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Journal of Virology, November 2001, p. 10231-10243, Vol. 75, No. 21
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.21.10231-10243.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Structure and Temporal Dynamics of Populations
within Wheat Streak Mosaic Virus Isolates
Jeffrey S.
Hall,1,2
Roy
French,1,3
T. Jack
Morris,2 and
Drake C.
Stenger1,3,*
Agricultural Research Service, U.S.
Department of Agriculture,1 School of
Biological Sciences,2 and
Department of Plant Pathology,3
University of Nebraska, Lincoln, Nebraska 68583
Received 28 March 2001/Accepted 1 August 2001
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ABSTRACT |
Variation within the Type and Sidney 81 strains of wheat streak
mosaic virus was assessed by single-strand conformation polymorphism (SSCP) analysis and confirmed by nucleotide sequencing.
Limiting-dilution subisolates (LDSIs) of each strain were evaluated for
polymorphism in the P1, P3, NIa, and CP cistrons. Different SSCP
patterns among LDSIs of a strain were associated with single-nucleotide
substitutions. Sidney 81 LDSI-S10 was used as founding inoculum to
establish three lineages each in wheat, corn, and barley. The P1,
HC-Pro, P3, CI, NIa, NIb, and CP cistrons of LDSI-S10 and each lineage at passages 1, 3, 6, and 9 were evaluated for polymorphism. By passage
9, each lineage differed in consensus sequence from LDSI-S10. The
majority of substitutions occurred within NIa and CP, although at least
one change occurred in each cistron except HC-Pro and P3. Most
consensus sequence changes among lineages were independent, with
substitutions accumulating over time. However, LDSI-S10 bore a variant
nucleotide (G6016) in NIa that was restored to
A6016 in eight of nine lineages by passage 6. This
near-global reversion is most easily explained by selection.
Examination of nonconsensus variation revealed a pool of unique
substitutions (singletons) that remained constant in frequency during
passage, regardless of the host species examined. These results suggest
that mutations arising by viral polymerase error are generated at a
constant rate but that most newly generated mutants are sequestered in virions and do not serve as replication templates. Thus, a substantial fraction of variation generated is static and has yet to be tested for
relative fitness. In contrast, nonsingleton variation increased upon
passage, suggesting that some mutants do serve as replication templates
and may become established in a population. Replicated mutants may or
may not rise to prominence to become the consensus sequence in a
lineage, with the fate of any particular mutant subject to selection
and stochastic processes such as genetic drift and population growth factors.
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INTRODUCTION |
RNA viruses have the common property
of high mutation rates due to an error-prone RNA polymerase (10,
12, 13). Because of this, RNA viruses can increase in genetic
diversity and undergo rapid evolutionary changes (11, 12, 34,
39). Sequence diversity among isolates of plant RNA viruses has
been well documented (1, 2, 9, 15, 24-28, 33, 45, 46, 55-59,
65). However, low levels of variation found within individual
isolates of many plant viruses imply that accumulation of fixed
differences can be a slow process (1, 22, 23, 25, 27, 28, 36, 44,
45, 52, 56). Thus, evolutionary and population genetic factors
favoring genetic stability such as purifying selection or episodes of
genetic bottlenecks must be in play for many plant viruses.
The deterministic quasispecies model is applicable when large pools of
virus are in direct competition (11-13, 37, 39, 60).
However, there are numerous examples where competition among viral
genomes appears to be limited to various degrees, resulting in
stochastic rather than deterministic outcomes (16, 20, 30, 34,
40, 41, 50). It has been proposed that no sharp boundary exists
between the deterministic and stochastic modes of population behavior
(48). Random drift cannot occur at the deterministic end
of the spectrum but gradually becomes increasingly likely as the
effective, or replicating, population size decreases (16, 38,
48). Selection, on the other hand, becomes less effective with a
decline in population size. One mechanism for reducing effective
population size is the introduction of genetic bottlenecks (35,
41, 46). Recently it was demonstrated that colonization of wheat
by strains of wheat streak mosaic virus (WSMV) is a discontinuous
process (17), suggesting that within-plant subdivision may
impose bottlenecks during systemic infection. If so, genetic drift
should be a prominent factor in WSMV evolution over time.
WSMV is the type species of the newly established genus
Tritimovirus within the family Potyviridae
(62). The monopartite genome (9,384 nucleotides) of WSMV
is translated as a polyprotein that is subsequently cleaved by
virus-encoded proteinases into 8 to 10 mature proteins. Three WSMV
strains (Type, Sidney 81, and El Batán 3) have been completely
sequenced (9, 62). Type and Sidney 81 have 97.6%
nucleotide sequence identity and are representative of American WSMV
isolates. El Batán 3, from Mexico (51), has diverged
from the American strains and retains ~79% nucleotide sequence
identity to Type and Sidney 81 (9). Examination of
variation among the three WSMV strains suggests that negative selection
operates to conserve certain regions of the genome, while much of the
divergence may be explained by genetic drift (9). Field
populations of WSMV are complex and consist of numerous genotypes that
occur in the same field but rarely (~2%) in the same infected plant
(33). Maintenance of genetic diversity within WSMV field
populations may be facilitated by three distinct mechanisms of genetic
isolation: cross-protection, subdivision of populations within infected
plants, and vector transmission bottlenecks (17). Thus,
sympatric viral lineages are genetically isolated from one another such
that stochastic processes may influence divergence.
In this report, variation within WSMV isolates was assessed after
passage through a genetic bottleneck imposed by
low-multiplicity-of-infection (MOI) inoculation and upon repeated
passage at high MOI. These experiments were designed to detect shifts
in the consensus sequence of a lineage at various points in time. A
single-strand conformation polymorphism (SSCP) assay was employed,
making it practical to screen for consensus sequence polymorphism in
multiple samples across large genomic regions. We further examined
nonconsensus variation existing after low-MOI bottlenecking and after
repeated passage at high MOI. Collectively, our results indicate that
populations of WSMV change over time and that there are multiple
processes affecting variation within plant virus populations.
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MATERIALS AND METHODS |
Generation of WSMV LDSIs.
