the phosphate of ATP with the phosphodiester bond between the penultimate nucleotide and the NRTI at the 3' end of the primer, thereby reducing the efficiency of excision. Thus, RT may not be able to structurally accommodate both K65R and TAMs without compromising the excision activity conferred by TAMs.
TAMs significantly diminished the resistance to ABC, ddC, and TNV conferred by K65R. There was also a trend toward diminished resistance to ddI and d4T. For the TAM41 combination, this effect was unlikely to be due to antagonism of excision, because the TAM41 mutations alone did not confer resistance to any of the NRTI studied. The TAM67 combination conferred low-level resistance to ABC and d4T; hence, antagonism of excision by K65R could have played some part in restoring susceptibility for these two NRTI. Preliminary biochemical studies suggest that TAMs may decrease NRTI discrimination by K65R through partial restoration of the catalytic rate of NRTI triphosphate incorporation (U. M. Parikh, N. Sluis-Cremer, and J. W. Mellors, Abstr. XIV Int. HIV Drug Resist. Workshop, abstr. 85, 2005). Having additional mutations in the finger region could restore molecular interactions needed for more efficient NRTI incorporation.
HIV-1 with the TAM67 combination showed significant resistance to 3TC and FTC, and this was not reduced by the addition of K65R. Naeger et al. have also reported that HIV-1HXB2 with D67N/K70R/T215Y has reduced susceptibility to 3TC (3.8-fold), but ATP-catalyzed removal of 3TC was inefficient, supporting a discriminatory mechanism rather than an excision mechanism of resistance (29). Our finding that K65R did not reduce TAM67-mediated 3TC and FTC also argues against an excision mechanism. Structural and biochemical studies are needed to better define how TAMs discriminate against 3TC triphosphate and FTC triphosphate incorporation.
Finally, susceptibility of virus with K65R and M184V was determined to assess whether the antagonism by K65R was specific for TAMs. We hypothesized that the K65R and M184V mutations, both of which increase discrimination against NRTI incorporation, would confer greater resistance together than either alone. This proved to be the case: the K65R/M184V double mutant exhibited significantly greater resistance to ddC, ddI, and ABC than did M184V (or K65R in the case of ABC) alone. Additionally, M184V is known to cause hypersusceptibility to TNV (47), and K65R reversed this. Pre-steady-state enzymatic analysis may explain this combined effect of K65R and M184V. Deval and colleagues showed that discrimination by K65R RT is due to a decreased catalytic rate of incorporation of NRTI triphosphates compared to wild-type RT, with little effect on binding affinity. Conversely, discrimination by M184V RT is due to decreased binding affinity of NRTI-TP compared to wild-type RT, with little effect on the catalytic rate of incorporation. The double mutant K65R/M184V has both decreased binding affinity and a decreased catalytic rate of incorporation of NRTI triphosphate compared to wild-type RT (8). This likely explains the greater resistance of the double mutant to most NRTI. Although viruses with K65R in combination with M184V have diminished replicative capacity and replicative fitness (8, 42), there is enough of an advantage for the double mutant to be frequently selected in patients. Indeed, over half of all isolates from the Virco database with K65R also have M184V, and preliminary work from our lab indicates that K65R and M184V do occur on the same genome in patients (D. Barnas, H. Z. Bazmi, C. J. Bixby, D. L. Koontz, J. Jemsek, and J. W. Mellors, Abstr. XIV Int. HIV Drug Resist. Workshop, abstr. 152, 2005).
To conclude, we have provided strong clinical and virological evidence that K65R and TAMs are counter-selected in patients because of antagonistic mechanisms of resistance. K65R negates resistance to AZT caused by TAMs, and TAMs negate resistance to TNV, ABC, and other NRTI conferred by K65R. Consequently, there is no advantage for HIV-1 to evolve both pathways of mutations. Our findings likely explain why K65R is frequently selected in patients on failing regimens that do not include AZT and is rarely found with failure of regimens that include AZT.
ACKNOWLEDGMENTS
We thank Paula McKenna, Han Vermeiren, and Brian Wasikowski, from Virco BVBA and xLeo Inc., for assistance with database queries.
This work was supported by grants from the National Cancer Institute (SAIC contract 20XS190A) and the National Institute of Allergy and Infectious Diseases (Virology Support Subcontract from the AACTG Central Group, grant U01AI38858).
