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Journal of Virology, September 2000, p. 8316-8323, Vol. 74, No. 18
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Response of Foot-and-Mouth Disease Virus to Increased
Mutagenesis: Influence of Viral Load and Fitness in Loss of
Infectivity
Saleta
Sierra,1
Mercedes
Dávila,1
Pedro
R.
Lowenstein,2 and
Esteban
Domingo1,*
Centro de Biología Molecular Severo
Ochoa, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain,1 and Molecular Medicine
Unit, Department of Medicine, University of Manchester, Manchester
M13 9PT, United Kingdom2
Received 20 March 2000/Accepted 20 June 2000
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ABSTRACT |
Passage of foot-and-mouth disease virus (FMDV) in cell culture in
the presence of the mutagenic base analog 5-fluorouracil or
5-azacytidine resulted in decreases of infectivity and occasional extinction of the virus. Low viral loads and low viral fitness enhanced
the frequency of extinction events; this finding was shown with
a number of closely related FMDV clones and populations differing by up to 106-fold in relative fitness in
infections involving either single or multiple passages in the absence
or presence of the chemical mutagens. The mutagenic treatments
resulted in increases of 2- to 6.4-fold in mutation frequency and up to
3-fold in mutant spectrum complexity. The largest increase
observed corresponded to the 3D (polymerase)-coding region, which is
highly conserved in nonmutagenized FMDV populations. As a result,
nucleotide sequence heterogeneity for the 3D-coding region became very
similar to that for the variable VP1-coding region in FMDVs multiply
passaged in the presence of chemical mutagens. The results suggest that
strategies to combine reductions of viral load and viral fitness
could be effectively associated with extinction mutagenesis as a
potential new antiviral strategy.
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INTRODUCTION |
RNA viruses replicate and evolve as
complex mutant distributions termed viral quasispecies as a result of
limited copying fidelity of viral replicases and retrotranscriptases
(2, 11, 13, 16, 28, 49). Error-prone replication predicts
the existence of an error threshold for the maintenance of genetic information (11, 16, 55, 58). The stability of a
quasispecies is determined by the selective superiority of the most fit
and most abundant genome
termed the master sequence (15,
17)and by the copying fidelity during genome replication
(11, 55, 58). An increase in the average error rate above a
critical threshold during template copying should result in the loss of genetic information in a process that has been referred to as violation
of the error threshold or entry into error catastrophe (11, 15,
55, 58). Such a critical transition has been equated with
"melting" of meaningful information through randomization of
nucleotide sequences (15, 55, 58). If applicable to viral infections, violation of the error threshold should result in a loss of
viral infectivity.
In line with theoretical predictions, a number of studies have
documented decreases in viral infectivity concomitant with increased
levels of mutagenesis during RNA genome replication. Ethyl
methanesulfonate, nitrous acid, 5-fluorouracil (FU), or 5-azacytidine
(AZC) increased no more than 1.1- to 2.8-fold the mutation frequencies
at defined single base sites of poliovirus and vesicular stomatitis
virus, even with levels of virus survival below 1% of the yield in the
absence of chemical mutagenesis (29). The high rate of
mutation during a single round of retroviral replication was increased
up to 13-fold by AZC (46). Although vesicular stomatitis
virus readily gained fitness upon large population passages in cell
cultures (43), such a gain was severely limited by the
presence of mutagenic agents (31). More recently, it was
shown that mutagenic nucleoside analogs produced a loss of replicative
potential of human immunodeficiency virus type (HIV-1) upon serial
passage in human cells, in a process that has been termed lethal
mutagenesis of HIV-1 (33, 34). These observations, together
with the generally high mutation rates during RNA genome replication
(2, 11, 13, 15, 28), have suggested that replication
fidelity of RNA viruses may already be close to the error threshold and
that such proximity may maximize virus adaptability (13, 15, 28,
29, 31).
There is little information on the effects of viral load, viral
fitness, and the types and numbers of mutations associated with a loss
of viral infectivity of RNA viruses associated with increased
mutagenesis. Such information is needed to assess the possible
development of antiviral strategies based on virus entry into error
catastrophe. In the present report, we have analyzed the effect of two
mutagenic base analogs, FU and AZC, on the mutation frequency, mutant
spectrum complexity, and survival of the important picornavirus
pathogen foot-and-mouth disease virus (FMDV) (48, 51).
