Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain1
Centro de Astrobiología (CSIC-INTA), Carretera de Ajalvir, km 4, 28850 Torrejón de Ardoz, Madrid, Spain2
Author for correspondence: Esteban Domingo. Fax +34 91 3974799. e-mail edomingo{at}cbm.uam.es
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Abstract |
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Introduction |
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In contrast to large population passages, repeated plaque-to-plaque transfers of RNA viruses result in fitness loss (Chao, 1990 ; Duarte et al., 1992
; Escarmís et al., 1996
; Yuste et al., 1999
). This is due to the accumulation of deleterious mutations as a result of repeated sampling (bottlenecking) of components of the mutant spectrum of the virus quasispecies. It must be stressed that a virus population may either gain or lose fitness depending on both the initial fitness of the population and the size of the genetic bottleneck, as shown by Novella et al. (1995c)
using vesicular stomatitis virus clones and populations of different initial fitness. In a study with the animal picornavirus foot-and-mouth disease virus (FMDV), a highly debilitated (low fitness) clone was derived by 22 successive plaque-to-plaque transfers of an FMDV clone termed
; the debilitated clone was termed
and its fitness value was 0·1 times that of
(Escarmís et al., 1996
). The consensus genomic nucleotide sequence of
differed from that of
in seven point mutations and in the acquisition of an internal polyadenylate tract as an extension of four adenylate residues at genomic positions 11191122, preceding the second functional AUG initiation codon (Fig. 1
). FMDV genome residues have been numbered according to Escarmís et al. (1996)
. One of the mutations was a deletion of a U residue at position 1056 (
U-1056), which rendered the region between the two AUG initiation codons non-functional with regard to protein-coding, since
U-1056 led to a termination codon at the position that would encode the eleventh residue of the large form of L protease (termed Lab) (Escarmís et al., 1996
). The internal polyadenylate at genomic positions 11191122 was the first genetic lesion to revert to the wild-type sequence in the process of fitness gain when
was subjected to large population passages, while
U-1056 did not revert, and served as a specific genetic marker for the
lineage (Escarmís et al., 1999
) (Fig. 1
).
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Methods |
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Molecular cloning.
RNA extracted from p50 was amplified by RTPCR using the thermostable Pfu polymerase (Promega), which has a proof-reading activity (Cline et al., 1996
). The synthesis of cDNA was carried out with 5 U AMV RT (Promega) in a final volume of 25 µl in a buffer containing 20 U RNasin (Promega), 10 mM TrisHCl, pH 8·3, 1·5 mM MgCl2, 50 mM KCl, 0·8 mM dNTPs, 200 ng oligodeoxynucleotide primer and about 7 ng FMDV RNA. For amplification with Pfu, the buffer recommended by the supplier was added to a final volume of 100 µl plus 200 ng of the second primer and 2·5 U of enzyme. The primers used contained restriction sites at their 5' ends to facilitate cloning in appropriately digested pGEM4Z. To amplify the internal ribosome entry site (IRES) and the region between the two initiation AUG codons, the primer for cDNA synthesis was complementary to positions 12001183 of FMDV RNA and contained a SacI restriction site; the second primer for PCR amplification corresponded to nucleotides 569587 of FMDV RNA and had a BamHI restriction site. To amplify the VP1-coding region, the primer for cDNA synthesis was complementary to positions 38883869 of FMDV RNA and contained a BamHI restriction site; the second primer for PCR amplification corresponded to nucleotides 31713192 of FMDV RNA and had a SacI restriction site. After treatment with phenol to inactivate the DNA polymerase, the DNA was recovered by ethanol precipitation (Sambrook et al., 1989
). After digestion of both the PCR-amplified products and the vector pGEM4Z with SacI and BamHI, the DNA was electrophoresed through 1% SeaPlaque agarose in 40 mM Trisacetate, 1 mM EDTA, pH 8·0, the appropriate bands were excised from the gel and the DNA was purified by using the Gene Clean II kit as indicated by the manufacturer (Bio 101). After ligation overnight at 16 °C, the ligation product was transformed in E. coli DH-5
and transformants were isolated and analysed following standard procedures (Sambrook et al., 1989
). Plasmid DNA was purified by using the Wizard Plus SV Minipreps kit (Promega). Two separate sets of molecular clones were obtained and analysed: 70 clones spanning the IRES and the region between the two AUG initiation codons and 70 clones corresponding to the VP1-coding region. Nucleotide sequencing was carried out in an ABI 373 automatic sequencer, as described previously (Escarmís et al., 1999
).
