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J Virol, March 1998, p. 2335-2340, Vol. 72, No. 3
Division of Biological Sciences, the
University of Montana, Missoula, Montana 598121;
Departments of Biochemistry and Radiation Oncology, Eccles
Institute of Human Genetics, University of Utah, Salt Lake City, Utah
841122; and
Georgia VA Research
Center for AIDS and HIV Infections and Department of Pediatrics,
Emory University School of Medicine, Decatur, Georgia
300333
Received 27 June 1997/Accepted 26 November 1997
Mutants of feline immunodeficiency virus (FIV) resistant to
( The emergence of drug-resistant
variants of human immunodeficiency virus type 1 (HIV-1) is believed to
be responsible for the failure of current antiviral chemotherapy to
halt the clinical progression of AIDS (2, 10, 48).
Drug-resistant mutants arise rapidly in HIV-1-infected individuals
treated with most of the currently approved drugs, including nucleoside
and nonnucleoside inhibitors of reverse transcriptase (RT) (12,
26, 27, 49, 50, 52, 53, 55) and the protease inhibitors (8,
21, 50). In addition, numerous mutants which are resistant to RT or protease inhibitors have been selected in vitro (13-15, 17, 22, 25, 39). In both laboratory and clinical isolates, resistance usually correlates with mutations in the RT- or protease-encoding regions, respectively, of the pol gene (50).
Therapeutic strategies using combinations of inhibitors have provided
the greatest success in slowing the clinical decline of HIV-1-infected
individuals. The most successful treatment protocol employs a triple
combination approach of simultaneous treatment with
( We have developed systems using the feline immunodeficiency virus (FIV)
as a model for examining the mechanisms of viral resistance to AIDS
therapy (36). The immune deficiency and neuropathogenesis resulting from infection of domestic cats with FIV are remarkably similar to AIDS in humans (1, 4, 11, 42-45, 61). FIV represents a particularly attractive model for studies of resistance to
inhibitors of HIV-1 due to the similarities between HIV-1 RT and FIV RT
with respect to physical properties, catalytic activities, and
sensitivity to the triphosphate forms of AZT, 2',3'-dideoxycytidine (ddC), 2',3'-dideoxyinosine (ddI),
2',3'-didehydro-3'-deoxythymidine (d4T), and 3TC (9, 33-35,
54). In addition, the first drug-resistant lentiviral variants
selected in vitro were AZT-resistant mutants of FIV (46). We
have subsequently reported FIV mutants resistant to ddI
(16), ddC (31), d4T (62),
( We have recently reported mutants of FIV selected with ( Chemicals.
Phosphonoformic acid (PFA), dCTP, dTTP, and ddC
were purchased from Sigma Chemical Co., St. Louis, Mo. AZT and the
5'-triphosphate of AZT (AZTTP) were provided by Glaxo Wellcome Co.,
Research Triangle Park, N.C.; d4T was provided by Bristol-Myers Squibb
Co., Wallingford, Conn.; 9-(2-phosphonylmethoxyethyl)adenine (PMEA) was
provided by Gilead Sciences, Inc., Foster City, Calif.; ddI was
provided by the Developmental Therapeutics Branch, Division of AIDS,
National Institute of Allergy and Infectious Diseases. 3TC, ( Cells and virus.
Virus produced from a molecular clone of
the Petaluma strain of FIV, 34TF10 (56), was used as
wild-type FIV for these studies. Wild-type and mutant strains of FIV
were grown and maintained in Crandell feline kidney (CrFK) cells with
L&M medium supplemented with 10% fetal bovine serum as previously
described (37, 54). Following selection, FIV mutants were
maintained in medium containing 3 µM 3TC, and all cultures were
replenished with fresh medium and drug every 2 days.
Focal infectivity assay.
Inhibition of FIV infection by
antiviral drugs was quantified by a focal infectivity assay as
described previously (46). Resulting data were plotted as
the percentage of control foci (no drug) versus inhibitor
concentration. Concentrations of drug required to inhibit focus
formation by 50% (50% effective concentrations [EC50s])
were obtained directly from the linear portion of these plots by using
a computer-generated regression line (46). Within an
experiment, each value represents the mean of four determinations. Results from three or more independent experiments were used to derive
the EC50 ± standard error.
Selection and plaque purification of 3TC-resistant mutants.
