Servicio de Virología Molecular, Centro Nacional de Biología Fundamental, Instituto de Salud Carlos III, Carretera Majadahonda-Pozuelo Km. 2, Majadahonda, 28220 Madrid, Spain1
Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain2
Author for correspondence: Cecilio López-Galíndez. Fax +34 91 5097918. e-mail clopez{at}isciii.es
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Abstract |
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Introduction |
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Potent antiviral treatment has brought about a breakthrough in the AIDS epidemic in developed countries. The principal target for anti-HIV therapy has been reverse transcriptase (RT). HIV-1 RT inhibitors can be divided into two groups (De Clercq, 1992 ): nucleoside analogues (Fischl et al., 1987
; Mitsuya et al., 1985
) and non-nucleoside RT inhibitors (NNRTI), like nevirapine (NVP). As opposed to what occurs with nucleoside analogues (Larder et al., 1989
, 1991a
), viruses resistant to NNRTI arise quickly in vivo (Richman et al., 1994
) and in vitro (Balzarini et al., 1993
; Larder et al., 1987
; Nunberg et al., 1991
). The loss of antiviral activity is associated with the acquisition of several mutations around the hydrophobic catalytic pocket in the p66 subunit of the RT, mainly at amino acid positions 103 (K
N), 106 (V
A), 181 (Y
C/I), 188 (Y
C) and 190 (G
A) (Kohlstaedt et al., 1992
; Korber et al., 1998
; Smerdon et al., 1994
). Some of these alterations have an effect not only on the resistant phenotype but also on enzymatic properties (Tantillo et al., 1994
).
One of the consequences of the error-prone replication of RNA viruses is the existence in any HIV-1 virus population of a swarm of related genomes (Meyerhans et al., 1989 ; Sabino et al., 1994
; Wain-Hobson, 1993
), termed quasispecies, prepared for the rapid dominance of different variants in response to different environments. These variants display different phenotypic and genotypic characteristics and they compete for the prevalence of the most fit variant (designated wild-type virus) in the virus population by Darwinian selection. The parameter that better measures the dominance of one virus is virus fitness and, in certain conditions, is related to the presence of a mutant in a virus population (Coffin, 1995
). Recently there have been interesting reports implicating virus fitness of the different HIV-1 antiviral-resistant variants in vivo with its dominance in virus population within patients (de Ronde et al., 2001
; Goudsmit et al., 1996
, 1997
; Harrigan et al., 1996
).
We report the genotypic and phenotypic characterization of HIV-1 NVP-resistant variants selected by in vitro passage of a clonal wild-type virus to increasing amounts of drug. The evolution of the distinct single and double mutants present in the virus population was examined during the passages. To further study the role that each single resistance mutation plays in antiviral resistance, we carried out a fitness study of single resistant variants, in both the presence and absence of the antiviral.
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Methods |
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An MT-2 cell line (Miyoshi et al., 1981 ) was used for the in vitro selection of HIV-1 NVP-resistant variants, for the evaluation of the replicative capacity of the selected virus, for susceptibility testing and for the competition assays. An MT-4 cell line (Miyoshi et al., 1981
) was used in a plaque assay to obtain biological clones. Both cell lines are maintained in RPMI 1640 (Bio-Whittaker) supplemented with 10% foetal bovine serum (Gibco) plus 1% antibiotics (Bio-Whittaker). HT4-6C, HeLa cells expressing the human CD4 receptor (Chesebro & Wehrly, 1988
), were used to determine drug susceptibility. This cell line is maintained in Dulbeccos modified Eagles medium (Gibco) containing 10% foetal bovine serum plus antibiotics (DMEM-10 or growth medium).
Test compound.
NVP (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyridol[3,2-b:2',3'-e][1,4]diazepin-6-one) stock solution was stored at -20 °C until use.
In vitro selection of HIV-1 NVP-resistant variants.