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VircoLabs, Durham, North Carolina
ABSTRACT
The K65R mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is selected in vitro by many D-nucleoside analog RT inhibitors (NRTI) but has been rarely detected in treated patients. In recent clinical trials, the K65R mutation has emerged frequently in patients experiencing virologic failure on antiretroviral combinations that do not include 3'-azidothymidine (AZT). The reason for this change is uncertain. To gain insight, we examined trends in the frequency of K65R in a large genotype database, the association of K65R with thymidine analog mutations (TAMs) and other NRTI mutations, and the viral susceptibility profile of HIV-1 with K65R alone and in combination with TAMs. Among >60,000 clinical samples submitted for genotype analysis that contained one or more NRTI resistance mutations, the frequency of K65R increased from 0.4% in 1998 to 3.6% in 2003. Among samples with K65R, a strong negative association was evident with the TAMs M41L, D67N, L210W, T215Y/F, and K219Q/E (P < 0.005) but not with other NRTI mutations, including the Q151M complex. This suggested that K65R and TAMs are antagonistic. To test this possibility, we generated recombinant HIV-1 encoding K65R in two different TAM backgrounds: M41L/L210W/T215Y and D67N/K70R/T215F/K219Q. K65R reduced AZT resistance from >50-fold to <2.5-fold in both backgrounds. In addition, TAMs antagonized the phenotypic effect of K65R, reducing resistance to tenofovir, abacavir, 2',3'-dideoxycytidine, dideoxyinosine, and stavudine. In conclusion, K65R and TAMs exhibit bidirectional phenotypic antagonism. This antagonism likely explains the negative association of these mutations in genotype databases, the rare emergence of K65R with antiretroviral therapies that contain AZT, and its more frequent emergence with combinations that exclude AZT.
INTRODUCTION
The lysine (K)-to-arginine (R) substitution at residue 65 (K65R) in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) results from a single G-to-A transition (AAA to AGA). This mutation was first identified in vitro by serial passage of HIV-1 in peripheral blood mononuclear cell cultures containing the nucleoside analog RT inhibitor (NRTI) 2',3'-dideoxycytidine (ddC) (49) and has subsequently been selected in vitro by many other NRTI (2, 9, 11, 15, 16, 39, 41).
The lysine at codon 65 in RT interacts with the phosphate of the incoming deoxynucleoside triphosphate (dNTP), positioning it properly for incorporation into the nascent DNA chain (19). The K-to-R substitution is believed to alter this positioning, favoring incorporation of the dNTP over NRTI triphosphate and resulting in NRTI resistance (13, 35, 38). To clarify the structural features of NRTI that influence their activity against HIV-1 with K65R, we recently analyzed a diverse panel of NRTI that varied by base component, pseudosugar structure, and enantiomer. The only NRTI that retain activity against HIV-1 with K65R are those having a 3'-azido component in the pseudosugar structure and either a thymine or adenine base (30). Of the eight NRTI currently approved by the U.S. Food and Drug Administration (FDA) for therapy of HIV-1 infection, only 3'-azidothymidine (zidovudine) (AZT) meets these criteria.
Resistance to AZT results from combinations of mutations collectively referred to as thymidine analog mutations (TAMs), which occur most often in two patterns: M41L/L210W/T215Y and D67N/K70R/T215F/K219Q (25, 48). These mutations improve the ATP-catalyzed primer unblocking activity of RT, resulting in the removal of the chain-terminating NRTI and resumption of DNA polymerization (1, 4, 26). This mechanism of resistance is different from that for K65R, which, as noted above, selectively decreases NRTI incorporation (35, 38).
The frequency of K65R emergence in patients treated with NRTI has depended on the antiretroviral treatment regimen. In trials of abacavir monotherapy (CNA2001 and CNA2002), K65R was selected in 13 of 127 (10%) patients (23). The frequency of K65R was lower (3 of 86 [3.5%]) in patients failing therapy with both abacavir and AZT. Of note, two of these three patients developing K65R received 4 weeks of abacavir monotherapy before the addition of AZT. Moreover, K65R was never (CNA2007) or rarely detected in patients treated with abacavir who had baseline virus with TAMs (23, 46). These observations suggested a protective effect of AZT and TAMs against the emergence of K65R, although the mechanism was not defined.