WSMV-Type (ATCC PV57; GenBank
accession no. AF285169) was isolated from wheat collected in Kansas
(32). WSMV-Sidney 81 (GenBank accession no. AF057533) was
recovered from wheat grown in western Nebraska (6). A
limiting-dilution method was used to resolve individual infectious
units. Limiting-dilution subisolates (LDSIs) were recovered from sets
of wheat (Triticum aestivum L. cv. Centurk) seedlings
inoculated with 10
5 dilutions of sap extract, in which
only ~1% of the plants became infected. Altogether, 22 Sidney 81 and
25 Type LDSIs were obtained. The probability of each LDSI being
initiated by >1 infectious unit was 
0.5% (5).
Systemically infected LDSI leaf samples were harvested 21 days
postinoculation and stored at 
80°C.
Passage inoculations.
Sidney 81 LDSI-S10 was used as the
founding inoculum for passage experiments. The first passage with
LDSI-S10 inoculum and all subsequent passage inoculations were
conducted at high MOI using a 101 dilution of sap
extract. Nine separate lineages (three each in wheat cv. Centurk, corn
[Zea mays L.] var. N28ht, and barley [Hordeum vulgare L.] cv. Black Hulless) were established at passage 1 and maintained for nine serial passages conducted at 3-week intervals. A
single passage in one lineage consisted of five plants grown in a
common pot. For each lineage at each passage, systemically infected
leaf samples were collected 21 days postinoculation (p.i.) and stored
at 80°C. To minimize differential selective pressures due to
environmental conditions, the passage experiment was conducted in a
growth chamber under controlled conditions (20°C with a 16-h photoperiod).
RT-PCR.
Total nucleic acids were isolated from frozen leaf
samples and viral RNA was reverse transcribed as described
previously (33). In addition to using the RCF1
primer (Table 1) to initiate cDNA synthesis, random primers were incorporated into reverse transcription (RT) reactions to ensure cDNA representative of the entire genome. Regions of the viral genome (Fig. 1) were amplified by PCR using primer
sets listed in Table 1. Variation among LDSIs was estimated by
examining four cistrons (P1, P3, NIa, and CP) amplified by RT-PCR.
LDSI-S10 and each of the nine lineages derived from this subisolate
were further characterized by RT-PCR amplification with all primer sets
listed in Table 1, resulting in ~96% coverage of the viral genome.
PCR was performed in a Perkin-Elmer 9600 thermocycler for 35 cycles
(except for NIa, for which 30 cycles were employed) using
Taq DNA polymerase, and the products were analyzed by SSCP
and sequencing (see below) to correlate haplotypes with consensus
sequences. To examine nonconsensus variation within the CP cistron of
LDSI-S10 and derived lineages, the Expand High-Fidelity System (EHFS)
PCR (Boehringer Mannheim) was used (30 cycles) to minimize PCR-induced
sequence errors.
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TABLE 1.
Primer sets used for RT-PCR and restriction endonucleases
used to prepare PCR products for SSCP analysis
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To determine the intrinsic error rate of each PCR system, plasmids of
known sequence served as template donors. Clones of
Sidney 81 LDSI-S9
NIa and LDSI-S10 CP served as template sources
for PCR with
Taq polymerase or the EHFS, respectively. Each template
donor plasmid was digested with
PvuII, and the fragments
containing
each WSMV insert (plus ~350 bp of flanking vector
sequence) were
gel purified. Each gel-purified
PvuII
fragment (1 ng) was used
as a template for PCR with the appropriate CP
or NIa primer set
(Table
1) under the same conditions described above.
The resulting
PCR products were gel purified and ligated into pGEM-T
(Promega),
and the ligation products were used to transform
Escherichia coli strain DH5
. A total of 10 (EHFS) or 20 (
Taq polymerase) clones
(defined as reclones) of each PCR
product were sequenced, and
all substitutions were attributed to
polymerase errors introduced
during
PCR.
SSCP analysis.
PCR products were digested with restriction
endonucleases to obtain fragments of appropriate lengths
(42) for SSCP analysis (Table 1; Fig.
1). Each restriction
endonuclease-digested PCR product was denatured in 25% formamide-5 mM
NaOH-0.5 mM EDTA at 99°C for 5 min and placed immediately on ice.
Single-stranded DNA fragments were separated on 10% polyacrylamide
gels for 16 h in 1X Tris-borate-EDTA buffer at 4 and
20°C. SSCP patterns were visualized by silver staining
(43). The accuracy of the method in detecting nucleotide
substitutions was determined using plasmids of known sequence (10 Type
LDSI CP inserts, and 10 Sidney 81 NIa reclone inserts).
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FIG. 1.
Physical map of the WSMV genome depicting locations of
RT-PCR products amplified for analysis of genotypic variation.
Arrangement of cistrons within the polyprotein open reading frame
bracketed by upstream (5'-UTR) and downstream (3'-UTR) untranslated
regions is presented at the top. The 3'-terminal polyadenylated tail of
variable length is designated An. Nucleotide coordinates
(vertically oriented numbers) correspond to the genomes of both Type
and Sidney 81 strains of WSMV, whose genomes are identical in length
and may be aligned without gaps. Genomic location and nucleotide
lengths of RT-PCR products are presented below the complete genome map.
Nucleotide sequence lengths analyzed for nucleotide polymorphism are
indicated in parentheses. The difference between total and analyzed
sequence lengths represents terminal regions where primers used in PCR
annealed.
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Cloning and sequencing.
The P1, P3, NIa, and CP cistrons of
each LDSI were screened for variation by SSCP. PCR products of
predominant and variant SSCP patterns were ligated into pGEM-T
(Promega, Madison, Wis.) and transformed into E. coli strain
DH5. PCR products (P1, HC-Pro, P3, CI-5', CI-3', NIa, NIb, and CP)
of LDSI-S10 were cloned into pGEM-T, as were PCR products displaying
variant SSCP patterns detected in all nine LDSI-S10-derived lineages at
passages 1, 3, 6, and 9. Additional PCR products displaying predominant
SSCP patterns for each cistron from various lineages and passages also were cloned into pGEM-T. Inserts of two plasmids derived from each PCR
product cloned were sequenced in both directions by primer walking
using universal and internal sequencing primers. Twenty additional
LDSI-S10 NIa clones were partially sequenced (nt 5896 to 6427) to
determine if two polymorphic sites appearing in more than one lineage
during the passage experiment were resident as minor components of the
LDSI-S10 population. Multiple EHFS-PCR CP clones of LDSI-S10 and three
lineages at passage 9 also were sequenced on both strands by primer
walking. Sequencing was performed at the Iowa State University DNA
Synthesis and Sequencing Laboratory, Ames, Iowa. Sequences were
compiled using Sequencher 3.1 (Gene Codes Corp., Ann Arbor, Mich.),
edited to remove terminal regions in which PCR primers annealed, and
aligned using Clustal X (64). A cladogram depicting
relationships among consensus sequence variants present at passage 9 was generated by neighbor-joining using Clustal X. Sequence data sets
were analyzed using the SITES program (18) and DnaSP v.3.0
(49). The probability that the amount of sequence variation observed in the LDSI-S10 data set was due to PCR error alone
was determined by generating 100,000 simulated samples using the
computer program MLPCR (67).
