From the *Section of Retroviral Therapeutics, Brigham and Women's Hospital, and Division of AIDS, Harvard Medical School, Boston, MA; and †ViroLogic, South San Francisco, CA.
Received for publication January 2, 2005; accepted May 19, 2005.
Supported by US Public Health Service grant AI42567 and by the Harvard Medical School Center for AIDS Research Virology Core (AI060354).
Reprints: Daniel R. Kuritzkes, Section of Retroviral Therapeutics, Brigham and Women's Hospital, 65 Landsdowne Street, Room 449, Cambridge, MA 02139 (e-mail: email@example.com).
Although present as polymorphisms in HIV-1 sequences from untreated patients, mutations at reverse transcriptase (RT) codon 207 are found at higher frequency in samples from zidovudine-treated patients. Introduction of the Q207D mutation into the RT of a zidovudine (ZDV)-resistant isolate by site-directed mutagenesis increased ZDV resistance 2.7-fold but had no effect on ZDV susceptibility when introduced into wild-type RT. Zidovudine-resistant recombinant HIV-1 with and without 207D showed comparable fitness in growth competition assays in the absence of ZDV, but this mutation enhanced the fitness of ZDV-resistant recombinants in the presence of drug. These results suggest that when present with other thymidine analogue resistance mutations, 207D serves as a resistance mutation that improves the fitness of ZDV-resistant HIV-1. Analyzing viral fitness can provide important insights into the role of polymorphisms in drug resistance.
Resistance to zidovudine (3′-azido-3′-deoxythymidine; ZDV; GlaxoSmithKline, Research Triangle Park, NC) emerges in a stepwise manner by the accumulation of thymidine analogue resistance mutations (TAMs) at reverse transcriptase (RT) codons 41, 67, 70, 210, 215, and 219.1-3 Polymorphisms at positions 211 and 214 contribute to ZDV resistance as well.4-6 The combined presence of 3 to 6 TAMs results in high-level (≥500-fold) ZDV resistance and confers cross-resistance to other nucleoside RT inhibitors.7
Previous work from our laboratory showed that substitutions at codons 44, 118, 207, and 208 are associated with higher levels of ZDV resistance in HIV-1 isolates that carry TAMs.8 Presence of a codon 207 mutation was associated with a 22.5-fold higher geometric mean 50% inhibitory concentration (IC50) for ZDV as compared with isolates that were wild-type at this position. Because mutations at codon 207 have not previously been associated with ZDV resistance, we further evaluated the effect of the Q207D mutation on ZDV susceptibility and viral replicative capacity in vitro.
Construction and Characterization of Recombinant Viruses
Infectious recombinant viruses expressing RT from wild-type (18A) or ZDV-resistant (18C) clinical isolates of HIV-1 (provided by Dr. Douglas Richman through the AIDS Research and Reference Reagent Repository Program) were generated as described by electroporating cloned RT genes into MT-2 cells together with pHIVΔRTBstEII, a full-length molecular clone of HIV-1NL4-3 from which the entire RT-coding region of pol has been deleted.9 The cloned RT sequences included approximately 100 bp of the protease- and integrase-coding regions of pol at the 5′ and 3′ ends of the molecule, respectively, to permit homologous recombination with the HIV-1NL4-3 recombination vector. This approach ensured that the entire cloned RT sequence was incorporated into the resulting recombinant viruses.
To introduce mutations into RT, a 2.2-kb fragment of HIV-1 pol encompassing the entire RT-coding region was cloned into vector pCR2.1 (Stratagene, La Jolla, CA) to generate plasmid pTA-RT. The M184V mutation (which confers lamivudine [3TC] resistance) and the Q207D mutation were introduced using QuikChange Site-Directed Mutagenesis Kit (Stratagene) following the manufacturer's protocol. Presence of the correct RT sequence and absence of adventitious mutations in the recombinant viruses were confirmed by sequencing. Drug susceptibility was determined using the PhenoSense HIV Assay (ViroLogic, South San Francisco, CA). Mean (±SD) IC50's were determined from results of at least 3 independent assays.
Construction of Recombinant Marker Viruses
A modification of the recombinant marker virus assay in which the green fluorescent protein (GFP) or Salmonella hisD genes served as sequence tags was used to determine relative fitness in growth competition assays.9 To construct appropriate recombination vectors, a segment of the GFP or hisD genes were cloned into an XhoI site in nef of pHIVΔRTBstEII to yield pHIVΔRTBstEIInef-hisD and pHIVΔRTBstEIInef-GFP. Infectious recombinant viruses were generated by co-transfecting the appropriate linearized plasmid together with the RT gene of interest as described here.