FIV mutants reported here were obtained by selection with 3TC alone
with a stepwise selection protocol as described previously (62). Briefly, virus generated from 34TF10 was initially
cultured in the presence of 1 µM 3TC and then subjected to five
additional rounds of infection in which the concentration of 3TC was
doubled with each subsequent round, ending in a sixth round of
infection at 32 µM 3TC. Each round of infection was initiated with
cell-free virus from the previous round of infection. The resulting
population, designated 3TR-1c, was resistant to 3TC and AZT (data not
shown). This stock was then plaque purified in 3 µM 3TC as previously described (47) to obtain 3TR-3c and 3TR-7c, which were then used for further analysis.
Enzymes and enzyme assays.
RT was purified from virions of
mutant FIV as previously described (34). Assays for RT
activity with poly(rA)-oligo(dT) or poly(rI)-oligo(dC) as
template-primer were also as reported elsewhere (33, 34).
Kinetic parameters were determined by using intercept values calculated
from double-reciprocal plots (9, 33). RT purified from
34TF10 virions (34) was used as the wild-type control.
Nucleic acid preparation and sequence analysis.
Total
cellular DNA containing proviral DNA was extracted from infected CrFK
cells and used for amplification by PCR as previously described
(31, 47, 54). PCR product was directly sequenced at the
Murdock Molecular Biology Facility with a Taq DyeDeoxy Terminator sequencing kit and analyzed on a model 373A automated DNA
sequencer (Applied Biosystems). Sequencing was performed in the forward
and reverse directions with primers at 250-bp intervals of the
RT-encoding region of the pol gene.
Site-directed mutagenesis.
In order to construct a molecular
clone of FIV containing the Pro-to-Ser mutation at codon 156 of RT, a
2,109-bp EcoRI-HindIII fragment,
corresponding to nucleotides 1871 to 3980 of the proviral portion of
pFIV-34TF10, was cloned into the pTZ18u phagemid vector (Bio-Rad
Laboratories) and mutagenized with the Muta-Gene in vitro mutagenesis
kit (Bio-Rad). The mutagenesis primer
5'-GATATATCAATGAACTTAA-3' was used to introduce
the Pro-156-Ser mutation (mutation underlined). Following sequence
analysis to confirm the presence of the desired mutation, an 869-bp
NsiI-PacI fragment corresponding to nucleotides 2674 to 3543 of pFIV-34TF10 was ligated into
NsiI/PacI-digested pFIV-34TF10 and transformed
into SURE-2 Ultracompetent Escherichia coli (Stratagene).
Clones were sequenced in order to verify the presence of the mutation
and the integrity of the pol gene, and 1 µg of the
resulting plasmid DNA was purified and used to transfect CrFK cells as
previously described (47) for the production of virus. All
constructs were also introduced into the J5 strain of E. coli JM109 for long-term storage and plasmid propagation.
Selection and plaque purification of 3TC-resistant FIV
mutants.
Virus produced from the 34TF10 molecular clone of FIV was
passaged in the presence of increasing stepwise concentrations of 3TC,
as described previously (62) and in Materials and Methods. This population, designated 3TR-1c, was plaque purified in order to
minimize heterogeneity within the mutant population. Two
plaque-purified mutants, designated 3TR-3c and 3TR-7c, were seven- to
eightfold resistant to 3TC (Fig. 1) and
were chosen for further phenotypic characterization.
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Novel Point Mutation at Position 156 of Reverse Transcriptase
from Feline Immunodeficiency Virus Confers Resistance to the
Combination of (
)-
-2',3'-Dideoxy-3'-Thiacytidine and
3'-Azido-3'-Deoxythymidine
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
)-
-2',3'-dideoxy-3'-thiacytidine (3TC) were selected by culturing virus in the presence of increasing stepwise concentrations of 3TC. Two
plaque-purified variants were isolated from the original mutant
population, and both of these mutants were resistant to 3TC.
Surprisingly, these mutants were also phenotypically resistant to
3'-azido-3'-deoxythymidine (AZT) and to the combination of 3TC and AZT.
Purified reverse transcriptase (RT) from one of these plaque-purified
mutants was resistant to the 5'-triphosphates of 3TC and AZT. DNA
sequence analysis of the RT-encoding region of the pol gene
amplified from the plaque-purified mutants revealed a Pro-to-Ser
mutation at position 156 of RT. A site-directed mutant of FIV
engineered to contain this Pro-156-Ser mutation was resistant to 3TC,
AZT, and the combination of 3TC and AZT, confirming the role of the
Pro-156-Ser mutation in the resistance of FIV to these two nucleoside
analogs. This represents the first report of a lentiviral mutant
resistant to the combination of AZT and 3TC due to a single, unique
point mutation.