Multiple virus passages in the presence of mutagens resulted in
increases of up to 6.4-fold in the mutation frequency at some genome
segments. Additionally, we document important effects of viral load and
viral fitness on virus extinction driven by increased mutagenesis.
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MATERIALS AND METHODS |
Cells, viruses, and infections.
The origin of baby hamster
kidney 21 (BHK-21) cells has been described previously (12,
57). Cells were grown in Dulbecco's modified Eagle's medium
(DMEM) (Gibco) supplemented with nonessential amino acids (Gibco) and
5% fetal calf serum (Gibco). A number of FMDV clones have been used in
studies with chemical mutagens. (i) FMDV C-S8c1 is a plaque-purified
derivative of natural isolate C1-Sta Pau-Spain 70, a
representative of the European subtype C1 FMDVs
(57). (ii) MARLS is a monoclonal antibody escape mutant obtained from FMDV C-S8c1p213 (C-S8c1 passaged 213 times in BHK-21 cells [1, 4]); MARLS includes substitution Leu-144 
Ser at antigenic site A located within the G-H loop of capsid protein VP1 (40). The replicative fitness of MARLS in BHK-21 cells
is about 130-fold higher than that of C-S8c1 (4; S. Sierra and C. Escarmís, unpublished results). (iii)
C229 is a highly debilitated clone derived from
multiple plaque-to-plaque transfers of FMDV C-S8c1 (19). The
replicative fitness of C229 in BHK-21 cells was about
10% that of C-S8c1 (19, 20). (iv) A number of low-fitness
subclones were obtained from clone H5 (derived from C-S8c1p113
[19]) which was passaged 91 times in plaque-to-plaque
transfers (termed H915 [19; Sierra
and Escarmís, unpublished]). For these subclones, relative
fitness values were estimated on the basis of the number of infectious
progeny produced per plaque; this estimate indicated that the fitness
of H915 subclones was about 103-fold lower
than the fitness of C229 (19; Sierra
and Escarmís, unpublished). A good correspondence between
relative fitness values obtained in growth competition experiments and
those estimated on the basis of infectious progeny production has been
previously documented (19).
Procedures for infection of BHK-21 cell monolayers with FMDV in liquid
medium and for plaque assays in semisolid agar medium have been
described previously (1, 12, 57). The standard viral
production assay consisted of the infection of 4 × 106 BHK-21 cells with FMDV at a multiplicity of infection
ranging from 10
5 to 10 PFU per cell. DMEM used for FMDV
infections contained 2% fetal calf serum. Infections were allowed to
proceed until cytopathology (cell detachment) was nearly complete (6 to
24 h postinfection). To control for the absence of contamination,
parallel passages of supernatants of mock-infected cells were carried
out throughout the experiments, with no signs of infectivity or
cytopathology in the cultures.
Mutagenic agents and mutagenesis treatments.
FU (Sigma) and
AZC (Sigma) were used as mutagenic base analogs. Both can be
incorporated into DNA and RNA, and both interfere with RNA processing
and translation (5, 21, 44, 45). They have been shown to be
mutagenic for a number of RNA viruses (14, 23, 26, 29, 32, 46,
50). To prepare DMEM containing FU or AZC, the appropriate amount
of analog was dissolved in the medium and sterilized by filtration to
yield solutions of 2.5 mg/ml, which were diluted as needed. Medium with
FU was stored at 4°C for a maximum of 15 days; medium with AZC was
freshly prepared for each experiment.
The effects of a number of doses and times of exposure to mutagens on
BHK-21 cells and FMDV production were tested in a series
of preliminary
experiments. In the standard assay for mutagenesis
(used for all
experiments, except when indicated otherwise), confluent
cell
monolayers were pretreated for 13 h with 200 µg of FU per
ml or
6 h with 10 µg of AZC per ml; then, the cells were washed
with
DMEM, infected with FMDV (adsorption for 1 h at 37°C), washed
for 1 min with 0.1 M phosphate buffer (pH 6.0) (to inactivate
unadsorbed virions), and washed again extensively with DMEM. The
infection was allowed to proceed in the presence of the same
concentration
of either FU or AZC for 20 to 24
h.
For serial passages in the presence of a mutagen, viral progeny were
used either undiluted or diluted 10-fold before the next
infection.
Virus was titrated after each passage. When no cytopathology
was
observed, at least three serial blind passages were carried
out in the
absence of a mutagen prior to viral detection tests.