V61 was passaged in 5x106 MT-2 cells in the presence of increasing concentrations of drug (Table 1) at an m.o.i of 0·01, following previous protocols (Gao et al., 1992
, 1994
; Larder et al., 1991a
, 1993). In order to obtain adequate intracellular levels of drug, cells are pre-incubated for 4 h. New medium containing the appropriate drug concentrations was added every 2 days to cultures. Passages were performed with the supernatant of the preceding passage, taking into account that virus titres do not change significantly during passages (Sánchez-Palomino et al., 1993
).
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Biological cloning.
Biological clones were obtained using an agarose MT-4 plaque assay as previously described (Harada et al., 1987 ; Sánchez-Palomino et al., 1993
). Well isolated plaques were picked at random 7 to 10 days post-infection and resuspended in 300 µl of culture medium (Yuste et al., 1999
). Virus stocks from NVP-resistant clones were obtained by infecting 5x106 MT-2 cells in the presence (for resistant clones) or absence (for wild-type virus) of low concentrations of drug (0·25 µM).
Genetic analysis of resistant variants.
Variants obtained by in vitro selection were analysed from HIV-1 proviral DNA of infected cultures. Total cellular DNA from infected cells was extracted by phenolchloroform followed by ethanol precipitation (Perucho et al., 1981 ) for large amounts of cells, or by the Instagene purification matrix (Biorad) for smaller numbers of cells. A 1951 bp fragment including the complete polymerase domain of the RT in the pol gene was amplified by PCR as described previously (Nájera et al., 1994
, 1995
) using Taq polymerase (Perkin Elmer). Primers 54RU (5' AGTTTGCCAGGAAGATGGAAACCA 3', positions 17191742), where the number corresponds to the position in the BH10 genome (GenBank accession no. M15654), and 53RD (5' GGCGAATTCACTAGCCATTGCTCTCCA 3', antisense, complementary to positions 36423669) were used. DNA from biological clones was amplified by using a nested PCR, performed with RT oligonucleotides 3RU and 20RD (Nájera et al., 1994
, 1995
). PCR products were purified with the High Pure PCR product purification kit (Promega) and sequenced with the fmol DNA cycle sequencing system (Promega), with primers 14RD (Nájera et al., 1994
, 1995
) or 15'RU (5' TAGATATCAGTACAATGTGCTTCCAC 3', positions 23332358), according to the manufacturers instructions.
Generation of mutant virus Y181C.
In order to obtain the mutant Y181C virus, in vitro mutagenesis was performed in the infectious molecular clone 89ESO61 using the Quikchange site-directed mutagenesis kit (Stratagene). Mutagenesis was carried out in the p61FA subclone (Olivares et al., 1997 ) with primers 283RU (5' CCAGACATAGTTATCTGTCAATACATGGACG 3', positions 24332463) and 283RD (complementary to above). The mutant Y181C virus was obtained by electroporation of the mutated plasmid in COS-1 cells as previously reported (Olivares et al., 1997
) and grown in MT-4 cells to obtain a stock. Mutation was confirmed by nucleotide sequencing of the complete RT region. Virus recovered was propagated in MT-2 cells and the IC50 of the wild-type and the Y181C viruses was established by the MTS method.
In vitro determination of relative fitness.
Fitness determination was performed by growth competition experiments as previously described (Chao, 1990 ; Escarmís et al., 1996
; Holland et al., 1991
; Yuste et al., 1999
). Cultures between the clone to be tested and the wild-type virus were carried out at an initial proportion of 1:1 for five passages and in three independent experiments. 5x104 MT-2 cells were infected at a m.o.i of 0·05, both in the absence and in the presence of 0·25 µM NVP. For the next passage, fresh MT-2 cells were infected with 5 µl supernatant of the preceding passage. RNA was extracted from 20 µl culture supernatant as described (Boom et al., 1990
), with a guanidinium isothiocyanate lysis buffer and glass milk, and resuspended in 50 µl TE buffer. After isolation of viral RNA, 10 µl of this RNA preparation was reverse-transcribed and amplified using the Titan one-tube RTPCR system (Boehringer Mannheim), according to the manufacturers instructions, with primers 54RU53RD and 3RU20RD. PCR products were purified as described above. Sequencing was performed with primers 15'RU or 14RD. Autoradiographic images of the sequencing gels were analysed on a HP50 scanner and the proportion of each variant in the competition was estimated by densitometry of the specific bands by using the PCBAS program (2.08 version, Isotopenmeßgeräte, 1993).