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
)--2',3'-dideoxy-3'-thiacytidine (3TC),
3'-azido-3'-deoxythymidine (AZT), and a protease inhibitor such as
indinavir (32). A central feature of this combination is the
unique interaction between 3TC and AZT. Mutants resistant to 3TC arise
during therapy through the acquisition of a Met-to-Val or Met-to-Ile
mutation at position 184 of RT, as first reported by Schinazi et al.
(51). However, these mutations at position 184 have been
demonstrated to phenotypically suppress AZT resistance mutations,
thereby providing a basis for sustained drug efficacy (28, 30,
58). The combination of AZT and 3TC represents a significant
improvement over conventional AZT monotherapy in suppressing virus load
and in delaying the resurgence of virus titers associated with the
emergence of drug resistance (28).
)--2',3'-dideoxy-5-fluoro-3'-thiacytidine [()-FTC]
(54), and the combination of AZT and ddI (16).
)-FTC which
are cross-resistant to 3TC due to a Met-to-Thr mutation at position 183 of RT (which corresponds to position 184 of HIV-1 RT)
(54). These FIV mutants are similar in phenotype to
the Met-184-Val and Met-184-Ile mutants of HIV-1, as all of these variants retain wild-type sensitivity to AZT. Here we report that selection with 3TC results in lentiviral variants resistant to 3TC and
to the combination of 3TC and AZT. These mutants may indicate a
possible mechanism by which HIV-1 can evade 3TC-AZT combination chemotherapy.
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
)-FTC,
and the 5'-triphosphate of 3TC (3TCTP) were synthesized and fully characterized by mass spectroscopy, nuclear magnetic resonance, and
high-pressure liquid chromatography as described previously (5, 7,
18). [5-3H]dCTP and
[methyl-3H]dTTP were obtained from Dupont-New
England Nuclear, Boston, Mass. GeneAmp PCR Core reagents were purchased
from Perkin-Elmer Cetus, Norwalk, Conn. The Taq DyeDeoxy
Terminator Cycle sequencing kit was purchased from Applied Biosystems,
Foster City, Calif. Restriction enzymes PstI,
HindIII, NsiI, and PacI were
obtained from Boehringer Mannheim, Indianapolis, Ind., and T4 DNA
ligase was obtained from Gibco BRL, Grand Island, N.Y. All other
chemicals were reagent grade or better.
RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (19K):
[in a new window]
FIG. 1.
Susceptibility to inhibition by 3TC of FIV 34TF10 ( 
)
and the plaque-purified 3TC-resistant mutants 3TR-3c (
) and 3TR-7c
(
). Results are from three or more experiments, with four
determinations per experiment. Bars represent standard errors of the
means.
)-FTC and ddC, as predicted from studies of mutants of FIV selected
with ()-FTC. Surprisingly, both 3TR-3c and 3TR-7c were also
cross-resistant to AZT (Table 1) and were
therefore phenotypically different from all previous mutants of HIV-1
or FIV selected with 3'-thiacytidine nucleosides. Additionally, both mutants showed wild-type susceptibility to ddI, PMEA, d4T, and PFA
(Table 1).
|
RT. Purified RT from 3TR-3c was compared to wild-type FIV RT for susceptibility to inhibition by the 5'-triphosphates of 3TC (3TCTP) and AZT (AZTTP). Inhibition by both 3TCTP and AZTTP was competitive with respect to dCTP and dTTP, respectively. Km and Ki values are summarized in Table 2. 3TR-3c RT was 8.7-fold resistant to inhibition by 3TCTP, based on comparison of Ki/Km ratios for the mutant and wild-type RTs. 3TR-3c RT was also resistant to AZTTP, with a twofold increase in Ki/Km ratio over the wild-type value.
|
Nucleotide sequence analysis. DNA sequence analyses of the RT-encoding regions of the pol genes from 3TR-3c and 3TR-7c were performed in both the forward and the reverse directions. The resulting sequences were compared to that of the 34TF10 molecular clone of FIV. Both plaque-purified mutants contained a C-to-T transition at position 2801 which results in a Pro-to-Ser mutation at codon 156 of FIV RT (Fig. 2). Both isolates shared additional mutations at positions 348 (Ile to Thr, T to C at position 3378) and 469 (Asp to Glu, T to A at position 3742) of RT. 3TR-7c was also shown to contain a unique mutation at amino acid 227 (Thr to Ala, A to G at position 3014) which was not present in 3TR-3c.