We define
extinction as the situation in which no infectivity
and no reverse
transcription (RT)-PCR-amplifiable material are
detected in the culture
supernatant after these three blind passages.
Populations passaged in
the presence of a mutagen are indicated
with the abbreviation of the
mutagen and passage number (e.g.,
C-S8c1FUp15 is C-S8c1 passaged 15 times in the presence of
FU).
The toxicity of mutagenic agents for confluent BHK-21 cell monolayers
was monitored by determining cell viability either by
fluorescence-activated cell sorter (FACS) analysis using propidium
iodide (
61) or by trypan blue exclusion after exposure to
the
base analogs. Cells were detached by trypsin treatment and added
to
the culture medium (which contained cells detached during culturing)
prior to centrifugation and viability measurements. Viable cells
comprised at least 75% of the total under the doses and times
of
exposure to mutagens used prior to
infection.
cDNA synthesis, PCR amplification, and nucleotide
sequencing.
Viral RNA was extracted by mixing 150 µl of medium
containing virus with 300 µl of Triazol reagent (Gibco) and
incubating the mixture for 5 min at room temperature. Then, 100 µl of
chloroform was added and mixed vigorously, and the mixture was
incubated for 10 min at room temperature. The RNA was recovered from
the aqueous phase by ethanol precipitation. cDNA synthesis and PCR amplification (RT-PCR) were performed as previously described (19). Prior to RT-PCR amplification, the amount of
FMDV-specific RNA was quantitated by dot blot hybridization as
previously described (8, 9) using purified FMDV C-S8c1 RNA
as a standard. The same amount of viral RNA (0.5 to 2 ng) was used for
each RT-PCR amplification to ensure an excess of template molecules for
copying by avian myeloblastosis virus reverse transcriptase and
amplification by Pfu DNA polymerase (Promega)
(6). For each RNA sample, one or several independent
amplification reactions were carried out, including a negative control
RT-PCR (excluding viral RNA). Two FMDV genomic regions were subjected
to RT-PCR amplification: residues 3193 to 3869 (spanning the VP1-coding
region) and residues 6609 to 8035 (spanning the entire 3D
[polymerase]-coding region). Numbering of residues is that used in
reference 20. The oligonucleotide primers used for
PCR amplification and nucleotide sequencing are based on primers which
have been previously described (1, 19) and which included
restriction enzyme sites for molecular cloning (BamHI and
SacI for the VP1-coding region and BamHI and
EcoRI for the 3D-coding region). Amplified DNAs were
digested with the appropriate restriction enzymes, ligated to plasmid
pGEM-4Z (Promega) digested with the same restriction enzymes, and
cloned in Escherichia coli DH5
. White colonies were grown
in Luria-Bertani medium, and plasmid DNA was obtained using a Wizard SV
Minipreps kit (Promega). DNA manipulations and cloning techniques were
carried out using standard procedures (53). Nucleotide
sequences were determined with PCR-amplified DNA or plasmid DNA using a
Big Dye Terminator Cycle Sequencing kit (Abi Prism; Perkin-Elmer) and
an automated sequencer (ABI373). Sequences were analyzed with a DNA
Star 4.0 pack.
The heterogeneity of the mutant spectrum of viral quasispecies was
quantitated by use of the mutation frequency, which is
the number of
mutations found relative to the number of nucleotides
sequenced. The
mutation frequency is calculated by dividing the
number of different
mutations found in a set of genomes (when
compared with the consensus
nucleotide sequence) by the total
number of nucleotides sequenced
(
11,
15). Another parameter
used to quantitate heterogeneity
is the normalized Shannon entropy,
which is a measure of the proportion
of identical sequences in
a distribution. The possible values of the
normalized Shannon
entropy range from zero to one. For example, a set
of 20 identical
genomic sequences is defined by the minimum normalized
Shannon
entropy of zero. A set of 20 genomes, each one differing in
nucleotide
sequence from any other genome in the set, is defined by the
maximum
normalized Shannon entropy of one, irrespective of the number
of mutations distinguishing each sequence from the others (reviewed
in
reference
60).
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RESULTS |
Serial passage of FMDV in the presence of FU or AZC may lead to
viral extinction.
In initial experiments, we monitored cell
viability and production of FMDV in a single round of infection of
BHK-21 cells with FMDV C-S8c1 in the presence of increasing
concentrations of FU or AZC (Fig. 1).
