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Journal of Virology, October 2000, p. 9546-9552, Vol. 74, No. 20
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Unusual Distribution of Mutations Associated with Serial
Bottleneck Passages of Human Immunodeficiency Virus Type 1
Eloisa
Yuste,1
Cecilio
López-Galíndez,2 and
Esteban
Domingo1,*
Centro de Biología Molecular "Severo
Ochoa," Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid,1 and Centro Nacional de
Biología Fundamental, Instituto de Salud Carlos III, Majadahonda,
28220 Madrid,2 Spain
Received 8 May 2000/Accepted 18 July 2000
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ABSTRACT |
Repeated bottleneck passages result in fitness losses of RNA
viruses. In the case of human immunodeficiency virus type 1 (HIV-1), decreases in fitness after a limited number of plaque-to-plaque transfers in MT-4 cells were very drastic. Here we report an analysis of entire genomic nucleotide sequences of four HIV-1 clones derived from the same HIV-1 isolate and their low-fitness progeny following 7 to 15 plaque-to-plaque passages. Clones accumulated 4 to 28 mutations
per genome, with dominance of A 
G and G A transitions (57% of
all mutations) and 49% nonsynonymous replacements. One clone
but not
three sibling clonesshowed an overabundance of G A transitions,
evidencing the highly stochastic nature of some types of mutational
bias. The distribution of mutations along the genome was very unusual
in that mutation frequencies in gag were threefold higher
than in env. Particularly striking was the complete absence
of replacements in the V3 loop of gp120, confirmed with partial
nucleotide sequences of additional HIV-1 clones subjected to repeated
bottleneck passages. The analyses revealed several amino acid
replacements that have not been previously recorded among natural HIV-1
isolates and illustrate how evolution of an RNA virus genome, with
regard to constant and variable regions, can be profoundly modified by
alterations in population dynamics.
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INTRODUCTION |
Retroviruses and in particular human
immunodeficiency virus type 1 (HIV-1) mutate and recombine at high
rates (14, 15, 39, 55, 60, 61, 70, 74). Rapid genetic
variation, together with the short replication times of HIV-1
(8), generates complex and highly dynamic mutant swarms
termed viral quasispecies (10, 18-22, 42, 66, 69). The
mutant spectra of viral quasispecies constitute reservoirs of
phenotypically relevant variants, as evidenced by the
nonsyncytial-to-syncytial switch in infected individuals or the rapid
selection of antibody-, cytotoxic-T-cell-, or inhibitor-resistant
mutants in viral populations in vivo (2-4, 32, 33, 36, 37, 41,
46, 47, 56, 59, 65). Although most individual mutations in mutant
swarms of RNA viruses may not be of immediate or even long-term
selective value for the virus (63), evolution of viral
quasispecies can be adaptive and may exert an influence in viral
pathogenesis (26, 34, 76; reviews in 10, 28,
29). The adaptive potential of viral quasispecies is manifested
by quantitatively important fitness variations as viruses evolve in
constant or changing environments (for recent examples and reviews, see
10, 11, 13, 31, 48, 50-52, 75).
Large-population passages under a constant environment tend to produce
fitness gains in viral populations (10, 11, 13, 25, 51, 52).
In contrast, bottleneck eventsexperimentally realized in an extreme
case by serial plaque-to-plaque transfers of virus on cell
monolayersoften lead either to average fitness losses (6, 17,
24, 78) or to limitations of fitness gains (23, 51,
52). The decrease in fitness mediated by repeated bottlenecks has
been interpreted as the result of an accentuation of Muller's ratchet
effect (45). According to this model, asexual populations of
organisms with a small population size will tend to incorporate
deleterious mutations unless compensatory mechanisms such as
recombination can restore mutation-free genomes (45). For
RNA virus quasispecies, accumulation of deleterious mutations is
expected from successive rounds of random sampling of genomes from the
mutant spectrum (reviewed in 10, 11).