|
Site-directed mutagenesis. Based on the three-dimensional crystal structure of HIV-1 RT and the high degree of homology between HIV-1 and FIV RTs in the areas surrounding positions 156 and 183 (corresponding to amino acids 157 and 184 of HIV-1 RT), we would predict that amino acid 156 lies near amino acid 183 in FIV RT. This prediction places Pro-156 in close physical proximity to a position in the YMDD motif which is known to confer resistance to 3TC when mutated in HIV-1 (Met-184) (6, 13, 14, 51, 52, 58), FIV (Met-183) (54), and hepatitis B virus (Met-550) (29). Therefore, we chose to substitute the Pro-156-Ser change into the FIV 34TF10 molecular clone by site-directed mutagenesis to determine the role of this mutation in resistance to AZT and 3TC. The resulting mutant, designated FIVPro156Ser, was eight- to ninefold resistant to 3TC, with an EC50 of 10.8 ± 1.1 µM. The site-directed mutant was also four- to fivefold resistant to AZT, with an EC50 of 7.5 ± 0.4 µM.
Resistance to the combination of 3TC and AZT. We have also determined the susceptibility of FIVPro156Ser to the combination of 3TC and AZT when present simultaneously and in equimolar concentrations in the focal infectivity assay. Inhibition data for the site-directed mutant were determined in parallel with the plaque-purified mutants (3TR-3c and 3TR-7c), wild-type 34TF10, and the site-directed 3TC-resistant mutants, FIVMet183Val and FIVMet183Thr. The results of these assays are shown in Fig. 3 and summarized in Table 3. FIVMet183Val and FIVMet183Thr displayed a twofold decrease in susceptibility to the combination of 3TC and AZT. These data are consistent with previous results which illustrate that these mutants are resistant to 3TC but remain sensitive to AZT. In contrast, FIVPro156Ser and the plaque-purified mutants 3TR-3c and 3TR-7c were seven- to eightfold resistant to the combination of 3TC and AZT. These results illustrate that the Pro-156-Ser mutation in FIV confers resistance to the combination of AZT and 3TC.
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), 3TR-3c (
), FIVMet183Val
(
), and FIVPro156Ser (
|
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The FIV mutants that we have selected with 3TC are unique in that they are not only resistant to 3TC but also resistant to AZT and to the combination of 3TC and AZT. These mutants carry a unique Pro-to-Ser mutation at position 156 of RT which is responsible for resistance to this drug combination. Previously reported 3TC-resistant mutants of HIV-1 and FIV contain mutations in the methionine codon of the YMDD motif of RT (Met to Val/Ile/Thr in HIV-1 and Met to Val/Thr in FIV) (6, 13, 14, 23, 51, 54, 58). These mutations confer resistance to 3TC but not to AZT. Similarly, mutations in HIV-1 previously reported to confer resistance to AZT do not confer resistance to 3TC. The results presented here represent the first report of a lentiviral mutant containing a novel point mutation in RT which confers resistance to 3TC and AZT individually and in combination.
We have recently reported mutants of FIV which were selected with
()-FTC and contained a Met-to-Thr mutation at position 183 of RT
(54). These mutants were resistant to ()-FTC and 3TC but
remained susceptible to AZT. It is intriguing that two closely related
nucleoside analogs would yield different patterns of drug resistance.
These genotypic and phenotypic differences may result from a single
chemical change from a 5-proton to a 5-fluoro on the pyrimidine base of
the oxathiolane nucleoside used for selection. Alternatively, these
differing outcomes may be the result of the differing selection
protocols used to obtain these mutants [high concentration of ()-FTC
versus stepwise selection with 3TC]. Further work with 3TC with a high
drug concentration for the selection is planned.
Previous studies of HIV-1 mutants resistant to the combination of AZT and ddI have illustrated that mutants resistant to combinations of antiviral drugs can arise as the result of point mutations which are not observed for mutants selected with either drug alone (19). The Pro-156-Ser mutation, shown here to confer resistance to the combination of 3TC and AZT in FIV, was also not predicted from the common mutations in HIV-1 or FIV yielding resistance to AZT or 3TC alone. Thus, point mutations which result in multidrug resistance can be quite different from those which confer resistance to monotherapy.
It has been noted elsewhere that the levels of viral resistance to 3TC observed for the FIV mutants resistant to 3TC alone (54), or the combination of 3TC and AZT, differ by an order of magnitude from the levels of resistance seen for HIV-1 mutants obtained both from clinical isolates and in vitro selections. These differences probably result from differences in the phenotypic assays used to determine the susceptibility profiles of FIV and HIV-1 mutants. The focal infectivity assay used to determine the drug susceptibilities of FIV isolates generates data resulting from the inhibition of a single round of viral replication and is based upon the direct quantitation of infectious virions. In contrast, the p24-based assays commonly used to determine drug susceptibilities of HIV-1 isolates are performed over multiple rounds of replication, and the relative level of resistance (fold resistance) is magnified over several cycles of replication.