More recently, K65R was identified frequently among patients on failing treatment regimens that excluded AZT (18, 24, 28, 34, 40). For example, four of seven (57%) patients in one study and two of six (33%) in another study had early virologic failure with K65R after initiation of dual nucleoside therapy with tenofovir (TNV) and didanosine (ddI) (24, 31) (Table 1). The frequency of K65R was even higher (44 to 92%) among patients experiencing virologic failure on triple nucleoside regimens that excluded AZT (7, 10, 22, 32; J. Jemsek, P. Hutcherson, and E. Harper, Abstr. 11th Conf. Retrovir. Opportunistic Infect. [CROI], abstr. 51, 2004) (summarized in Table 1). The reason for the higher frequency of K65R in these studies was not known.
To investigate this, we examined changes in the prevalence of K65R and TAMs in a large genotype database, analyzed associations of K65R with TAMs from the same database, and determined the resistance profile of K65R in combination with TAMs against the eight FDA-approved NRTI.
MATERIALS AND METHODS
Clinical samples and database queries. To obtain frequency data, a large database of 62,222 genotypes (VircoBVBA, Mechelen, Belgium) was searched for the presence of NRTI mutations defined by the International AIDS Society-USA (IAS-USA) (20). The genotypes analyzed were from samples and sequences submitted between 1998 and 2003 for routine clinical testing. Samples originating from clinical trials were excluded. Patient treatment histories were not available. Genotype analysis of plasma samples was performed by Virco as described previously (17). A mutation was considered to be present in a plasma sample if it was detected in the sequencing reaction either as a pure mutant or as a mixture with another variant at the same position. To examine the associations of K65R with other NRTI mutations, 66,224 genotypes in the database were searched by first selecting the subset of genotypes that had one or more of the IAS-USA-listed mutations associated with resistance to NRTI (20) and then dividing them into subgroups of those with K65R and those without K65R. Chi-square analysis was performed with GraphPad software (GraphPad Software, Inc., San Diego, Calif.). All genotype data for this study were obtained without patient identifiers.
Chemicals. (–)--2',3'-Dideoxy-3'-thiacytidine (lamivudine) (3TC), -l-2',3'-dideoxy-5-fluoro-3'-thiacytidine (emtricitabine) (FTC), and -D-3'-deoxy-2',3'-didehydrothymidine (stavudine) (d4T) were kindly provided by Raymond Schinazi, Emory University. 9-[(R)-2-(Phosphonomethoxy)propyl]adenine (tenofovir; TNV) was provided by Gilead Sciences (Foster City, Calif.). AZT was obtained from TriLink Biotechnologies (San Diego, Calif.) and Sigma Chemical Corporation (St. Louis, Mo.). 2',3'-Dideoxyinosine (ddI; didanosine) and 2',3'-dideoxycytidine (ddC; zalcitabine) were also obtained from Sigma Chemical Corporation (St. Louis, Mo.). (1S,cis)-4-[2-Amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol sulfate (salt) (abacavir) (ABC) (2:1) was obtained from GlaxoSmithKline (Research Triangle Park, N.C.). The compounds were dissolved in dimethyl sulfoxide or sterile water as 10 mM or 30 mM stock solutions and stored at –20°C. Compounds were diluted immediately before use to the desired concentration in Dulbecco's modified Eagle medium, Phenol Red free (Gibco-BRL, Grand Island, N.Y.).
Generation of mutant recombinant HIV-1. Mutations in RT were introduced by site-directed PCR mutagenesis (QuikChange; Stratagene, La Jolla, Calif.). Silent 5' XmaI and 3' XbaI restriction sites in the pxxRT clone (37) facilitated subcloning of the mutated RT fragment into the infectious pxxHIV-1LAI clone. Mutants encoding the following changes in RT were made: K65R alone, M184V alone, K65R/M184V, M41L/L210W/T215Y (TAM41), M41L/L210W/T215Y + K65R (TAM41/65R), D67N/K70R/T215F/K219Q (TAM67), and D67N/K70R/T215F/K219Q + K65R (TAM67/65R). Stock viruses were prepared by electroporating (Gene Pulser; Bio-Rad, Hercules, Calif.) 5 to 10 μg of pxxHIV-1LAI plasmid DNA into 1.3 x 107 MT-2 cells (AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases, National Institutes of Health). Seven days after transfection, cell-free supernatant was harvested and stored at –80°C. The genotype of stock viruses was confirmed by extracting RNA from virions (QIAamp kit; QIAGEN, Valencia, Calif.), treating the extract with DNase I (Roche, Indianapolis, Ind.), amplifying the full-length coding region (amino acids 1 to 560) of RT by RT-PCR, purifying the PCR product (Wizard PCR Purification System; Promega, Madison, Wis.), and sequencing the PCR product with the Big Dye terminator kit, version 3.1, on an ABI 3100 automated DNA sequencer (Applied Biosystems, Foster City, Calif.).