Growth Competition Assays
Pairwise growth competition assays were performed as described.9,10 In brief, recombinant marker viruses being tested were mixed together at a 50:50 ratio and inoculated onto MT-2 cells at a multiplicity of infection of 0.001 infectious units/cell in the absence or presence of ZDV (Sigma, St. Louis, MO). Culture supernatants were harvested at intervals over 10 days and the proportion of GFP- and hisD-tagged viruses determined by quantitative real-time RT polymerase chain reaction (PCR) with an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). Growth competition experiments were performed twice, once with the mutant gene linked to the hisD marker and once as the reciprocal experiment with the mutant gene linked to the GFP marker; each plot represents a single experiment.
Susceptibility to ZDV and 3TC remained unaffected by the introduction of 207D into a wild-type background (Table 1). Introduction of 207D into the ZDV-resistant background of strain 18C, however, resulted in a 2.7-fold increase in ZDV IC50 (P = 0.06); 3TC susceptibility remained unchanged (Table 1). As expected, introducing 184V into the 18C RT conferred 3TC resistance but enhanced ZDV susceptibility 17-fold (Table 1). Introduction of 207D into 18C184V increased the IC50 for ZDV approximately 2-fold but did not counteract significantly the ZDV-sensitizing effect of the 184V mutation (Table 1).
The effect of the 207D mutation on replicative fitness was tested in a series of pairwise growth competition assays in the absence or presence of ZDV at concentrations of 5.0 μM (for 18C and 18C207D) or 0.2 μM (for 18C184V and 18C184V207D). The choice of ZDV concentrations was based on the IC50's determined in drug susceptibility assays. In the absence of ZDV, introduction of 207D into the wild-type 18A backbone did not alter fitness as compared with wild-type (Fig. 1A and B). The relative fitness of 18C also remained unchanged with the introduction of 207D (Fig. 1C and D). By contrast, in the presence of 5.0 μM of ZDV, a recombinant virus carrying the 207D mutation in the context of other TAMs (18C207D) showed a distinct growth advantage in competition assays (Fig. 1E and F). This growth advantage was not apparent when the 207D mutation was present together with the 184V mutation (Fig. 2).
In this study, we showed that the Q207D mutation in HIV-1 RT increases resistance to ZDV in the presence of several TAMs and increases relative fitness in the presence (but not in the absence) of the drug. Although the effect of the 207D mutation on ZDV susceptibility was relatively modest (2.7-fold), the magnitude of this effect is comparable to that of the L210W substitution, which increased the IC50 for ZDV by ≤2-fold when present together with the 41L and 215Y mutations in clinical isolates and mutants constructed by site-directed mutagenesis.3 Thus, this naturally occurring polymorphism should be added to the growing list of mutations implicated in antiretroviral drug resistance.
In a previous study, we found that the geometric mean IC50 for ZDV of clinical isolates carrying a mutation at codon 207 was more than 20-fold greater than that of isolates that were wild-type at this position.8 Several factors could account for the difference between the results obtained with clinical isolates and the recombinant viruses studied here. The earlier study tested drug susceptibility of biologically cloned virus isolates in activated peripheral blood mononuclear cells, whereas the current study tested recombinant viruses in the PhenoSense assay.11 Moreover, in the previous study, isolates carrying the 207D mutation also carried other RT mutations, such as G196E, V118I, and E44D/A, that were also associated with increased levels of ZDV resistance.8 Another possibility is that differences between the RT backbone of the clinical isolates and the RT studied by site-directed mutagenesis could influence the degree to which the 207D mutation affects ZDV resistance.
In most cases, mutations that confer resistance to antiretroviral drugs do so at the cost of reduced replicative fitness. For example, the M184V mutation confers high-level 3TC resistance but substantially reduces viral replication capacity and fitness in the absence of drug.9,12 Likewise, emergence of L74V during didanosine (ddI) treatment results in ddI resistance but also impairs virus replication due to diminished RT processivity.13 In other cases, mutations emerge that improve replicative fitness without substantially altering the level of drug resistance.14,15
The data presented here suggest that the 207D mutation contributes to increased ZDV resistance and fitness of HIV-1 in the appropriate genetic background and under selective conditions. These results are consistent with the observation that mutations at codon 207 are found in approximately 10% of RT sequences obtained from treatment-naive patients, but in nearly 30% of sequences from ZDV-treated patients.8 These results also demonstrate the potential utility of growth competition assays performed in the absence and presence of drug as a way of probing the contribution of polymorphisms to viral fitness. We did not study the 207E mutation, which can be found as an alternative polymorphism at this position. As the D and E substitutions are similar, it would be interesting to determine whether 207E has the same effect on fitness of ZDV-resistant mutants as 207D.