In retroviruses, decreases in fitness as a result of serial bottleneck
passages were first documented with HIV-1 following plaque-to-plaque
passages on MT-4 cells (78). In this virus, fitness losses
were unexpectedly drastic when compared with the fitness losses
experienced by other RNA viruses, such as bacteriophage 
6,
vesicular stomatitis virus, or foot-and-mouth disease virus (FMDV) subjected to similar passage regimens (6, 17, 24). Only 4 out of 10 HIV-1 clones could produce viable progeny after 15 plaque-to-plaque transfers, and 3 of the 4 survivors displayed important decreases in fitness (78). Very little is known
about the numbers and types of mutations which accompany fitness
decreases of RNA viruses when they are subjected to sequential
bottleneck passages. In the case of the animal picornavirus
pathogen FMDV, debilitated clones showed some unusual genetic
lesions (infrequent or absent in populations evolved without
intervening bottlenecks; 24, 25). Such lesions included
mutations that resulted in amino acid replacements at internal sites of
the viral capsid and a unique elongation of five adenylate residues
which resulted in an internal polyadenylate tract of variable length
preceding the second functional AUG initiation codon of the FMDV genome (24, 25). No information on genetic lesions associated with fitness losses in retroviral genomes is available. Here we report complete HIV-1 genomic sequences of HIV-1 clones subjected to plaque
transfers that led to severe fitness losses. The results reveal a broad
spectrum of mutations associated with fitness decrease and an
unexpected distribution of mutations along the HIV-1 genome.
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MATERIALS AND METHODS |
HIV-1 clones.
The origin and passage history of the
biological clones of HIV-1 used in the present study have been
previously described (78). Briefly, virus clones were
isolated by plating a natural isolate of HIV-1, termed S61, on MT-4
cells. Virus populations D1, G1, I1, and K1 are from randomly chosen,
individual plaques. After the first plating, viruses from individual
plaques (in the range of 102 to 105 PFU) were
diluted in 300 µl of culture medium and plated on fresh MT-4 cells,
and this process was repeated a number of times (Fig. 1). Plaques appeared 7 to 10 days after
infection. Fitness of the clonal populations of HIV-1 was determined by
growth-competition experiments in MT-4 cells, and populations were
analyzed by the heteroduplex tracking assay as previously described
(78).
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FIG. 1.
Scheme of passages of HIV-1 clones subjected to
plaque-to-plaque transfers in MT-4 cells. Clonal populations (HIV-1
isolated from individual plaques) are depicted as filled squares. The
experimental procedures and the origins of natural HIV-1 isolate S61
and clones B1 to K1 are given in reference 78 and in Materials and
Methods. HIV-1 clones are indicated by letters followed by a number
which gives the total number of plaque-to-plaque transfers undergone by
the clone. Infectious virus could not be rescued from viral populations
B15, E13, G7, H13, and J15 as described in reference 78.
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DNA extraction, PCR amplification, and nucleotide
sequencing.
DNA was extracted using an Instagene purification
matrix (Bio-Rad) according to the manufacturer's instructions. To
determine the consensus nucleotide sequence of the entire HIV-1 genome
in individual virus clones, a collection of overlapping sets of
oligonucleotide primers was used. They either have been previously
described (46, 47) or were designed for the present
experiments (Table 1). HIV-1 DNA was
amplified using nested PCR. The first amplifications (external primers)
were carried out using the GeneAmp PCR kit (Perkin-Elmer) and resulted
in the copying of 1,235-, 4,253-, and 6,686-bp fragments, comprising
residues 1 through 1235, 546 through 4799, and 2975 through 9661, respectively; residue numbers correspond to those of the genome of
HIV-1 isolate HXB2 (35). Internal amplifications yielded
fragments of 500 to 1,500 residues, which were used for
nucleotide sequence determination. For amplification of short
regions of gag (positions 1337 to 1598) and
env (positions 7071 to 7333), a single PCR
amplification was carried out. Both external and internal
amplifications involved 35 cycles with temperatures chosen according to
the composition of the oligonucleotide primers (78). Before
sequencing, the PCR mixture was digested with exonuclease I and shrimp
alkaline phosphatase (Amersham Life Sciences). Nucleotide sequences
were determined on the two cDNA strands, with an ABI 373 automatic
sequencer. Multiple sequence alignments were obtained using the CLUSTAL
W program (71).