Concentrations of FU of 100 to 1,000 µg/ml resulted in a reduction in
virus progeny production of 50- to 100-fold (Fig. 1B), with a BHK-21
cell viability of 75% after 36 h of exposure to the base analog
(Fig. 1A). A similar decrease in FMDV production was obtained in the
presence of 10 µg of AZC per ml (Fig. 1D), with a BHK-21 cell
viability of 25% after 24 h of exposure to the drug (Fig. 1C). To
investigate whether additional losses of infectivity could be induced
by extended mutagenic treatments, FMDV C-S8c1 was serially passaged in
the presence or absence of FU or AZC and with or without a 1/10
dilution of viral infectivity prior to each infection. The results
(Fig. 2) show a decline in virus titer
and occasional episodes of viral extinction when virus progeny were
diluted to titers in the range of 103 to 104
PFU/ml. Viral extinction was dependent on the presence of a mutagen, since the same preextinction populations (Fig. 2) survived and produced
titers of 104 to 106 PFU/ml in parallel
infections in the absence of a mutagen. These results suggest that a
small population size contributes to FMDV extinction in the presence of
FU or AZC.
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FIG. 1.
BHK-21 cell viability and FMDV C-S8c1 production in the
presence of FU (A and B) and AZC (C and D). The origin of BHK-21 cells
and FMDV C-S8c1 and procedures for cell growth, mutagenesis treatment,
quantification of cell viability, and infection with FMDV are detailed
in Materials and Methods. Percent viabilities are calculated relative
to those in parallel untreated BHK-21 cell cultures. Absolute
viabilities for untreated BHK-21 cells were at least 95%. Time in the
cell viability panels (A and C) refers to the time elapsed between the
addition of a mutagen to cells and the determination of cell viability.
In the experiments with FU, cell viability was quantitated by FACS
analysis (5,000 events per sample); in the experiments with AZC, trypan
blue exclusion (200 to 500 cells per sample) was used. Control assays
showed that differences in percentages of viable cells after exposure
to the same doses of mutagens in independent experiments and with the
two procedures to determine cell viability did not exceed 20%.
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FIG. 2.
Infectivity values upon passage of FMDV C-S8c1 in the
absence or presence of FU or AZC. Conditions for mutagenic treatment
and for determination of FMDV infectivity are detailed in Materials and
Methods. Filled circles indicate passages of undiluted virus in the
presence of mutagens, and squares correspond to passages in which the
virus was diluted 10-fold prior to each infection (to a titer 1/10 the
one indicated on the ordinate), irrespective of the virus titer
obtained. Empty circles indicate serial passages of undiluted virus in
the absence of mutagens (the same experimental series plotted on the
two panels). No extinction of FMDV C-S8c1 has ever been observed in
serial passages of viruses, even with 100- or 1,000-fold dilutions
intervening between passages, in the absence of mutagens
(56). Preextinction populations are indicated by arrows. The
preextinction population C-S8c1AZCp10 was split into two sublineages;
one was extinguished at passage 11, and the other could be further
passaged and was extinguished at passage 21. Loss of infectivity was
ascertained by three additional blind passages, with no evidence of
infectivity or RT-PCR-amplifiable FMDV RNA in the culture supernatant
from the third passage. Preextinction populations were tested in
infections of BHK-21 cells in the absence of a mutagen; in all
instances, titers produced were 104 to 106
PFU/ml. All titrations were done in triplicate. Standard deviations
(not included in the plots) never exceeded 50%.
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Low fitness favors extinction by increased mutagenesis.
To
investigate the effect of viral fitness on FMDV production in the
presence of mutagens, clones C229 and MARLS (both
derived from C-S8c1 and with fitnesses of 0.1 and 130 relative to
C-S8c1, respectively [19; Sierra and
Escarmís, unpublished]) were compared with C-S8c1 with regard
to virus production in the presence or absence of FU or AZC. For the
high-fitness FMDV MARLS, the decrease in virus production attributable
to the mutagenic treatment was 2- to 11-fold, whereas for the
low-fitness C229 clone, the decrease was 87- to
446-fold (Fig. 3A). The effect of FU also
was tested on preextinction population C-S8c1FUp15 (described in Fig.
2) and on six highly debilitated subclones of H915
(Fig. 3B). Decreases in virus yield of 102- to
104-fold were observed for four subclones, and an
irreversible loss of infectivity was seen for C-S8c1FUp15 (Fig. 3B).