Single-replication-cycle drug susceptibility assay. Single-replication-cycle drug susceptibility assays were done as previously described (30). Briefly, threefold serial dilutions of an inhibitor were added in triplicate to P4/R5 cells (provided by Ned Landau, Salk Institute, La Jolla, Calif.), which is a HeLa cell line stably transfected with a Tat-activated -galactosidase gene under the control of an HIV long terminal repeat promoter. Cells were infected with the amount of site-directed mutant that would yield a relative light unit (RLU) value of 100 in the no-drug, virus-infected control wells. A cell lysis buffer and luminescent substrate for -galactosidase (Gal-Screen; Tropix/Applied Biosystems, Foster City, Calif.) were added to each well at 48 h postinfection, and RLU were determined with a luminometer (ThermoLabSystems, Waltham, Mass.). Fold resistance was determined by dividing the concentration of compound required to inhibit virus replication by 50% (IC50) for wild-type HIV-1 by the IC50 for mutant HIV-1. IC50 values from at least three independent experiments were log10 transformed and compared with a two-sample Student t test. P values of less than 0.05 were considered statistically significant.
RESULTS
Changes in the frequency of occurrence of K65R and TAMs. We analyzed a large genotype database to investigate changes over time (1998 to 2003) in the frequency of NRTI resistance mutations. Of the 62,222 genotypes analyzed, the only NRTI mutation that increased in frequency was K65R, rising from 0.4% in 1998 to 3.6% by the beginning of 2003 (Table 2). By contrast, other NRTI mutations, including TAMs (M41L, K70R, L210W, T215F/Y, and K219E/N/Q) and M184I/V, decreased in frequency during the same time period. Of the TAMs, T215Y had the greatest decrease, a 19% change from 1998 to 2003, followed by M41L (17% decrease) and L210W (13% decrease) (Table 2). The frequencies of the 69-insert mutation, Q151M, and Y115F were low and changed little (1.1%) during this time period (Table 2).
Negative association between K65R and TAMs. Because of the divergent trends in the frequency of K65R and TAMs, we queried the database to determine whether a negative association exists between K65R and TAMs. As a control, we also queried the prevalence of K65R with Q151M, in which a positive association has been reported (34, 40, 44, 45; C. Amiel, A. Kara, V. Schneider, G. Pialous, W. Rozenbaum, and J. C. Nicolas, Abstr. 11th Conf. Retrovir. Opportunistic Infect., abstr. 627, 2004). Of the 65,535 genotypes from 1998 to 2003 that had at least one NRTI-associated mutation but not K65R, 32,356 (49.4%) had M41L (Table 3). Of the 689 genotypes that had K65R, only 71 (10.3%) also had M41L. This frequency of both K65R and M41L in the same genotype is significantly lower than expected (P < 0.0001). Significant negative associations were also noted for other TAMs, including D67N, L210W, T215Y, T215F, and K219Q/E (Table 2). Only K70R was not negatively associated with K65R; the subset of genotypes with K70R in the group with K65R was similar to that of the group without K65R (26.7% versus 27.2%; P = 0.775). By contrast, K65R was positively associated with Q151M, as reported previously. Only 2.4% of genotypes without K65R had Q151M, compared to 55% of samples with K65R.
K65R antagonizes resistance of TAMs to AZT. To gain a better understanding of the negative association between K65R and TAMs, we generated site-directed mutants with K65R alone (HIV-165R) and mutants with two different combinations of TAMs: M41L/L210W/T215Y (HIV-1TAM41) and D67N/K70R/T215F/K219Q (HIV-1TAM67) Additionally, we created mutants with K65R in combination with both sets of TAMs: M41L/L210W/T215Y with K65R (HIV-1TAM41/65R) and D67N/K70R/T215F/K219Q with K65R (HIV-1TAM67/65R). Both pathways of TAMs conferred high-level resistance to AZT, with a >50-fold change in IC50 for HIV-1 with the TAM41 combination or the TAM67 combination. The addition of K65R to TAMs significantly decreased this AZT resistance, to 2.2-fold for HIV-1TAM41/65R and to 1.5-fold for HIV-1TAM67/65R (Table 4). This reduction in AZT resistance provides clear evidence of phenotypic antagonism of TAMs by K65R.