The newly determined nucleotide sequences have been deposited in
the EMBL sequence database with accession numbers
AF256204,
AF256205,
AF256206,
AF256207,
AF256208,
AF256209,
AF256210, and
AF256211.
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RESULTS |
Mutation accumulation as a result of bottleneck transfers.
HIV-1 clones underwent severe fitness losses as a result of serial
plaque-to-plaque transfers in MT-4 cells (78). To determine the types and numbers of mutations accumulated during bottleneck transfers, the entire genomic nucleotide sequence of four HIV-1 clones
(D1, G1, I1, and K1) and their derivatives after 15, 7, 15, and 15 plaque-to-plaque passages, respectively (termed D15, G7, I15, and K15,
respectively [Fig. 1]), were obtained. The comparison of genomic
nucleotide sequences of each passaged clone relative to the
corresponding initial clone showed that transitions were 2.8-fold more
frequent than transversions and that A G and G A accounted for
20% and 36%, respectively, of all mutation types; nonsynonymous
mutations represented 49% of the total; an insertion of one nucleotide
was present in clone I15 (Table 2).
Mutation frequencies varied up to sevenfold among lineages (range,
4.4 × 10
4 to 3.1 × 103
substitutions per nucleotide [Table 2]). There was no obvious correlation between fitness decrease and mutation types or frequencies; for example, clone G7, which was debilitated to the point of not allowing a reliable determination of fitness value (78),
showed a mutation frequency that was sevenfold lower than that of I15 (Table 2).
Unusual distribution of mutations.
Mutation frequencies in
env are generally higher than in gag and
pol when natural HIV-1 isolates are compared (35, 44, 57, 58). In contrast, the HIV-1 clones debilitated by serial plaque transfers showed average mutation frequencies that were threefold higher for gag than env (Fig.
2). No mutations were found in tat,
vpu, and rev. Most remarkable was the absence of mutations in the regions encoding variable loops of gp120, particularly the V3 loop. The asymmetric distribution of mutations was visualized by
dividing the HIV-1 genome into three arbitrary regions of similar length: region 1, residues 1 through 3028; region 2, residues 3029 through 6065; and region 3, residues 6066 through 9035. Taking into
account all mutations scored for clones D15, G7, I15, and K15 (as
quantitated in Table 2), we found mutation frequencies for regions 1, 2, and 3 of 2.5 × 103, 9.0 × 104, and 6.6 × 104 substitutions per
nucleotide, respectively. The results suggest that plaque-to-plaque
transfers of HIV-1 lead to accumulation of mutations at multiple sites
of the HIV-1 genome, following a pattern which is quite different from
that observed in the natural evolution of HIV-1 in infected hosts.
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FIG. 2.
Location of mutations found in HIV-1 clones D15, G7,
I15, and K15, relative to their parental counterparts. The upper part
indicates HIV-1 genes and regulatory regions based on the compilation
of Korber et al. (35). The four horizontal bars in the center of the
figure indicate the positions of mutations along the genome (9.1 kb) in
the four clones analyzed (from top to bottom, D15, G7, I15, and K15
[described in Materials and Methods]); vertical lines within these
bars indicate one, two, or three mutations, according to thickness.