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FIG. 3.
Effect of viral fitness on the decrease in infectivity
in a single round of infection in the absence or presence of FU or AZC.
Empty columns indicate viral yield in the absence of a mutagen; black
columns indicate yield in the presence of FU (200 µg/ml); grey
columns indicate yield in the presence of AZC (10 µg/ml). (A) FMDV
populations MARLS, CS8c1, and C229 (with relative
fitness values of 10, 1, and 0.1, respectively; described in Materials
and Methods) were tested by infecting 4 × 106 BHK-21
cells with 5 × 105 PFU of virus. (B) In this
experiment, 3 × 105 BHK-21 cells were infected with
2 × 102 PFU of the indicated virus. C-S8c1 and MARLS
were the same preparations as those used in panel A, diluted in DMEM;
C-S8c1FUp15 is the preextinction population shown in Fig. 2;
H915-1, H915-2, H915-3,
H915-4, H915-5, and
H915-6 are highly debilitated clones derived from
C-S8c1 (19; Sierra and Escarmís,
unpublished; see Materials and Methods). Empty columns indicate viral
yield in the absence of a mutagen; filled columns indicate yield in the
presence of FU (200 µg/ml). Yields of H915-5 and
H915-6 in the presence of FU were undetectable, but
virus reemerged after one blind passage in the absence of the mutagen.
Extinction of C-S8C1FUp15 was confirmed by blind passages, as detailed
in the legend to Fig. 2. Titrations were done in triplicate, and
standard deviations are indicated. Procedures for chemical mutagenesis
and determination of viral infectivity are described in Materials and
Methods.
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In serial passages in the absence or presence of mutagens (Fig.
4), extinction of low-fitness
C
229 was more frequent than extinction of high-fitness
MARLS. In particular,
extinction of C
229 in FU was
rapid even with undiluted virus. Again, when a preextinction
population
was used to infect BHK-21 cells in the absence of a
mutagen,
10
3 to 10
6 (depending on fitness values) PFU of
progeny virus were produced.
Thus, low fitness contributes to FMDV
extinction in the presence
of FU or AZC.
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FIG. 4.
Serial passage of MARLS (top panels) and
C229 (bottom panels) in the absence or presence of FU
or AZC. FMDV MARLS and C229 are described in Materials
and Methods. MARLS has a 1,300-fold-higher relative fitness than
C229 (19; Sierra and
Escarmís, unpublished). The experimental design is identical to
that described in the legend to Fig. 2. Empty circles correspond to
serial passages of undiluted virus in the absence of a mutagen. Filled
circles correspond to passages of undiluted virus, and squares
correspond to passages in which the virus was diluted 10-fold prior to
infection, irrespective of the virus titer obtained. Preextinction
populations are indicated by arrows. Loss of infectivity was
ascertained by absence of infectivity and RT-PCR-amplifiable material
after three blind passages, as detailed in Materials and Methods and
the legend to Fig. 2.
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Treatment of FMDV with FU and AZC results in nonuniform increases
in mutation frequency.
Consensus nucleotide sequences and
sequences from molecular clones (mutant spectrum) were determined for
FMDV C-S8c1 at passages 1 and 25 in the absence or presence of FU and
at passages 1 and 10 (a preextinction population) in the presence of
AZC. The maximum increase in the mutation frequency (Table
1) was 6.4-fold, observed for the
3D-coding region of virus passaged in FU relative to the population
passaged in the absence of the mutagen; for other populations and
genomic regions, the increase in the mutation frequency ranged between
2- and 4-fold. Shannon entropies of 1 or near 1 were attained in
populations subjected to multiple mutagenesis passages, indicating an
increased algorithmic complexity of the mutant spectrum (see Discussion). The most abundant types of mutations after multiple passages in FU were base transitions (mainly U C, C U, and G
A). With AZC treatment, preextinction population C-S8c1AZCp10 preferentially showed transitions C U, G A, and A G (Table 2). Thus, increases in mutation frequency
and in mutant spectrum complexity which were nonuniformly distributed
along the FMDV genome were observed in FMDV populations that were
passaged in the presence of chemical mutagens and that underwent
occasional extinction.
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TABLE 1.
Quantification of genetic heterogeneity in the mutant
spectrum of FMDV progeny in the absence or presence of the
mutagenic base analog FU or AZCa
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TABLE 2.