TAMs antagonize K65R resistance to ABC, TNV, and ddC. To analyze the effect of TAMs on the phenotype of K65R, we evaluated the in vitro susceptibilities of wild-type HIV-1, HIV-165R, HIV-1TAM41, HIV-1TAM67, HIV-1TAM41/65R, and HIV-1TAM67/65R to several FDA-approved NRTI, including zalcitabine (ddC), didanosine (ddI), stavudine (d4T), ABC, and TNV (Table 5). HIV-1TAM41 had wild-type susceptibility to d4T, ddC, ddI, ABC, and TNV (0.8- to 1.2-fold change; P > 0.05). HIV-1TAM67 exhibited decreased susceptibility to d4T and ABC (2.3- and 2.0-fold, respectively; P < 0.01) but not to other NRTI. As expected (30), HIV-165R exhibited reduced susceptibility to all five NRTI, ranging from 2.7-fold to 5.0-fold (Table 5). The most striking antagonism of K65R by TAMs was observed for ABC, ddC, and TNV. The addition of the TAM41 combination significantly reduced resistance of HIV-1 with K65R to ABC, from 4.2-fold to 2.6-fold (P < 0.005), and the addition of the TAM67 combination significantly reduced resistance to ABC from 4.2-fold to 2.4-fold (P < 0.005). Similarly, both combinations of TAMs reduced resistance of HIV-1 with K65R to ddC from 5.0-fold to 2.2-fold for HIV-1TAM41/65R (P = 0.04) and from 5.0-fold to 2.1-fold for HIV-1TAM67/65R (P = 0.04). Only the TAM67 combination significantly diminished resistance of K65R to TNV (2.8-fold to 1.0-fold; P < 0.005); TNV susceptibility was unchanged by the TAM41 combination (2.4-fold for HIV-1TAM41/65R compared to 2.8-fold for HIV-165R) (Table 5).
For the other two NRTI tested, trends for antagonism of K65R by TAMs were observed. Resistance of K65R to ddI (2.7-fold) was reduced slightly more by the TAM67 combination (to 1.8-fold) than by the TAM41 combination (to 2.1-fold), but these differences from K65R without TAMs were not significant (P > 0.05). Similarly, resistance of HIV-165R to d4T (3.8-fold) was diminished by both sets of TAMs (HIV-1TAM41/65R, 1.5-fold; HIV-1TAM67/65R, 2.1-fold), but these differences were also not significant (P > 0.05) (Table 5).
TAMs do not antagonize K65R resistance to 3TC or FTC. We also tested 3TC and FTC to determine whether TAMs antagonized resistance of K65R to these compounds. HIV-165R was highly resistant to both 3TC (58-fold) and FTC (21-fold). The addition of the TAM41 combination to K65R did not alter susceptibility to 3TC (58-fold versus 65-fold; P = 0.90) and marginally increased resistance to FTC (from 21-fold to 57-fold; P = 0.04). The addition of TAM67 to K65R did not significantly increase resistance to 3TC (from 58-fold to 79-fold; P = 0.58) or to FTC (from 21-fold to 44-fold; P = 0.16) (Table 6).
K65R enhances resistance of M184V to ABC, ddI, and TNV. Finally, to assess whether the observed antagonism is specific to K65R and TAMs, recombinant mutants with both M184V and K65R (HIV-165R/184V) were generated to examine their combined effects (Table 7). K65R significantly increased resistance of HIV-1 with M184V alone (HIV-1184V) to ddC (from 1.8-fold to 4.9-fold), ddI (from 1.5-fold to 3.4-fold), and ABC (from 2.7-fold to 11-fold) (P < 0.001). Of note, HIV-165R reversed hypersusceptibility of HIV-1184V to TNV (0.5-fold), restoring HIV-165R/184V to wild-type susceptibility (1.1-fold) (P < 0.001). There was no discernible effect of the double mutant K65R/M184V on susceptibility to the other NRTI, including 3TC, FTC, AZT, and d4T. Resistance to 3TC and FTC was maximal at the highest drug concentration tested (90 μM) for HIV-1 with M184V alone or with K65R and M184V.