Mutations were found at positions 35, 171, 377, 379, 570, 584, 760, 807, 833, 988, 1128, 1161, 1188, 1351, 1467, 1545, 1578, 1596, 1810, 1863, 1875, 1937, 1961, 1966, 2145, 2174, 2329, 2668, 2804, 3068, 3114, 3129, 3945, 4458, 5239, 5270, 5342, 5422, 5684, 5686, 6588, 6655, 6670, 7962, 8095, 8890, 8900, and 8989 according to the numbering of HIV-1
isolate HXB2 (35). The gp120-coding region of env has been
enlarged to depict the positions of variable loops V1 to V5; two
mutations affected the V1-coding region. The two shaded rectangles
correspond to genome positions 1337 to 1598 (gag [left
shaded rectangle]) and 7071 to 7333 (env [right shaded
rectangle]), which have been sequenced for a number of additional
HIV-1 clones to confirm the asymmetric distribution of mutations (see
text). The mutations found in these additional clones are not included
in this scheme. The bottom part shows the three arbitrary regions into
which the HIV-1 genome was divided to illustrate the bias in the
distribution of mutations along the genome. Procedures used for
nucleotide sequence determination are described in Materials and
Methods, and the oligonucleotide primers are listed in Table 1.
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Conservation of the V3 loop.
The unusual distribution of
mutations found in D15, G7, I15, and K15 and the absence of mutations
in the region encoding the variable V3 loop of gp120 prompted us to
extend nucleotide sequence determinations to additional HIV-1 clones
subjected to serial bottleneck events. These additional analyses
included two clones from each of the lineages D15, G7, and K15, one
clone from I15, and clones from independent lineages which were
previously described (78), including three clones from F15
and one clone from H13 (Fig. 1). The analysis involved nucleotide
sequences of residues 7071 to 7333 (which include the V3-coding region)
and residues 1337 to 1598 (within the p24-coding region). The
comparison of nucleotide sequences with those of the corresponding
initial clones fully confirmed the bias in the distribution of
mutations; in all, 13 replacements were found in the gag
region analyzed (which represents a mutation frequency of 4.5 × 103 substitutions per nucleotide), while no replacements
were found in the V3-coding region (mutation frequency, <3.5 × 104 substitutions per nucleotide). The statistical
significance of the biased distribution of mutations was evaluated by
comparing the expected versus the actual number of mutations in
gag, pol, and env for regions 1, 2, and 3 into
which the genome was arbitrarily divided (Fig. 2), as well as for the
short gag and env stretches for the additional
clones from populations D15, F15, G7, H13, I15, and K15. These results
(Table 3) indicate high statistical significance of the biased mutant distribution. The degree of statistical significance of the biased distribution did not vary when
either all G A mutations or just G A mutations found in clone
I15 were excluded from the calculations (in all cases, P < 0.001 [
2 test]). Therefore,
accumulation of mutations that were associated with serial bottleneck
events and with fitness loss of HIV-1 affected genomic regions that are
less variable during the natural evolution of the virus.
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TABLE 3.