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RESULTS |
Accuracy of SSCP for detection of nucleotide substitutions.
SSCP has been used to characterize variation in both nuclear genes and
virus populations (7, 19, 37, 43, 61, 68). This technique
is dependent on a bulk property of PCR products, so that nucleotide
variation due to misincorporation by Taq DNA polymerase does
not interfere with the analysis. Electrophoresis at two different
temperatures has been reported to increase the accuracy of this
technique in detecting single nucleotide polymorphisms to ~98%
(68). To evaluate accuracy as applied to WSMV, SSCP analysis was performed with PCR products amplified from 20 cloned templates of known sequence. Of the 20 plasmids examined, 12 had one or
more unique nucleotide changes (defined as sporadics by Smith et al.
[58]) whereas 8 did not. Each clone free of sporadics yielded an SSCP pattern identical to the original PCR product from
which it was cloned. All but one clone bearing sporadic substitutions yielded novel SSCP patterns, as expected. Based on this analysis, SSCP
was estimated to be 95% (19 of 20) accurate for detection of
nucleotide substitutions (data not shown). Thus, each unique haplotype
identified by SSCP could be correlated with specific sequences for all
47 LDSIs.
Low-MOI inoculation generates LDSIs with different consensus
sequences.
Four regions of each LDSI genome were amplified by
RT-PCR (Fig. 1), with haplotypes detected by SSCP for the CP cistron
presented in Fig. 2 and summarized for
all four cistrons in Table 2. The predominant haplotypes for each cistron have been designated A (Sidney
81) and D (Type). Three different haplotypes each were detected in the
Sidney 81 CP and P1 cistrons. Sidney 81 haplotypes B (CP) and C (P1)
were present only together in the same LDSIs. Two Sidney 81 haplotypes
were detected in NIa, while no variation was found in P3 of Sidney 81. Type had two variant haplotypes that each occurred once, one in the CP
cistron and one in the P3 cistron. The occurrence of haplotype
configurations across the four cistrons is shown in Table 2. For Sidney
81, five distinct linkage patterns were detected, with 55% of the
LDSIs bearing at least one cistron with a variant haplotype. In
contrast, 92% of Type LDSIs displayed the predominant haplotype for
all four cistrons. Sidney 81 LDSI-S9 yielded an SSCP pattern for NIa
that appeared to be a mixture of haplotypes A and B (Table 2), an interpretation supported by the sequences of two clones (Table 3). To preclude stochastic variation
among samples arising during early stages of amplification,
RT-PCR-SSCP analysis was repeated for all variant haplotypes as well
as several of the predominant haplotypes for each cistron. In every
case, the SSCP patterns were consistent (data not shown).
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FIG. 2.
SSCP analysis of the WSMV coat protein cistron.
Presented are SSCP profiles for different haplotypes (A to E) observed
among LDSIs of the Sidney 81 and Type strains. Denatured, restriction
endonuclease-digested PCR products were electrophoresed under
nondenaturing conditions in 10% polyacrylamide gels at 4 and 20°C.
Alteration of the SSCP pattern at one or both electrophoresis
temperatures corresponded to single-nucleotide substitutions in variant
haplotypes (B, C, and E) relative to the predominant SSCP patterns
observed among Sidney 81 (A) or Type (D) LDSIs.
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PCR products derived from five LDSIs per cistron (representing all
haplotypes) were cloned for both Type and Sidney 81. For
all variant
SSCP patterns observed among the LDSIs, both clones
derived from a PCR
product contained the same nucleotide substitution
at one position
(Table
3). In every case, a shift in SSCP pattern
at one or both
electrophoresis temperatures resulted from a single-nucleotide
change
in the consensus sequence. Of seven polymorphic nucleotide
sites found
in the two LDSI populations, five were transitions
and two were
transversions. One polymorphism was in the 3'-untranslated
region of
Sidney 81. Within the polyprotein coding region, four
synonymous and
two nonsynonymous changes occurred (Table
3).
Nonsporadic substitutions present in the LDSI sequence data set were
associated with unique haplotypes as identified by SSCP,
whereas
sporadic substitutions were not. Among 84,620 nucleotides
sequenced
from LDSI clones, 79 sporadic changes were noted (Table
4). All polymerases have intrinsic error
rates, and if these
are not accounted for, an overestimation of
variability is the
result (
3,
58). Although SSCP and
sequencing were sufficient
to identify differences in consensus
sequences among LDSIs, this
procedure was not suitable for
discrimination of nonconsensus
variations resident within
LDSIs from
Taq polymerase errors introduced
during
PCR. Because sporadic rates among the LDSI data sets and
the NIa
reclone experiment were similar (Table
4), partitioning
of sporadic
substitutions as real (produced in vivo by viral polymerase)
or
artifactual (produced in vitro by RT or
Taq polymerase)
variation
was not attempted.
Passage at high MOI also results in consensus sequence change.
The linkage pattern (BABA) determined for the P1, P3, NIa, and CP
cistrons of LDSI-S10 was shared by two other LDSIs (Table 2). The
LDSI-S10 P3 and CP cistrons displayed the predominant haplotype
observed among Sidney 81 LDSIs that corresponded to Sidney 81 sequence
AF057533. Although LDSI-S10 P1 was characterized by SSCP as haplotype
B, sequencing revealed that haplotype A also was present as a minority
of the population. The LDSI-S10 NIa haplotype B represented a variant
(G6016) that also occurred in two other LDSIs (Table 3).