DISCUSSION
This is the first study to demonstrate bidirectional phenotypic antagonism between K65R and TAMs. Two independent lines of evidence support the existence and relevance of this antagonism: (i) clinical and epidemiological observations of increasing K65R frequency and negative association with TAMs, and (ii) a systematic analysis of mutants containing K65R with two different clinically relevant combinations of TAMs against all eight FDA-approved NRTI.
From our database analyses, we observed that the frequency of K65R increased while that of all individual TAMs decreased over the same time period. We had first noticed this increase in 2003 (U. M. Parikh, D. L. Koontz, J. L. Hammond, L. T. Bacheler, R. F. Schinazi, P. R. Meyer, and J. W. Mellors, Abstr. XII Int. HIV Drug Resist. Workshop, abstr. 136, 2003). Since then, other studies have observed increases in the frequency of K65R (5). For instance, Valer and colleagues found that the prevalence of K65R increased from 0.6% in 1999 to 11.5% in 2004 in their database of 1,846 HIV-infected patients in Spain (40). In a retrospective analysis, Winston and colleagues noticed a significant increase in K65R, from 1.7% in 2000 to 4% in 2002, from 997 regimen-failing patients from Chelsea and Westminster Hospital, United Kingdom (45). Kagan and colleagues also noticed a significant increase in K65R, from 0.94% to 4.6% (P < 0.0001) from 1999 to 2003 in the Quest Diagnostics clinical database (21). The reason for the increasing frequency of K65R is uncertain, but antagonism between K65R and TAMs is likely to be involved. It is possible that the increasing use of TNV (which selects K65R) after its approval in 2001, without concomitant use of AZT, contributed to the rise in K65R as well as to the decrease in TAMs. In our study, however, patient treatment information was not available to directly test this hypothesis.
Using a large sample size (66,224 genotypes), we found that K65R shows a strong negative association with specific TAMs, including M41L, D67N, L210W, T215F/Y, and K219Q/E. TAMs facilitate the excision reaction by increasing binding of ATP through substitutions at 210 and 215 (6, 48). M41L and D67N may enhance the effect of T215F/Y by altering the interaction of the 3'-azido group of AZT with the dNTP binding pocket, favoring excision (33). In the database, we found that K70R was not among the TAMs negatively correlated with K65R. The crystal structure of RT predicts that, like K65R, K70R may interact with the phosphate of the incoming dNTP (19). In addition, K70R, in the absence of other TAMs, may have minor discrimination activity against AZT and ddI (36). Thus, in the absence of other TAMs, K70R and K65R may not be antagonistic. The combinations of NRTI used and their order of use, which was likely to be variable among patients with samples in the database, may also have contributed to the associations or lack of associations observed between K65R and specific TAMs.
We further investigated these two observations—the divergent prevalence trends of K65R and TAMs and the negative association of K65R with TAMs—by analyzing the phenotypic effects of both K65R and TAMs on HIV-1 susceptibility to the eight FDA-approved NRTI. We found that K65R significantly diminishes AZT resistance (from >50-fold to <2.5-fold) in the context of two clinically relevant combinations of TAMs (M41L/L210W/T215Y and D67N/K70R/T215F/K219Q) (Table 4). Reversal of resistance to AZT by K65R was first reported by Bazmi and colleagues in a different, clinically uncommon TAM background (2). Our study extends the finding that K65R antagonizes TAMs. M184V, which also has an antagonistic effect on TAMs, has been shown to diminish the primer-unblocking activity of TAMs. M184V may alter the position of the primer terminus, causing a reduction in the efficiency of the excision reaction (3, 12). Biochemical studies have also shown that RT with K65R excises NRTI less efficiently than wild-type RT (43) and that K65R diminishes the excision activity of TAMs (27; U. M. Parikh, D. L. Koontz, N. Sluis-Cremer, J. L. Hammond, L. Bacheler, R. F. Schinazi, and J. W. Mellors, Abstr. 11th Conf. Retrovir. Opportunistic Infect., abstr. 54, 2004). The mechanism for antagonism of TAMs by K65R may be different from that of M184V. Preliminary modeling of RT suggests that K65R alters the alignment of the phosphate of ATP with the phosphodiester bond between the penultimate nucleotide and the NRTI at the 3' end of the primer, thereby reducing the efficiency of excision. Thus, RT may not be able to structurally accommodate both K65R and TAMs without compromising the excision activity conferred by TAMs.