We have previously described AZT-resistant mutants of FIV which revert very rapidly (within a single round of infection) in the absence of AZT (47). For both the Pro-156-Ser mutants described here, and for Met-183-Thr variants of FIV resistant to 3TC alone (54), the viral isolates remained significantly resistant to 3TC following three rounds of infection in the absence of drug. However, with each of these 3TC-resistant mutants of FIV, a decrease in EC50 was observed by the third round of infection in the absence of drug, suggesting that wild-type virus may have begun to emerge in the populations. This is most likely due to a selective disadvantage of the variants when replicating in the absence of 3TC. Studies of HIV-1 isolates resistant to 3TC have shown that Met-184-Val, Met-184-Thr, and Met-184-Ile mutants may have impaired replication rates relative to that of wild-type HIV-1 in primary peripheral blood mononuclear cell cultures (3, 23). We have noted that 3TC-resistant mutants of FIV also replicate slower and yield consistently lower titers than wild-type FIV when cultured on CrFK cells (data not shown). Detailed comparisons of the replication kinetics of these viruses in CrFK cells and in primary human lymphocyte cultures are in progress.
The Pro-156 position of FIV RT corresponds to the Pro-157 of HIV-1 RT
(Fig. 2). Structural models of HIV-1 RT based on crystallographic data
have shown that Pro-157 is located within the N-terminal portion of the
E helix (positions 155 to 174 of the p66 subunit) and is proximal to
Met-184 (20, 24, 41). Based on the 57% identity and 87%
homology of amino acid sequence within this region (residues 148 to 162 of HIV-1 RT [Fig. 2]) and the overall high degree of homology between
HIV-1 and FIV RTs (38, 56), we predict that the Pro-156
residue of FIV RT is proximal to Met-183 in the active site of the FIV
enzyme. Therefore, we speculate that the resistance to AZT and 3TC
conferred by the Pro-156-Ser mutation may result from an alteration in
deoxynucleoside triphosphate binding and/or template-primer positioning
during reverse transcription (41, 57, 60).
Studies of RTs from 3TC-resistant HIV-1 variants may provide clues to the biochemical consequences resulting from mutations near the active site of the enzyme. The Met-184-Val HIV-1 mutant displays an increased fidelity of nucleotide insertion in gel-based, kinetic assays (40, 59). Additionally, RTs from both the Met-184-Val and Met-184-Ile mutants of HIV-1 produce shorter cDNA products in vitro than does the wild-type enzyme, suggesting that these mutant RTs may be less processive enzymes (3). Due to its location, the Pro-156-Ser mutation in FIV reported here may also have effects on the fidelity and/or processivity of the mutant enzyme.
Combination chemotherapy involving the use of AZT and 3TC, often in conjunction with a protease inhibitor, has provided a substantial therapeutic advantage over conventional monotherapy. The data presented here illustrate that lentiviral mutants resistant to the combination of 3TC and AZT can be selected in vitro and may represent a potential mechanism for the development of multiple drug resistance during combination therapy for HIV-1. In addition, these data suggest that it may not be necessary for HIV-1 to accumulate a large number of point mutations in order to become resistant to the triple combination of 3TC, AZT, and a protease inhibitor. However, the mutants reported here appear to be replication impaired in cell culture and therefore might be expected to result in lower viral loads in vivo. The role of such mutants in pathogenesis will be addressed experimentally with the FIV cat model.
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ACKNOWLEDGMENTS |
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This work was supported by Public Health Service grants AI28189 (T.W.N.) and AI38755 (B.D.P. and T.W.N.) from the National Institute of Allergy and Infectious Diseases and by the Department of Veterans Affairs and the Georgia Research Center on AIDS and HIV Infection (R.F.S.).
We thank Robert M. Lloyd, Jr., for providing technical information and Joan Strange and the Murdock Molecular Biology Facility for DNA sequence analysis and oligonucleotide synthesis.
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FOOTNOTES |
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* Corresponding author. Present address: Center for Comparative Medicine, University of California, Davis, CA 95616. Phone: (530) 752-3414. Fax: (530) 752-7914. E-mail: twnorth{at}ucdavis.edu.
Present address: Departments of Biochemistry and Radiation
Oncology, Eccles Institute of Human Genetics, University of Utah, Salt
Lake City, UT 84112.
Present address: Bayer Corporation, Clayton, NC 27520.
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Abstract
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Materials & Methods
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References
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J Virol, March 1998, p. 2335-2340, Vol. 72, No. 3
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