Statistics.
Standard statistical procedures used were those described in the package Hypothesis tests of the program Mathematica 3 (Wolfram Research).
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Results |
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Insertions and deletions (indels) were found in the IRES and in the region between the two AUGs (Table 3). G-1118, the site of a mutational hot spot for G
A transitions, was deleted in one molecular clone. A rich repertoire of adenylate insertions was found within positions 11191122, including one molecular clone with 20 additional adenylate residues, a length which is very close to the average present in the dominant sequence of the parental clone
(Escarmís et al., 1996
).
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Point replacements, insertions and deletions may reflect quasispecies memory or be predictive of dominant genomes
Virus quasispecies may contain memory genomes in the form of minority components of the mutant spectra that reflect their past evolutionary history (Ruiz-Jarabo et al., 2000 ). The clonal analysis of
p50 reinforces the previous evidence of quasispecies memory and, furthermore, reveals that some mutations may anticipate a future, dominant sequence in quasispecies evolution. Specifically, A-3653
C, which results in the amino acid replacement K-149
T in VP1, was present in two biological clones and two molecular clones from
p50, and also in genomes that had been dominant at passages 0 and 20, and which became dominant again at passage 100 (Escarmís et al., 1999
). Mutation C-3650
A, which results in the amino acid replacement T-148
K in VP1, was found in two biological clones and two molecular clones and it became dominant at passage 100 (Escarmís et al., 1999
) (compare Fig. 2
and Table 2
). The clonal analysis of
p50 extends the previous study on memory genomes harbouring an internal polyadenylate tract as a memory marker (Ruiz-Jarabo et al., 2000
) by 70 additional clones. Despite their debilitating effect on virus fitness (Escarmís et al., 1999
), additional adenylate residues were detected in 17% of the clones analysed (Table 3
). Thus, at a passage in which the virus population was actively gaining fitness, sequence analysis of individual clones revealed mutations that anticipated the future evolution of the population and other mutations that were remains of its evolutionary history.
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Discussion |
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The genetic heterogeneity in the IRES of p50 was comparable to that in the VP1-coding region (Table 1
). Application of the M-fold program (included in the GCG package) indicated that 50% of all point substitutions and the C insertion within residues 810814 found in the IRES did not alter its predicted secondary structure, because these substitutions are located in loops or bulges (Pilipenko et al., 1989
; Martínez-Salas et al., 1996
). One of the molecular clones [MOL (IA)56; Table 4
] included C-766
U and C-886
U, which are predicted to convert G:C into G:U base pairs in domains 3 and 4, respectively, with a total
G of +3·8 kcal/mol (15·9 kJ/mol). The most drastic mutation was U-799
C, present in one biological clone and one molecular clone, which is predicted to disrupt a stem in a very conserved hammerhead structure present in domain 3 with
G=+5·5 kcal/mol (23·0 kJ/mol) relative to the same IRES domain with the consensus nucleotide sequence. The remaining mutations are predicted to cause little modification in the secondary structure but various degrees of alteration of base pairing. The calculated differences in
G never exceeded 2·8 kcal/mol (11·7 kJ/mol). Mutations that are not in predicted loops or bulges in the IRES are underlined in Table 2
.