The finding that the 207D mutation altered ZDV susceptibility only in viruses carrying multiple TAMs suggests that the mechanism by which this mutation contributes to ZDV resistance must depend on interactions made possible by TAM-induced alterations in RT. It will be interesting to determine the biochemical and structural mechanisms by which this mutation exerts its effect.
The authors thank Dr. Douglas Richman for providing HIV-1 isolates 18A and 18C through the AIDS Research and Reference Reagent Program.
1. Boucher CAB, O'Sullivan E, Mulder JW, et al. Ordered appearance of zidovudine resistance mutations during treatment of 18 human immunodeficiency virus-positive subjects. J Infect Dis. 1992;165:105-110.
2. Larder BA, Kemp SD. Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT). Science. 1989;246:1155-1158.
3. Harrigan PR, Kinghorn I, Bloor S, et al. Significance of amino acid variation at human immunodeficiency virus type 1 reverse transcriptase residue 210 for zidovudine susceptibility. J Virol. 1996;70:5930-5934.
4. Kemp S, Shi C, Bloor S, et al. A novel polymorphism at codon 333 of HIV-1 reverse transcriptase can facilitate dual resistance to zidovudine and L-2′,3′-dideoxy-3′-thiacytidine. J Virol. 1998;72:5093-5098.
5. Nijhuis M, Schuurman R, deJong D, et al. Lamivudine-resistant human immunodeficiency virus type 1 variants (184V) require multiple amino acid changes to become co-resistant to zidovudine in vivo. J Infect Dis. 1997;176:398-405.
6. Stürmer M, Staszewski S, Doerr H-W, et al. Correlation of phenotypic zidovudine resistance with mutational patterns in the reverse transcriptase of human immunodeficiency virus type 1: interpretation of established mutations and characterization of new polymorphisms at codons 208, 211, and 214. Antimicrob Agents Chemother. 2003;47:54-61.
7. Whitcomb JM, Parkin NT, Chappey C, et al. Broad nucleoside reverse-transcriptase inhibitor cross-resistance in human immunodeficiency virus type 1 clinical isolates. J Infect Dis. 2003;188:992-1000.
8. Stoeckli TC, Ma Whinney S, Uy J, et al. Phenotypic and genotypic analysis of biologically cloned human immunodeficiency virus type 1 isolates from patients treated with zidovudine and lamivudine. Antimicrob Agents Chemother. 2002;46:4000-4003.
9. Lu J, Kuritzkes DR. A novel recombinant virus assay for comparing the relative fitness of HIV-1 reverse transcriptase variants. J Acquir Immune Defic Syndr. 2001;27:7-13.
10. Lu J, Sista P, Giguel F, et al. Relative replicative fitness of human immunodeficiency virus type 1 mutants resistant to enfuvirtide (T-20). J Virol. 2004;78:4628-4637.
11. Petropoulos CJ, Parkin N, Limoli K, et al. A novel phenotypic drug susceptibility assay for human immunodeficiency virus type 1. Antimicrob Agents Chemother. 2000;44:920-928.
12. Larder BA, Kemp SD, Harrigan PR. Potential mechanism for sustained antiretroviral efficacy of AZT-3TC combination therapy. Science. 1995;269:696-699.
13. Sharma PL, Crumpacker C. Attenuated replication of human immunodeficiency virus type 1 with a didanosine-selected reverse transcriptase mutation. J Virol. 1997;71:8846-8851.
14. Nijhuis M, Schuurman R, de Jong D, et al. Increased fitness of drug resistant HIV-1 protease as a result of acquisition of compensatory mutations during suboptimal therapy. AIDS. 1999;13:2349-2360.
15. Kosalaraksa P, Kavlick MF, Maroun V, et al. Comparative fitness of multi-dideoxynucleoside-resistant human immunodeficiency virus type 1 (HIV-1) in an in vitro competitive HIV-1 replication assay. J Virol. 1999;73:5356-5363.
© 2005 Lippincott Williams & Wilkins, Inc.