In the study with the Y181C mutant obtained by direct mutagenesis, the proportion of each molecular species during competition passages was calculated by heteroduplex tracking assay (HTA) (Delwart et al., 1994 ). Briefly, 10 µl of RNA preparation was reverse-transcribed and amplified using the Titan one-tube RTPCR system (Boehringer Mannheim) with primers 226RU (5' CAGTTCCCTTAGATAAAGAATGGAGGAAGTACACTGC 3', positions 22572293) and 250RD (5' CCATTTATCAGGATGGAGTTCCCAACCCATCCAAAGG 3', antisense, complementary to positions 25882625) in the first PCR and primers 15'RU and 248RD (5' GGATGGAGTTCATAACCCATATAAAGGAATGGAGG 3', antisense, complementary to positions 25802615) in the second PCR. cDNA of a wild-type clone obtained in the second PCR was labelled with [
-32P]dTTP (3000 Ci/mmol) in an asymmetric PCR using primer 15'RU. About 10000 c.p.m. of this radioactive PCR probe was mixed with 50100 ng of unlabelled second-round PCR product from the competing viruses in annealing buffer (0·1 M NaCl, 10 mM TrisHCl pH 7·8 and 2 mM EDTA). The DNA mixtures were denatured at 94 °C for 2 min and then quickly cooled. Heteroduplexes were then resolved on a denaturing 15% polyacrylamide15% urea gel in TBE (88 mM boric acid and 2 mM EDTA) at 15 mA for 18 h. Autoradiograms were obtained by exposing gel on a Fuji 2000 instrument for 2 h. The quantification of the ratio of the wild-type cDNA (homoduplex) to the cDNA of the mutant virus (heteroduplex) was determined by densitometry with the help of the PCBAS program.
Fitness vectors were derived from the ratio of the competing clone to the wild-type virus in each passage in relation to the initial proportion (Rn/Ro), as in previous reports (Chao, 1990 ; Escarmís et al., 1996
; Holland et al., 1991
; Yuste et al., 1999
). The vector was obtained by linear regression and the slope represents the relative fitness value of the corresponding virus clone. In this procedure the vector of the reference clone is established as zero (relative fitness 1), a negative value represents a virus less fit than the wild-type and positive value a more fit virus.
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Results |
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Fitness determination
In order to further characterize the single resistant variants virus fitness was determined. Trying to minimize the biological and virus differences among variants, clones with V106A, Y181C and G190A mutations were derived from the same passage (number 6). Clone 6 displayed the V106A change, clone 12 the Y181C mutation, and clone 13 the G190A transition. A wild-type clone from the control culture of V61 was obtained from the same culture passage and it was used as the reference virus in the competition experiments. Fitness estimation was first evaluated by looking at the dominance of the virus in the competitions. This analysis provided a fitness order among the variants, which was the same in the three independent experiments. For more detailed studies the relative fitness values were established from vector slope, obtained as indicated above, and the result of one of these competition experiments is shown in Fig. 2. Fitness values of all three resistant variants in the presence of drug gave values higher than the wild-type clone and the relative fitness order was: Y181C>V106A>G190A>wild-type. In the absence of the drug, the Y181C mutant displayed a fitness value higher than the wild-type and fitness was Y181C>wild-type>V106A
G190A. Values generated in the three competition experiments were not identical but the fitness order of the variants was always maintained.