SSCP analysis performed on HC-Pro, CI5', CI3', and NIb PCR products
defined haplotypes for these LDSI-S10 cistrons. Sequence comparisons of
LDSI-S10 clones for each of these additional PCR products revealed no
nonsporadic differences between LDSI-S10 and Sidney 81 sequence
AF057533.
SSCP analysis of all eight PCR products for each of the nine lineages
derived from LDSI-S10 was performed at passages 1, 3,
6, and 9 (Table
5). Sequences of cloned PCR products that
displayed
variant SSCP patterns revealed nonsporadic substitutions,
whereas
sequences of cloned PCR products with SSCP patterns identical
to that of LDSI-S10 did not. Nonsporadic nucleotide substitutions
corresponding to variant haplotypes identified by SSCP (Table
5) are
specified in Table
6. The passage data
set (including
LDSI-S10) consisted of 191,299 nucleotides sequenced.
The total
number of nucleotides screened for substitutions by SSCP was
considerably
larger.
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TABLE 5.
Haplotypes determined by SSCP and verified by sequencing
of two clones at select passages for nine lineages derived
from LDSI-S10
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After nine passages, no WSMV lineage maintained the original LDSI-S10
haplotype pattern (Table
5). Shifts in consensus sequence
(i.e., new
alleles) were detected in all cistrons except HC-Pro
and P3 (Table
6).
However, most changes in consensus sequence
occurred within two
cistrons, with CP yielding 12 allelic shifts
at 11 sites and NIa having
15 events at 7 sites. While the barley
BC lineage had only one change,
most lineages accumulated multiple
changes in consensus sequence. The
wheat WA and corn CB lineages
accumulated the largest number of
nucleotide changes (five each)
in their respective consensus sequences
after nine passages. The
total accumulation of nucleotide substitutions
in consensus sequences
at each passage is illustrated in Fig.
3. Several changes in predominant
haplotypes occurred in passage 1 (P1 of corn CB; NIa and CI of
corn
CC), although most consensus sequence changes were detected
in passages
6 and 9. Some variant haplotypes gained transitory
prominence, only to
be subsequently replaced by the original haplotype
sequence (NIa of
wheat WB and NIb of barley BA), or disappeared,
only to reappear in
later passages (CI of corn CC). Several variant
SSCP patterns were
mixtures of haplotypes (NIa of wheat WB passage
6), in which two
alleles at the same polymorphic site were detected
by nucleotide
sequencing and were probably present at similar
frequencies such that
both contributed to the novel SSCP pattern
observed.
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FIG. 3.
Histogram of cumulative substitutions in consensus
sequence summed for nine lineages derived from an LDSI of WSMV Sidney
81. The total bar height corresponds to the frequency of all
substitutions in consensus sequences sampled after 1, 3, 6, and 9 passages. Black regions of bars correspond to all consensus sequence
substitutions other than the near-global reversion
(G6016 A) which occurred in eight of nine lineages
(hatched region of bars). The line denotes the linear-expectation best
fit to the observed data (excluding G6016A) that
intersects the origin (no mutations at time of limiting dilution
inoculation).
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Consensus sequence changes shared by more than one lineage derived
from LDSI-S10.
Consensus sequence divergence accrued by and
surviving in passage 9 is illustrated in Fig.
4. While most lineages accumulated unique
changes in consensus sequence, several exceptions were noted. Eight of
the nine lines had a common substitution relative to LDSI-S10. This
change within NIa (G6016A) was at a known polymorphic site in the LDSI data set and restored the consensus sequence of all
but one lineage to the allele that predominated among Sidney 81 LDSIs
(Tables 2 and 5). Since both alleles were present in the Sidney 81 LDSIs (with one LDSI a known mixture), it is possible that
A6016 also was present in LDSI-S10 as a minority variant. However, a partial sequence (nt 5896 to 6427) determined for 20 additional clones of the LDSI-S10 NIa PCR product did not detect A6016.
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FIG. 4.
Cladogram reflecting consensus sequence substitutions
accrued by and surviving in nine WSMV lineages after nine passages in
wheat, corn, or barley. The founding inoculum for all lineages was
derived from LDSI-S10. The tree was drawn manually, with common nodes
reflecting consensus sequence substitutions shared by more than one
lineage at passage 9. Branch lengths correspond to the number of
nucleotide substitutions (1 through 5) in the consensus sequence of
each lineage relative to LDSI-S10 after nine passages.
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A second example of a substitution shared among lineages was
U
6418C in NIa. This change occurred in passage 1 of corn
CC
and remained stable in this lineage through passage 9. C
6418 also
was detected as a mixture with
U
6418, resulting in a novel SSCP
pattern seen only in wheat
WB at passage 6. However, C
6418 was
not detected in wheat
WB at passage 9 and the SSCP pattern corresponded
to haplotype A
(bearing only the near-global substitution G
6016A).
All
20 additional NIa clones of LDSI-S10 partially sequenced (nt
5896 to
6427) had U
6418 and not C
6418.
While wheat lineages WB and WC both contained the adjacent
substitutions C
8764 U and A
8765C, first
detected at passage 6,
these two shared substitutions became the
consensus sequence only
transiently at passage 6 in wheat WC. At
passage 9, both alleles
at positions 8764 and 8765 were detected in
both lineages, such
that the haplotypes observed were due to mixtures.
Neither U
8764 or C
8765 was detected among 17 LDSI-S10 CP clones of an EHFS-PCR
product.
Polymorphism revealed by sequencing but not detected by SSCP.
Although SSCP is a powerful tool for detecting nucleotide
substitutions, the procedure is not 100% accurate. Therefore, some consensus sequence nucleotide substitutions may have escaped notice. To
investigate this possibility, a number of PCR products displaying the
predominant haplotype in the LDSI data set or the same haplotype as
LDSI-S10 in the passage data set were cloned and sequenced for each
cistron. Within the LDSI data set, one discrepancy was noted. The
LDSI-S10 P1 PCR product was characterized as haplotype B by SSCP (Table
2). However, the sequences of two clones indicated that both haplotypes
A (C961) and B (U961) were present. The
simplest explanation is that both alleles were present in LDSI-S10 but that U961 comprised the majority such that haplotype B
dominated the SSCP pattern.