Types of mutations in the mutant spectrum of FMDV C-S8c1
progeny in the absence or presence of the mutagenic base analog FU
or AZCa
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DISCUSSION |
Nature and location of chemically induced mutations in the FMDV
genome.
In the present study, we have reported the effect of two
mutagenic base analogs, FU and AZC, previously used to generate mutants of other RNA viruses (14, 23, 29, 32, 46, 50), on genomic
sequences and infectivity of FMDV. With FU, the most abundant mutation
type found in the mutant spectrum of treated populations was transition
U C, which affected a total of 11 sites; only 1 site in untreated
populations included this mutation (Table 2). This mutation is expected
from the ambiguous reading of fluorouridine (as complementary to
adenosine) once incorporated into plus-strand RNA molecules acting as
replicative intermediates (52, 63). The second most abundant
transitions (C U and G A) are expected to occur when
fluorouridine takes the place of cytidine, often when concentrations of
cytidine are low (63). In AZC-treated populations, the most
frequent mutations were A G, G A, and C U transitions.
Transition G A was also reported as an abundant type of AZC-induced
mutation in a retroviral vector (47). However, in the latter
study, as well as in bacteria (7, 10), C G and G C
transversions were the most frequent AZC-induced mutations, while in
our analyses, these transversions were infrequent (Table 2). The
Pfu polymerase used for the RT-PCR procedure is about 10 times more accurate than the Taq polymerase, an enzyme which yielded a basal error rate of <104 substitution per
nucleotide in a similar number of amplification cycles with FMDV RNA as
a template (41). Therefore, although the possibility cannot
be excluded that one particular mutation could be due to
misincorporation during the in vitro RT-PCR amplification rather than
to spontaneous or chemical mutagenesis during FMDV replication, the
mutation frequency values observed (Table 1) ensure that the vast
majority of mutations were present in FMDV RNA.
Two complementary types of measurements have been carried out to
characterize the extent of heterogeneity in the mutant spectra
of FMDV
quasispecies: mutation frequencies and normalized Shannon
entropies
(
60). The former describes the numbers of mutations
present
in the mutant spectrum, without attending to their distribution
among
individual genomes. The latter is a measure of algorithmic
complexity
in that it reflects the number of genomes with a different
nucleotide
sequence in a given mutant spectrum. We chose to compare
two regions of
the FMDV genome: the capsid protein VP1- and the
3D-coding regions
(Table
1). In the VP1- and 3D-coding regions,
59% (excluding two point
deletions; Table
2) and 36%, respectively,
of mutations found in
populations treated with FU or AZC were
nonsynonymous (led to an
amino acid replacement; Table
3). A
comparison of these nonsynonymous replacements with those found
among natural FMDV type C isolates and laboratory variants
indicated
that most replacements found in populations treated with FU
or
AZC were unique to mutagenized populations (Table
3). Except
for 3D
substitution I-334
M, which affected
-strand 2 (located
within
motif C, three residues before the catalytic triad GDD
[
25]), all other 3D substitutions were located in
loops linking
helices and
strands, assuming that amino acids
assigned to
structural motifs in poliovirus 3D (
25) are
located in the equivalent
amino acids of aphthovirus 3D
(
37).
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TABLE 3.
Amino acid replacements found in VP1 and 3D of the
mutant spectrum of FMDV populations subjected to
chemical mutagenesis
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The 3D-coding region is very conserved among aphthoviruses and did
not show any increase in mutation frequency after FMDV
C-S8c1 was
subjected to 25 passages in the absence of mutagens
(Table
1).
Treatment with FU resulted in an interesting equalization
of mutation
frequencies for the VP1-coding region (2.4 × 10
3
substitutions per nucleotide) and the 3D-coding region (1.8 ×
10
3 substitutions per nucleotide). These comparisons
(summarized
in Table
1) led to similar conclusions when mutation
frequencies
were calculated relative to the consensus sequence of each
particular
population or to the sequence of the initial C-S8c1 clone.
Therefore,
rather than large increases in average mutation frequencies,
the
mutagenic treatment was noted as inducing increases in mutation
frequencies in a genomic region (encoding 3D) which generally
undergoes
little variation and which maintains a mutant spectrum
of limited
complexity. Modest increases in mutational load in
otherwise conserved
regions may be critical to drive the virus
to extinction. Mutagenic
treatments may override mechanisms that
normally tend to reduce the
heterogeneity of particular genomic
regions. This point is currently
under further
investigation.