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A second question that the analysis of the mutant spectrum of p50 allows to be addressed is the comparison between the expected and actual number of genomes harbouring one, two, three, four or five mutations (Table 4
). Considering the analysis of biological clones for the IRES and the VP1-coding region (a length of 1061 nucleotides), the maximum mutation frequency is 6·5x10-4 substitutions per nucleotide, which gives an expected mean number of mutations within these regions of 0·69 (6·5x10-4x1061). The expected proportions of clones with no mutations or one, two, three, four or five mutations in the 1061 nucleotides analysed in biological clones are respectively 50, 35, 12, 2·7, 0·46 and 0·07% (calculated according to the Poisson distribution PK=mKe-m/K!, where PK is the probability of a genome having K mutations and m is the mean number of mutations per genome); the actual experimental values were respectively 50, 37, 10, 1·4, 0 and 1·4%. The same calculation for the VP1-coding region among molecular and biological clones indicates that the predicted proportions of sequences with no mutations or one, two, three, four or five mutations are 62, 30, 7·3, 1·2, 0·14 and 0·01%; the actual values were 64, 26, 7·9, 2·1, 0 and 0%. In all cases, there is good agreement between the expected and actual distribution of mutations among the clones analysed (in both cases: 0·7<P<0·9;
2, 1 degree of freedom), further supporting the conclusion that molecular and biological clones provided an indistinguishable representation of the
p50 quasispecies.
Analysis of the mutant spectrum of p50 has detected the presence of memory genomes in virus quasispecies, documented previously in this and in another evolutionary lineage of FMDV (reviewed in Domingo, 2000
; Ruiz-Jarabo et al., 2000
). The memory markers identified were the replacement K-149
T in VP1 (Table 2
) and the heterogeneous internal polyadenylate tract dominant in
p0, which contained an average of 19 additional adenylate residues (Escarmís et al., 1996
) (Fig. 1
). One molecular clone from
p50 included 20 additional adenylates (Table 3
) and thus represents an accurate memory genome of the ancestral
p0 population, while other genomes with smaller numbers of adenylates must be regarded as derivatives of the founder genomes.
Additions or deletions of adenylate residues within positions 11191122, the hot-spot G-1118A transition and the rare deletion of G-1118 can occur as a result of misalignment of the growing RNA strand or the template strand (Ripley, 1990
) during viral RNA synthesis (Fig. 3
). Misalignment mutagenesis events tend to occur in repeated sequences (Streisinger et al., 1966
; Ripley, 1990
; Denver et al., 2000
; Funchain et al., 2000
) and they may be favoured by the low stability of the poly(A)·poly(U) duplex. Homopolymeric poly(A)·poly(U) displays low melting temperature and a tendency to form a triple helix as the salt concentration is raised (Saenger, 1984
; and references therein). Obviously, other molecular mechanisms are possible to account for the repertoire of point substitutions and indels in
p50 (Ripley, 1990
). The presence of
U-1056 prevents the expression of Lab and hence may explain both the higher mutation frequency observed in the region located between the two AUG initiation codons and the high frequency of the transition G-1118
A. This mutation would lead, in the absence of
U-1056, to the replacement G-27
E in protease Lab. Since G-27 in Lab is conserved among European FMDV isolates (Ryan & Flint, 1997
), its substitution by E may be deleterious when Lab is functional.
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In conclusion, a reliable characterization of the mutant spectrum of a virus quasispecies at the nucleotide sequence level can be obtained through the analysis of biological clones or molecular clones. The results revealed the great complexity of a mutant spectrum of a clonal population in the process of fitness gain in a constant biological environment. The distribution of mutations provided evidence of substructuring within the mutant spectrum of the p50 quasispecies. Some mutants reflected the past evolutionary history of the population, while other mutants anticipated those that will become dominant at a later stage of the evolutionary process. This observation may be of practical relevance, in that the quantification of mutations related to variations in B cell or T cell epitopes and to resistance to antiviral agents, which may be present in different proportions in the mutant spectrum, may guide decisions on alternative immunotherapeutic or antiviral regimens. There is increasing evidence that fitness values, virus load and quasispecies complexity may be relevant to the pathogenic potential of viruses (Rowe et al., 1997
; Farci et al., 2000
; Quiñones-Mateu et al., 2000
) and to the response of an infected host to antiviral treatment (Pawlotsky et al., 1998
). The present study encourages quasispecies composition analyses at the nucleotide sequence level for diagnostic and therapeutic purposes.
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Acknowledgments |
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Received 23 November 2000;
accepted 16 January 2001.