Examination of nonconsensus sequence variation.
In addition to
consensus sequence shifts, the amount and pattern of sequence variation
within lineages may have changed over time. To examine sequence
heterogeneity present within viral populations before and after nine
passages, the CP cistrons of LDSI-S10 and lineages wheat WB, barley BA,
and corn CC at passage 9 were amplified using EHFS-PCR, cloned, and
sequenced. After 30 cycles of EHFS-PCR with a DNA template of known
sequence, the observed mutation frequency was 2.2 × 104/nt. The error rate calculated for EHFS-PCR (0.72 × 105/nt/cycle) was 5.6-fold lower than the error rate
obtained with Taq polymerase (0.4 × 104
nt/cycle). These PCR error rates were similar to those reported by
others (3, 57, 66). Error due to RT was accounted for by
assuming a maximum misincorporation rate of 0.36 × 104/nt (21), to give a combined in vitro
mutation frequency estimated to be 2.6 × 104/nt.
Note that although error introduced by RT occurs during the first stage
of amplification, RT errors are independent and therefore still yield
primarily sporadic substitutions after PCR amplification, as
exemplified by the absence of nonsporadic substitutions in the LDSI-S10
population (Fig. 5) and the entire data set of Schneider and Roossinck
(52). Sixteen sites of nucleotide substitutions were found
in the LDSI-S10 population sample. Employing a novel maximum-likelihood
test of template homogeneity devised by Weiss and von Haeseler
(67), the probability that these were all due to PCR
misincorporation was estimated to be less than 0.02%. Thus, 63 to 76%
of sporadic substitutions present in sequence data sets obtained using
RT followed by EHFS-PCR could not be accounted for by in vitro
polymerase error and therefore probably represented substitutions (here
termed singletons) due to viral polymerase error (Table
7; see Fig. 6).
None of the singletons observed in LDSI-S10 EHFS-PCR-derived CP clones
were present in any of the three passage 9 populations
examined (Fig.
5 and data not shown). Nonetheless, after
nine
passages, corrected mutation frequencies in three lineages
passaged
in different hosts did not significantly differ from that of
LDSI-S10
(Table
7). There was, however, a qualitative difference in the
composition of mutants before and after passage, as exemplified
by Fig.
5. All polymorphic sites in the LDSI-S10 sample were singletons
whereas
nucleotide substitutions after nine passages included
both singletons
and nonsingletons shared among several clones
(Table
7; Fig.
5).
Nonsingletons were shared among two independent
RT-PCR samples from a
single lineage at passage 9 (Fig.
5), demonstrating
that they are not
in vitro artifacts. Although the proportion
of unique substitutions
(sporadic substitutions) in a population
may be designated as resulting
from viral polymerase (singletons)
or PCR error, the origin of any
individual sporadic mutant cannot
be determined. However, polymorphism
shared among clones (nonsingletons)
is unlikely to be an artifact of
PCR (
58), particularly if the
same substitution appears in
independent RT-PCR products from
a sample (Fig.
5). Thus, the shared
substitutions probably represent
viral polymerase-induced errors that
have replicated and reached
detectable frequencies. Among the shared
substitutions, five were
transitions (four C to U and one A to G) and
four were transversions
(two each A to U and A to C). Three of these
were silent, and
four resulted in amino acid substitutions (data not
shown).
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FIG. 5.
Nucleotide sequence variation in the WSMV coat protein
cistron and 3'-untranslated region before (LDSI-S10) and after (wheat
WB) nine passages in wheat. Sequences were determined from multiple
clones derived from RT-PCR products amplified with the EHFS employing a
proofreading polymerase. Only positions at which polymorphisms were
observed are shown. WSMV genomic coordinates corresponding to each
polymorphic site are listed above each sequence data set. Substitutions
present within clones are identified by specific nucleotide changes
from the LDSI-S10 (S10) consensus sequence. Dashes indicate
positions within clones that are identical to the LDSI-S10 consensus
sequence. Note that all nonsingleton substitutions in the wheat WB
passage 9 data set occur in clones derived from two independent PCR
products (I and II).
|
|
Allelic frequencies of polymorphic sites within sampled clones of
LDSI-S10 and each passage 9 population are presented in
Fig.
6. For example, if a nucleotide
substitution was found in
three clones of a sample, it was placed in
allele size class 3.
The distributions are folded in that for
i shared substitutions
from a sample size of
n,
it is the smaller of
i or
n i that
is plotted.
For comparison, expected values for three theoretical
sampling
distributions are presented in Fig.
6: (i) a Poisson
distribution, (ii)
the neutral infinite-sites model (
63,
66),
and (iii) the
Luria-Delbrück sampling distribution (
29,
31,
47)
applied to nucleotide variation (
54). Only LDSI-S10 was
in
concordance with a Poisson distribution. All of the passage
9 samples
had ranges of allele size classes inconsistent with
the Poisson
distribution yet had singleton (allele size class
of 1) frequencies in
excess of those expected for populations
conforming to model ii or iii.
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 6.
Distribution of alleles size classes of variants
detected among EHFS PCR clones of the coat protein cistron and
3'-untranslated region of WSMV before (LDSI-S10) or after nine passages
in wheat, barley, or corn. Black bars indicate the frequency of allele
size classes corrected for the proportion of singletons estimated to be
derived from in vitro RT-PCR error (open bars). Shaded or hatched bars
denote allele size class frequencies expected for each of three
theoretical distributions. The expected frequencies for the
Luria-Delbrück distribution were calculated using equations 17 and 16 in reference 47, and those for the neutral,
infinite alleles distribution were calculated using equation 51 in
reference 62. Note that the LDSI-S10 variation is composed
only of singletons whereas the passage 9 data sets contain both
singletons and nonsingletons. Two novel size classes (classes 8 and 14)
appear in the total passage 9 data set and represent cases where the
majority allele within a single data set (wheat WB allele size class 5 and corn CB allele size class 7) become the minority allele in the
total data set.
|
|
| |
DISCUSSION |
Limiting dilution serves as a bottleneck to restrict genetic
diversity.