Prospects for viral extinction as a therapeutic tool.
The
results of increased mutagenesis and loss of infectivity reported here
for FMDV indicate that extinctions were stochastic, not systematic.
This conclusion is illustrated by both extinction and survival after
identical passages of C-S8c1AZCp10 (Fig. 2). Low viral loads favored a
loss of infectivity of FMDV populations which had already accumulated
mutations due to a history of passage in the presence of a mutagen
(Fig. 2). Extinctions were not due exclusively to small population
sizes, since passage of small FMDV C-S8c1 populations in the absence of
a mutagen did not lead to extinction (56) and preextinction
populations always yielded large numbers of progeny virus in the
absence of a mutagen. Furthermore, extinction was very unlikely even
after many serial plaque-to-plaque transfers of C-S8c1
(16; C. Escarmís et al., unpublished results).
A second, important influence on FMDV extinction was viral fitness. Low
fitness favored viral extinction by increased mutagenesis
(Fig.
3 and
4). This information on the effects of viral load
and viral fitness is
relevant to a potential application of extinction
mutagenesis as an
antiviral strategy in vivo. Administration of
antiviral inhibitors
often results in a decrease in viral load
that may be sustained in the
case of combination therapy, as documented
in many studies on HIV-1
(
18,
22,
24,
54). Also, selection
of inhibitor-resistant
variants may involve transient decreases
in viral fitness
(
42). Viruses would appear to be vulnerable
to extinction
mutagenesis at these low viral loads and low fitness
intervals. Our
results with a number of FMDV clones and populations
suggest that FU
was more efficient than AZC in driving viral extinction
(Fig.
4), in
agreement with its increased mutagenic potential,
with respect to AZC,
reported for other viruses (
14,
50).
Although FU is used in
anticancer therapy (
45), it would be
desirable to design new
drugs to target the fidelity properties
of RNA replicases or
retrotranscriptases, a notion supported by
mounting evidence that
structural alterations of these enzymes
may increase or decrease their
copying fidelity properties (e.g.,
references
38 and
62). In spite of obvious difficulties in
the
implementation of extinction mutagenesis as a valid antiviral
strategy
(
11), nonretroviral riboviruses would seem to be the
most
vulnerable to enhanced mutation rates, since their replication
and
survival depend on an RNA-dependent RNA replication process
in infected
organisms, with no insertion of DNA copies of the
viral genome into
cellular DNA. In this respect, studies done
with additional RNA viruses
to find parallels or differences with
the conclusions drawn from the
FMDV results would be of great
interest.
Given the pertinacious adaptability of RNA viruses and the
high-frequency isolation of escape mutants (resistant to antibodies,
cytotoxic lymphocytes, and antiviral agents) whenever an effective
selective constraint is in operation (
11,
28,
41,
42),
it is
important to explore whether entry into error catastrophy
(lethal
mutagenesis) could be developed into a new antiviral strategy,
as has
been suggested by several authors (
11,
28,
29,
33,
34). In
the present report, we have shown, using chemical mutagens
as model
drugs, that such an approach can also be effective with
the important
animal pathogen FMDV (
48,
51). The observations
on the
influence of viral load and viral fitness encourage the
design of
virus-specific, fidelity-reducing drugs that could be
applied in
combination with immunotherapy and inhibitors of viral
replication.
Research in this direction is currently in
progress.
| |
ACKNOWLEDGMENTS |
We are indebted to C. Escarmís and N. Pariente for
valuable discussions, A. T. Larregina for help with FACS analyses,
and L. Hall, E. Madueño, and R. Gutiérrez for help
with sequencing.
Research in Madrid was supported by grants PM97-0060-C02-01 and EU PSS
0884 and Fundación Ramón Areces. S.S. was supported by a
predoctoral fellowship from CAM. Work in Manchester was supported by a
Sir Henry Wellcome award for innovative research (053995) to P.R.L. and
E.D. and an International Research Collaboration grant from the
Wellcome Trust (049862) to P.R.L. and E.D.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centro de
Biología Molecular Severo Ochoa, Universidad Autónoma de
Madrid, Cantoblanco, 28049 Madrid, Spain. Phone: 34-91-397 8485. Fax:
34-91-397 4799. E-mail: edomingo{at}cbm.uam.es.
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Abstract
Introduction