TAMs significantly diminished the resistance to ABC, ddC, and TNV conferred by K65R. There was also a trend toward diminished resistance to ddI and d4T. For the TAM41 combination, this effect was unlikely to be due to antagonism of excision, because the TAM41 mutations alone did not confer resistance to any of the NRTI studied. The TAM67 combination conferred low-level resistance to ABC and d4T; hence, antagonism of excision by K65R could have played some part in restoring susceptibility for these two NRTI. Preliminary biochemical studies suggest that TAMs may decrease NRTI discrimination by K65R through partial restoration of the catalytic rate of NRTI triphosphate incorporation (U. M. Parikh, N. Sluis-Cremer, and J. W. Mellors, Abstr. XIV Int. HIV Drug Resist. Workshop, abstr. 85, 2005). Having additional mutations in the finger region could restore molecular interactions needed for more efficient NRTI incorporation.
HIV-1 with the TAM67 combination showed significant resistance to 3TC and FTC, and this was not reduced by the addition of K65R. Naeger et al. have also reported that HIV-1HXB2 with D67N/K70R/T215Y has reduced susceptibility to 3TC (3.8-fold), but ATP-catalyzed removal of 3TC was inefficient, supporting a discriminatory mechanism rather than an excision mechanism of resistance (29). Our finding that K65R did not reduce TAM67-mediated 3TC and FTC also argues against an excision mechanism. Structural and biochemical studies are needed to better define how TAMs discriminate against 3TC triphosphate and FTC triphosphate incorporation.
Finally, susceptibility of virus with K65R and M184V was determined to assess whether the antagonism by K65R was specific for TAMs. We hypothesized that the K65R and M184V mutations, both of which increase discrimination against NRTI incorporation, would confer greater resistance together than either alone. This proved to be the case: the K65R/M184V double mutant exhibited significantly greater resistance to ddC, ddI, and ABC than did M184V (or K65R in the case of ABC) alone. Additionally, M184V is known to cause hypersusceptibility to TNV (47), and K65R reversed this. Pre-steady-state enzymatic analysis may explain this combined effect of K65R and M184V. Deval and colleagues showed that discrimination by K65R RT is due to a decreased catalytic rate of incorporation of NRTI triphosphates compared to wild-type RT, with little effect on binding affinity. Conversely, discrimination by M184V RT is due to decreased binding affinity of NRTI-TP compared to wild-type RT, with little effect on the catalytic rate of incorporation. The double mutant K65R/M184V has both decreased binding affinity and a decreased catalytic rate of incorporation of NRTI triphosphate compared to wild-type RT (8). This likely explains the greater resistance of the double mutant to most NRTI. Although viruses with K65R in combination with M184V have diminished replicative capacity and replicative fitness (8, 42), there is enough of an advantage for the double mutant to be frequently selected in patients. Indeed, over half of all isolates from the Virco database with K65R also have M184V, and preliminary work from our lab indicates that K65R and M184V do occur on the same genome in patients (D. Barnas, H. Z. Bazmi, C. J. Bixby, D. L. Koontz, J. Jemsek, and J. W. Mellors, Abstr. XIV Int. HIV Drug Resist. Workshop, abstr. 152, 2005).
To conclude, we have provided strong clinical and virological evidence that K65R and TAMs are counter-selected in patients because of antagonistic mechanisms of resistance. K65R negates resistance to AZT caused by TAMs, and TAMs negate resistance to TNV, ABC, and other NRTI conferred by K65R. Consequently, there is no advantage for HIV-1 to evolve both pathways of mutations. Our findings likely explain why K65R is frequently selected in patients on failing regimens that do not include AZT and is rarely found with failure of regimens that include AZT.
ACKNOWLEDGMENTS
We thank Paula McKenna, Han Vermeiren, and Brian Wasikowski, from Virco BVBA and xLeo Inc., for assistance with database queries.
This work was supported by grants from the National Cancer Institute (SAIC contract 20XS190A) and the National Institute of Allergy and Infectious Diseases (Virology Support Subcontract from the AACTG Central Group, grant U01AI38858).
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