Limiting-dilution inoculation was a stringent
bottleneck, yielding LDSIs with a 
99.5% probability of having been
initiated by a single infectious unit (5). However, a
single infectious unit does not necessarily equate with a single
virion. Clearly, some variation resident among LDSIs of Sidney 81 (probably representing preexisting variation in Sidney 81 prior to
limiting-dilution inoculation) was excluded from LDSI-S10 and, with two
exceptions (A6016 and C961), did not reappear
during the passage experiment. Reappearance of A6016 on
passage was most probably due to selection. C961, unlike
A6016, was detected as a minor variant in LDSI-S10, demonstrating that this polymorphism was present in the common inoculum
(LDSI-S10) used to establish all lineages subsequently passaged.
Because it was not possible to examine the LDSI-S10 population for
polymorphism until after the establishment of systemic infection, it is
not known whether A6016 and C961 were excluded by limiting-dilution inoculation and subsequently regenerated after
infection (or passage). Thus, we cannot determine whether LDSI-S10 was
derived from only one or a few founding genotypes.
Superficially, an infectious clone may appear to provide a better
method to achieve a bottleneck with a defined sequence as
a starting
point. However, the use of an infectious clone as a
transcription
template does not provide a uniform source of inoculum.
Schneider and
Roossinck (
52) report a composite mutation frequency
of
4.5 × 10
5/nt in a control experiment using a cloned DNA
template for in
vitro transcription followed by RT and PCR (15 cycles
with
Pfu polymerase). If only 20% of the composite mutation
frequency was
due to in vitro transcription, the RNA polymerase error
rate equals
~1 × 10
5/nt. In vitro transcription
using an RNA polymerase with an error
rate of ~1 × 10
5/nt would result in many single-substitution mutants
(for WSMV,
a total of 28,152 are possible) represented in the initial
inoculum,
with an average of ~0.1 nucleotide substitution per viral
genome
transcribed. Although the frequency of each individual mutant
is
low, collectively the sum of all mutants generated would average
~1
mutation per 10 complete genomes transcribed. Note that the
use of a
35S promoter construct to deliver the cloned virus genome
as a DNA
template for in vivo transcription (as for tobacco mosaic
virus [TMV]
in reference
52) does not eliminate variability
(which in
this case is equal to the error rate of the host RNA
polymerase) but,
rather, only delays the time when variation is
generated. Although
selection for infectious genomes occurs on
inoculation and is expected
to reduce primary variation, this
would occur with both cloned and
uncloned
inocula.
Use of an infectious clone does not preclude rapid establishment of
heterogeneity (
52) after multiple rounds of virus
replication
and systemic movement in planta. Note that all LDSI-S10
mutants
were singletons (Fig.
5) and that the corrected singleton
mutation
frequency per nucleotide obtained for LDSI-S10 (4.3 × 10
4) (Table
7) was similar to the mutation frequency per
nucleotide
(albeit uncorrected for RT-PCR error) of cucumber mosaic
virus
(CMV) (4.6 × 10
4) or TMV (5.2 × 10
4) on systemic infection established with transcripts
derived from
cloned DNA (
52). While neither method
guarantees exclusion of
all variation (an absolute bottleneck), both
limit genetic diversity
at inoculation and yield similar levels of
variation after systemic
infection. Finally, because the purpose of the
LDSI and passage
experiments was to monitor changes in the consensus
sequence at
specific points in time, it did not matter whether all or
only
some of the variation arose after a bottleneck
event.
Variant haplotypes among LDSIs probably reflect preexisting
variations in Type and Sidney 81.
Differences in haplotype
patterns among LDSIs are most easily explained as preexisting
variations resident within Type and Sidney 81 that were biologically
separated by limiting-dilution inoculation. This explanation is
particularly relevant to the four variant haplotypes identified among
Sidney 81 LDSIs, each of which was observed in at least two LDSIs
(Table 2). Over half of the genomes resident within the Sidney 81 culture bore at least one substitution, relative to the consensus
sequence corresponding to the predominant haplotype pattern. However,
since variation among Sidney 81 LDSIs was determined for only four
cistrons, representing ~45% of the genome, this represents a minimum
estimate and does not account for singleton variation, which could not
be distinguished from PCR artifacts in the LDSI experiment.
Fewer variant haplotypes were separated as LDSIs by limiting-dilution
inoculation of the Type culture (Table
2). This difference
could have
resulted from random probability of sampling and/or
arbitrary choice of
genomic regions examined in the LDSI experiment.
Differences in
within-isolate genetic diversity also were observed
for citrus tristeza
virus (
25). It is unlikely that the Type
genome encodes a
viral polymerase with greater fidelity or is
subject to greater
negative selective pressure than Sidney 81;
however, these hypotheses
cannot be excluded. Given the results
of the passage experiment, in
which nonsingleton variation increased
over time, the difference in
variant haplotype pattern frequencies
among LDSIs of Sidney 81 and Type
may reflect differences in the
population growth histories of the two
cultures. Although the
laboratory propagation histories of the two WSMV
strains are not
completely known, Type was subjected to a bottleneck
event by
mite transmission (
4) prior to storage as dried
infected tissue
and was passaged in the laboratory at high MOI fewer
times than
was Sidney 81 prior to the beginning of our experiments.
Thus,
sampling at different stages of population expansion following
a
genetic bottleneck could readily explain differences in the
polymorphism frequency between Type and Sidney
81.
In the LDSI experiment, a deliberate bottleneck was introduced. The
outcome measured was essentially that of joint probability;
i.e., given
low MOI, how often were differences in the consensus
sequence of WSMV
detected? Type and Sidney 81 vary by about 0.02/nt
(~2%), confirming
that WSMV can tolerate multiple substitutions,
and the preponderance of
synonymous substitutions within and between
these isolates are
compatible with the idea that most of the observed
polymorphisms among
consensus sequences are essentially neutral
(
9).
Nonsingleton variation uncovered within the two LDSI populations
was
limited, with mean pairwise differences (
) ranging from 0
to 7.7 × 10
4/nt among the four cistrons examined. The average
across all
four cistrons was 2.9 × 10
4/nt (Sidney 81) or
0.4 × 10
4/nt (Type). These are 2 orders of
magnitude smaller than the nucleotide
diversity (
2 × 10
2) estimated for WSMV field populations (
8,
33).
Selection explains one shared substitution during passage.