Expected versus actual number of mutations in different
genomic regions of HIV-1 clones subjected to serial plaque
transfers
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DISCUSSION |
The results reported here describe the numbers and types of
mutations associated with fitness loss of HIV-1 as a result of the
operation of Muller's ratchet (Table 2; Fig. 2). For clones D15, G7,
and K15, the average mutation frequency (1.3 × 104
substitutions per nucleotide, measured relative to the genomic nucleotide sequence of their respective parental clones D1, G1, and K1)
was 12 mutations per genome (range, 10 to 15), with a predominance of
transitions (75% of all mutations) over transversions. These figures
are similar to those of previous determinations of the number of
mutations accompanying Muller's ratchet in the FMDV genomean average
of six mutations per genome, with 77% of these transition mutations
(24). However, clone I15 displayed a different pattern in
that its mutation frequency corresponded to 28 mutations per genome
with an overabundance of G A transitions (43% of all
mutations [Table 1]). G A is one of the substitutions that
have been associated with hypermutagenesis in HIV-1 (72, 73) and a number of other viruses (reviewed in 5,
43). In clone I15, G A transitions were distributed rather
uniformly along the genome. Several possible mechanisms have been
proposed to explain biased hypermutagenesis (5, 43),
including alterations in intracellular deoxynucleotide pools. The
sequence context of 67% of the G A transitions in clone I15 was
GpA or GpG, which suggests a possible influence of low dCTP levels
during minus cDNA synthesis in the origin of this mutation type
(43). Our results with debilitated HIV-1 clones emphasize
the stochastic nature of the G A mutation bias because it was
observed in only one of four clones derived from the same viral
isolate, and these clones were subjected to identical treatment during
the serial plaque transfers (Fig. 1). If the mechanism of nucleotide
pool bias was in operation (43), it must have been triggered
either by extremely subtle perturbations in the intracellular
environment or by differences among the genomes of the four clones,
differences that existed initially or that were generated in the course
of passaging. In the latter case, the mutational biases must be
subjected to indeterminations derived from the dynamics of mutant
generation within the quasispecies swarms (reviewed in 10, 20).
A dominance of G A transitions was also found in an analysis of
mutations in a lacZ
-based reporter gene, which was
constructed to study a single cycle of HIV-1 replication
(39). However, there are important differences in the mutant
repertoire found following a single cycle of replication and in our
clones subjected to serial plaque transfers. In the former study, G A, C T, and T C mutations occurred at frequencies of 1.7 × 103, 7.1 × 104, and 1.3 × 104 substitutions per nucleotide, respectively, while in
our study these same mutations occurred at frequencies of 4.9 × 104, 2.7 × 104, and 1.9 × 104, respectively (Table 2). After a single-cycle
replication, T G, T A, and A G each occurred at a frequency
of 6.5 × 105 substitutions per nucleotide, while in
our clones the values for these substitutions ranged from 2.7 × 104 to 2.7 × 105 substitutions per
nucleotide. In addition, A C, C A, C G, and G T
mutations found in HIV-1 clones (Table 2) were not represented among
the mutations found after a single infectious cycle (39).
These variations in mutation types and frequencies probably arose not
only from differences in the number of replication cycles but also from
the sequence context in the template being copied in a different
biological environment (43).
The most unexpected finding in the analysis of low-fitness HIV-1 clones
was the distribution of mutations along the genome (Fig. 2), with a
statistically significant accumulation of mutations in gag
and the first third of the genome, relative to env, which appears as the most conserved genomic region in all clones examined (Fig. 2; Table 3). This is in sharp contrast to results with natural
HIV-1 isolates (35, 44, 57, 58) and with large-population passages of HIV-1 clonesderived also from natural isolate S61in cell culture (64); in all of these analyses,
gag and pol showed more nucleotide and amino acid
sequence conservation than env. Several mechanisms could
contribute to this striking difference in the distribution of
mutations. In the course of plaque-to-plaque transfers, fitness gains
or purifying selection is probably diminished since competition among
genomes from the quasispecies is limited to the period of plaque
development (10, 25). In this view, the higher variation
normally seen in env relative to gag and pol would be essentially due to selection for immune
evasion, for adaptation to alternative cellular receptors, increased
particle stability, etc. Evidence for selection in vivo has been
obtained for HIV-1 and for simian immunodeficiency virus (2, 3,
26, 34, 48, 54, 62, 77). However, even if replicative
optimization of the mutant spectrum was limited as a result of
bottlenecking, the deficit in mutations in env remains to be
explained. There are at least two possibilities (which are not mutually
exclusive): env may have a lower tolerance for mutations
than gag and the 5' third of the genome under the cell
culture environment in which the passages were carried out, or HIV-1
genome replication may be more error prone in the process of copying
gag than when copying env. Constraints to accept
replacements in surface proteins were documented through functional and
structural studies with FMDV (12 and references
therein). The mutant repertoire in viral quasispecies could be strongly
influenced by tolerance to nucleotide and amino acid substitutions,
including silent replacements in coding regions (12, 38,
40, 67). In HIV-1, constraints in surface proteins could come
about from the need always to use the cellular receptors presented by
MT-4 cells in culture and to enter this same cell type monotonously in
an invariant cell culture environment. Purifying selection during
plaque growth, which must include many cycles of replication, may have
contributed to the observed bias in the distribution of mutations along
the genome.