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Discussion |
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Phenotypic assays performed in HeLa CD4+ cells and MTS assays showed a 100-fold reduction in NVP susceptibility from passage 5. Mutations detected in RT from this passage have been demonstrated to confer NVP resistance by site-directed mutagenesis (Richman et al., 1991 ). At passage 15 an increase in IC50 values from 0·1 to above 50 µM in HeLa assay or from 0·2 to above 100 µM using the MTS method (>500x) (Table 1
) was coincident with the major presence of double variants. In general, successive accumulation of mutations caused an increase in phenotypic resistance (Larder et al., 1991b
). The appearance of double mutants in our study could be the result of the incorporation of new mutations into single mutants or to recombination between single mutants, as previously described in the development of resistance to RT inhibitors (Gu et al., 1995
; Kellam & Larder, 1995
; Moutouh et al., 1996
). However, we could not discriminate between these two possibilities because of the lack of specific genetic markers. Studies with other antivirals have shown that in vivo and in vitro resistance is a progressive process, associated with the accumulation of different mutations in the RT region of the pol gene (Boucher et al., 1992
; Molla et al., 1996
). These data reflect the existence within patients of multiple competing resistant variants (Richman et al., 1994
; Havlir & Richman, 1996
), which results in the prevalence of the most fit virus as a function of the resistant phenotype and other virus factors.
Fitness values obtained from NVP-resistant viruses V106A and G190A displayed relative fitness lower than the wild-type in the absence of drug, as anticipated. In contrast, fitness value obtained with the Y181C NVP-resistant variant in the absence of the drug was greater than the wild-type virus. This result was somewhat unexpected and was in contrast with general concepts which relate wild-type virus with the best adapted and most fit variant present in any environment (Eigen & Biebricher, 1988 ). In the absence of the drug, any resistance mutation is expected to have a negative effect on fitness, the resistance cost (Havlir & Richman, 1996
), and resistant mutants replicate more poorly than wild-type (Coffin, 1996b
). Why this mutation is not present as the wild-type if it confers a fitness advantage to the virus? This could be related to the need for a threshold level for a variant to prevail in a virus population (de la Torre & Holland, 1990
), to clonal interference (Miralles et al., 1999
) or to frequency-dependent selection (Ayala, 1971
). The fact that the Y181C mutant displays a positive fitness is supported by different evidence. The cost of resistance mutations is generally low, as shown by the maintenance of resistant variants without obvious reversion during repeated passages in the absence of drug (Borman et al., 1996
; Gao et al., 1992
) but also in patients that interrupted NVP treatment, even after 20 months without therapy (Havlir & Richman, 1996
). In vitro we detected the appearance of the Y181C mutation in an AZT-resistant virus after 11 passages in the absence of the drug, which became the major form by passage 16 (Nájera et al., 1994
). Also, the Y181C residue is found naturally in HIV-2 viruses (Shih et al., 1991
) and in isolates of subtype O (Descamps et al., 1997
; Quiñones-Mateu et al., 1997
). Also, it has been estimated that the Y181C resistant variant was present in around 7 to 133 per 10000 copies of HIV RNA in plasma from patients before drug treatment (Havlir et al., 1996
). Similarly, there are recent reports on the superior fitness value of the Q151M multiresistant variant in the absence of drugs (Kosalaraksa et al., 1999
).
The positive value of fitness of the Y181C virus (Fig. 2) is small but, due to the very high number of replication cycles in HIV-1, this increase in fitness could suffice for the dominance of the variant in virus populations (Coffin, 1996a
). Fitness values are always relative to the system in which they are determined, generally in vitro conditions (i.e. T cell lines). These fitness values cannot be directly translated to other systems, particularly to in vivo situations, in which different cells lines and changing environments are operating.
In summary, by in vitro culture of a wild-type virus we have obtained NVP-resistant variants with different combinations of resistant mutations. Single resistant mutants were obtained at early passages but were replaced by double mutants at passage 15. Regarding virus fitness, we have found that the Y181C mutant is more fit than wild-type virus in the absence of the drug. This result shows that resistance mutations not only give an advantage for the resistant phenotype but also could give a better fitness to the virus with a positive effect on virus evolution.
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Acknowledgments |
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References |
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Received 13 June 2001;
accepted 11 September 2001.