The
near-global reversion (G6016A) represented the sole
example of a change most consistent with selection. Since both alleles at this polymorphic site yield synonymous codons (Fig. 3), a selective advantage of A over G at this position could reflect RNA structural features or codon bias during translation. The eight lineages with
A6016 at passage 9 have a common node in a phylogenetic
analysis (Fig. 4). Since G6016 was present in all 22 LDSI-S10 NIa clones sequenced, A6016 was either absent from
LDSI-S10 or present at low frequency (<5%). If A6016 was
absent from LDSI-S10, then the substitution G6016A
occurred de novo in each lineage, such that the common node shared by
all lineages (except corn CC) represents a point of convergence rather
than divergence. Regardless of whether A6016 occurred at a
low level in LDSI-S10 or was generated de novo in each lineage, a
selective advantage of A over G at position 6016 remains the simplest
explanation for reversion to A6016 in eight of nine
lineages by passage 6.
LDSI-S10 was known to be a mixture at nucleotide position 961 in P1,
with both U and C present. Throughout the passage experiment,
the
haplotype associated with C
961 was detected by SSCP only
once
in corn CC at passage 6 (Table
5). Sequences of two clones from
each passage 6 P1 PCR product for all nine lineages indicated
that only
the two clones derived from corn CC had C
961. Thus,
unlike
A
6016, there did not appear to be selective pressure for
reversion to C
961, the nucleotide most common at this
position
among Sidney 81
LDSIs.
It was anticipated that changes in consensus sequence driven by
host-specific selection might be revealed as identical changes
in all
lineages passaged in the same host species. Although the
adjacent
substitutions C
8764U and A
8765C occurred
in two wheat
lineages, it is unlikely that this represents an example
of host-specific
selection, since U
8764 or
C
8765 did not appear in all three wheat
lineages and did
not successfully displace the original alleles
in any lineage at
passage 9 (Tables
5 and
6). Thus, no clear
example of host-specific
selection was observed, a result similar
to those reported with TMV
virus passaged in different host species
(
23). While a
signature of negative selection (fewer substitutions
in a certain
region of the genome) was detected among divergent
strains of WSMV
(
9), too few substitutions accrued during passage
to
perform similar analyses. Although the reversion G
6016A
was
probably due to selection, occurrence in all three host species
suggests that selective forces operating here did not vary among
the
three host species
tested.
Most newly generated mutations are sequestered in virions and have
yet to be tested for fitness.
Variation within a plant virus
isolate may be separated into two classes: (i) new mutants that have
yet to serve as replication templates and (ii) older mutants that have
been replicated. The RNA genome contained in each virion was
transcribed independently from a minus-strand template by the viral
polymerase with some probability of nucleotide misincorporation. By
definition, any "new" mutation appearing in a sample will be a
singleton. The sample frequency of all singletons representing new
mutations is dependent solely on the viral polymerase error rate.
Because most positive strands become encapsidated and thereby are
removed from the pool of replicating templates (14), the
bulk of viral RNA sampled for variation represents end products of
replication. If this is so, most variation is sequestered and is not
part of the effective population (that fraction of genomes giving rise to progeny). Thus, a substantial fraction of variation generated de
novo in a plant cell has not been subject to selection, the essential
cis-acting requirements of encapsidation notwithstanding. Therefore, the virion population could include deleterious or lethal
mutations that have yet to be tested for fitness. The observation that
3 of 16 satellite tobacco mosaic virus (STMV) cDNA clones bearing
nucleotide substitutions yielded noninfectious transcripts (27) supports the "sequestered and yet to be tested"
hypothesis. A corollary to this hypothesis is that the error rate of
the viral polymerase will be no greater than the per-nucleotide
frequency of singletons observed in a population.
Although most new mutants are sequestered by encapsidation and are not
immediate objects of selection, there remains an evolutionary
advantage
for high viral RNA polymerase error rates. Constant
production of
mutants ensures that alternative genotypes are poised
to take advantage
of new environments or selective conditions.
A fraction of new mutants
must serve as replication templates,
otherwise populations could not
evolve. Furthermore, encapsidated
genomes are available to initiate new
infections upon transmission,
such that the pool of sequestered
variation is not an evolutionary
dead
end.
Replicated mutants have been subjected to evolutionary
processes.
Only mutants that grow (serve as replication templates
and move) are subjected to selection and drift within a plant, and those that survive lead to a distribution of shared "older"
polymorphisms in the sample. Examples of older mutants identified in
this study include the variant haplotypes observed among Sidney 81 LDSIs and nonsingleton variants detected after passage. In
contradistinction to most other organisms with DNA genomes, RNA virus
growth is not semiconservative and has a significant linear component
(14) that is not accounted for by population growth models
based on binary (exponential) growth. The linear aspect of virus growth should have a profound quantitative effect on population divergence and
polymorphism, which warrants future theoretical examination.
The distribution of allele size classes (Fig.
6) expected would be a
composite of the Poisson distribution (singletons) and
another
distribution reflective of underlying parameters of mutant
growth,
selection, and drift. Differences in allele size class
distribution
before and after passage were due to the accumulation
of nonsingleton
mutants. All variants in LDSI-S10 were singletons,
consistent with a
Poisson distribution. This is not unexpected,
since bottlenecking by
limiting-dilution inoculation should have
eliminated most preexisting
variation. On passage at high MOI,
the accumulation of nonsingleton
variants shifted the population
away from a Poisson distribution,
although the frequency of singletons
remained higher than that
predicted from population models for
which probability distributions
may be calculated: the constant-sized,
infinite-sites model
(
64), or a model incorporating population
growth
(
28,
30,
51). The latter predicts more singleton
sites
than does the infinite-site model but clearly not as many
as were
actually observed (Fig.
6). Moreover, singleton sites
were randomly
distributed among WB passage 9 clones, which also
contained sites with
moderate allele class sizes (Fig.
5). It
is difficult to imagine a
population model that retains some sites
with moderate allele class
sizes while predicting many more singleton
sites unless two different
distributions are superimposed. The
excess of singletons is consistent
with the hypothesis that most
genomes in this allele size class
represent end products of
replication.