A possible molecular basis for a difference in accuracy during copying
of different genome segments of HIV-1 is not obvious. The lowest
number of mutations was seen in the genomic region copied
immediately after the first strand transfer in the synthesis of
minus-strand DNA by reverse transcriptase (RT) (reviewed in reference
68). Since processivity of RT is limited, it could be proposed that as synthesis proceeds inside the nucleocapsid, accuracy may decrease as a result of environmental alterations (ionic
composition, deoxynucleoside triphosphate pools, etc.
[43]). It has been suggested that misalignment
mutagenesis could be more frequent during dissociation and reinitiation
of RT-catalyzed reactions (reviewed in 1). However,
examination of the sequence context of each mutation suggests that the
frequency of mutations which may have occurred as a result of
template misalignment (1, 53) is not significantly different
for regions 1, 2, and 3, into which we have divided the HIV-1
genome (52, 64, and 63%, respectively). Other effects on fidelity or,
more likely, a combination of factors outlined in previous paragraphs
may converge to produce the unusual distribution of mutations
seen in HIV-1 clones as a result of operation of Muller's ratchet.
A total of 8 out of 20 nonsynonymous replacements found among the HIV-1
clones analyzed have not been recorded in current sequence data banks
(35; Table 4). An
attractive possibility is that replacements in gag could
have multiple effects in RNA-protein interactions, nucleocapsid
assembly, and protein stability (16, 30) and that such
effects could contribute to fitness loss. It must also be considered
that Vif, Vpr, and Nef are dispensable functions for HIV-1 replication
in some permissive cell lines, including MT-4 (7, 49).
Therefore, the accumulation of nonsynonymous replacements in vif,
vpr, and nef genes (Fig. 2) could be of little consequence for plaque formation in MT-4 cells. However, an evaluation of the influence of amino acid replacements (individually or in combination) in viral fitness would require analysis of the effects of
candidate mutations when introduced into infectious clones or
examination of possible reversions and fixation of compensatory mutations upon fitness recovery of the debilitated HIV-1 clones. These
studies are now in progress.
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TABLE 4.
Amino acid replacements associated with
nonsynonymous mutations during plaque-to-plaque transfers of
HIV-1
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In conclusion, some mutations associated with the operation of
Muller's ratchet in HIV-1 have not been previously reported among
natural isolates evolved over more than two decades (35; Table 4). Furthermore, the mutations were distributed along the viral
genome, unlike mutations in natural HIV-1 isolates, and env
was the most conserved genomic region. An interesting possibility is
that, in vivo, HIV-1 is not subjected to as severe bottleneck events as
in experiments designed to accentuate Muller's ratchet. The
observations reported here add to the complexities inherent in the
relationships between occurrence of mutations and what can be
eventually observed upon examination of genomic nucleotide sequences.
The HIV-1 mutational pattern could be made to vary with
respect to hundreds of sequences recorded in data banks simply by
changing the passage regimen, without intervening, externally applied,
selective forces.
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ACKNOWLEDGMENTS |
Work at Centro de Biología Molecular "Severo Ochoa" was
supported by grants FIS98/0054-01 and PM97-0060-C02-01 and that at Centro Nacional de Biología Fundamental was supported by grants FIS00/0266 and FIS98/0054-02. E.Y. was supported by a postdoctoral fellowship from Comunidad Autónoma de Madrid.
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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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Top
Abstract
Introduction