The two classes of mutations (singleton and nonsingleton) observed in
our data set are not unique to WSMV. Heterogeneity within
STMV and CMV
satellite RNA also partitioned into these two classes
(
26-28). The lack of nonsingleton variation observed
(
52) on
passage of TMV, CMV, or cowpea chlorotic mottle
virus may have
resulted from examination of only a small portion (12 to
16%)
of each genome and/or characterization of only a single lineage
for each of the three viruses studied. Nonuniform distribution
of
nucleotide substitutions accumulating in WSMV (Table
6) or
vesicular
stomatitis virus (
34) after multiple passages clearly
indicates that the amount of variation detected may differ depending
on
which region and proportion of the genome is examined. Alternatively,
the relative titer may affect the ratio of singletons to nonsingletons,
with high-titer viruses such as TMV, CMV, and cowpea chlorotic
mottle
virus having higher ratios of singletons to nonsingletons
relative to a
lower-titer virus such as WSMV. For the relative-titer
explanation to
hold, the number of genomes participating in cell-to-cell
movement
would be roughly equivalent regardless of the titer but
the total
number of genomes produced within individual cells would
differ
significantly. In this scenario, higher-titer viruses experience
more
stringent bottlenecks, not because fewer viral genomes move
but simply
because a greater percentage do not. Note that this
explanation would
not affect the singleton frequency per nucleotide,
which does not
correspond to relative virus titer as long as the
majority of
singletons do not serve as replication templates.
Nonetheless, the
consensus sequence of TMV can change on passage
(
23),
demonstrating that fundamental properties of population
growth are
similar among divergent plant virus
species.
Host species effects on mutant accumulation.
The singleton
frequency per nucleotide remained constant after passage in wheat
(Table 7), a result in congruence with that observed (52)
for TMV and CMV passaged in Nicotiana benthamiana. Because
viral polymerases have relatively high error rates and lack
proofreading capabilities (13, 14), it is not surprising that singletons were the most common allele size class observed. That
the singleton frequency per nucleotide did not significantly differ on
passage suggests that given a constant rate of singleton production
(the viral polymerase error rate), repeated bottlenecks at each
movement event dictate that the bulk of the "quasispecies cloud"
must be regenerated within each cell. Mutant frequencies at passage 9 for lineages passaged in barley or corn were not significantly higher
than for lineages passaged in wheat (Table 7), implying that the three
cereal host species do not provide differential environments affecting
the accumulation of mutants. However, passage of TMV and CMV in
different solanaceous hosts did result in altered mutant frequencies
(53), indicating that certain host-virus combinations may
differentially affect mutant accumulation.
Changes on passage are consistent with genetic drift.
By
examining nearly the entire WSMV genome, we measured changes in
consensus sequence during serial passage at high MOI and under constant
environmental conditions. From these results, it was clear that WSMV
populations are dynamic, with genotypes arising, gaining predominance,
losing dominance to other sequences, or serving as the foundation for
the accumulation of additional changes.
Variation within the LDSI-S10 population was dominated by singletons.
Upon passage, changes in SSCP patterns facilitated the
identification
of changes in consensus sequence due to point mutations.
At passage 9, changes in consensus sequence were reflected by
a majority of clones
bearing the point mutation associated with
the altered SSCP pattern
(Fig.
5 and data not shown). However,
not all clones examined in a
lineage at passage 9 contained the
point mutation which rose to
prominence. Thus, mutations do not
necessarily need to be fixed to give
rise to SSCP differences
between populations, but they need only
achieve a threshold
frequency.
Stochastic processes are now recognized as contributing to viral
evolution (
9,
17,
20,
30,
34,
38,
48). Most
changes in
consensus sequence during passage were unique to individual
lineages
and appear nondeterministic. That we observed consensus
sequence shifts
in populations of ~10
12 viral particles per plant implies
either strong selection, transmission
bottlenecks, or population
subdivision within the host plant.
The different populations were
maintained in a constant environment,
and only one consensus sequence
change (G
6016A) appears to have
been driven by
selection.
High-MOI inoculations might be expected to preclude plant-to-plant
transmission bottlenecks. However, the signature of bottlenecking
was
clearly evident in the distribution and composition of variation
during
passage at high MOI. Genotypic complexity among nonsingletons
within
each lineage at passage 9 was limited. For example, within
wheat WB
passage 9 (Fig.
5), clones appear to be from two groups
or sublineages,
suggesting that genetic bottlenecks must have
occurred despite a
high-MOI inoculation regime. Several pairs
of polymorphic sites (e.g.,
A
8248U and A
8403G) were linked in
the
majority of clones in which they were present (Fig.
5). This
is
consistent with a stepwise accumulation of mutations in a sublineage
that increases in frequency by fortuitously passing through
bottlenecks.
A simple and plausible explanation for bottlenecking during
transmission at high MOI is subdivision of viral populations within
a
plant. Population subdivision within mixed infections of WSMV
strains
has been documented (
17). Although that study examined
the
distribution of Type and Sidney 81 in plants simultaneously
inoculated
with a mixture of the two strains, variants of a single
strain should
subdivide in a similar fashion. Subdivision probably
results from a
small number of genomes participating in movement,
followed by
exclusion via cross-protection at the cellular level
(
17).
One consequence of subdivision is that different regions
of a
systemically infected plant may harbor different proportions
of
variants such that an inoculum harvested from a given region
of
systemically infected tissue may be randomly enriched for a
specific
genotype. Subdivision will enhance the probability of
fixation (i.e.,
genetic drift) of a mutant upon passage, even
at high MOI. Over
multiple passages, this process is repeated,
resulting in accrual of
mutations in a stepwise manner. Because
the process of drift is
essentially random, the outcome within
any single lineage is not
predetermined. The stochastic processes
of bottlenecking, subdivision,
and drift actually reduce variation
within any given lineage, yet the
random nature of these processes
facilitates divergence among
genetically isolated lineages within
the constraints of
selection.
| |
ACKNOWLEDGMENTS |
We acknowledge the excellent technical assistance of Melissa
Morris and the support and advice of Casey Carmelea, Kempton Horken,
and Il-Ryong Choi.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: USDA-ARS, 344 Keim Hall, University of Nebraska, Lincoln, NE 68583. Phone: (402)
472-2710. Fax: (402) 472-2853. E-mail:
dstenger{at}unlnotes.unl.edu.
| |
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Journal of Virology, November 2001, p. 10231-10243, Vol. 75, No. 21
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.21.10231-10243.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
