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Negative Effect of Genetic Bottlenecks on the Adaptability of Vesicular Stomatitis Virus
doi:10.1016/j.jmb.2003.12.002
J. Mol. Biol. (2004) 336, 61–67
Negative Effect of Genetic Bottlenecks on the Adaptability of Vesicular Stomatitis Virus I. S. Novella* Department of Microbiology and Immunology, Medical College of Ohio, 3055 Arlington Avenue, Toledo, OH 43614-5806, USA
Muller’s ratchet is a principle of evolutionary genetics describing mutant accumulation in populations that are repeatedly subjected to genetic bottleneck. The immediate effect of Muller’s ratchet, overall loss of fitness, has been confirmed in several viral systems belonging to different groups. This report shows that in addition to fitness loss, genetic bottlenecks also have longer-term effects, namely changes in the capacity of viral populations to adapt. Thus, vesicular stomatitis virus strains with a history of genetic bottleneck have lower adaptability than strains maintained at relatively large population sizes. This lower adaptability is illustrated by their reduced ability to regain fitness and by their inability to outcompete wildtype populations in situations where the initial fitness of the bottlenecked mutant is the same or even higher than the initial fitness of the wild-type. q 2003 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: quasispecies; VSV; experimental evolution; genetic bottleneck; Muller’s ratchet
Introduction RNA viruses are excellent systems to study evolutionary processes.1,2 With mutation rates of 1023 –1025 substitutions per nucleotide and round of replication (or an average of about one mutation per genome copied3), RNA viruses generate highly polymorphic populations known and quasispecies. Quasispecies theory was originally developed by Manfred Eigen and colleagues,4 – 8 and it can be viewed as a special case of the mutation– selection balance model for extremely high mutation rates.9 One of the most important features of quasispecies populations is the dominance of group selection, in that selection acts at the level of the whole populations rather than of individual virions. Since the original publication of the theoretical models, several groups have provided experimental support for quasispecies as a good descriptor of RNA virus populations,10 – 15 although some controversy can still be found in the literature.10 – 13 One of the genetic principles that has been studied most thoroughly with RNA viruses is Muller’s ratchet.14 Muller’s ratchet describes the accumulation of deleterious mutations and overall fitness loss that occurs after repeated genetic Abbreviations used: VSV, vesicular stomatitis virus; wt, wild-type; m.o.i., multiplicity of infection. E-mail address of the corresponding author: [email protected]
bottleneck, and its operation has been demonstrated in several viral species.15 – 18 Vesicular stomatitis virus (VSV) was the first mammalian virus in which Muller’s ratchet was demonstrated,16 and work from John Holland’s laboratory has provided a better understanding of this process.19 – 22 VSV is one of the preferred models to study RNA virus evolution.23 VSV is the prototype of the Rhabdoviridae family,24 which also includes rabies virus. It has a non-segmented, negative-stranded, 11 kb genome that contains five open reading frames (ORFs). Three polypeptides (N, P and L) are required for RNA synthesis and two (M and G) have structural roles. The M and P ORFs also code for additional products that are less well characterized.25,26 VSV is an arbovirus with a very wide host range in both groups of its hosts: mammals and insects. Replication in mammalian cells is normally cytolytic, and leads to complete cell killing with titers of 109 –1010 plaque-forming units (PFU)/ml within 12 –48 hours, depending on the viral strain, size of infecting population and cell type. In contrast, insects and insect cells become persistently infected with little or no obvious cell damage, and titers are significantly lower. Two reports have described a VSV strain, called MARM C, having an initially neutral relative fitness but which consistently loses in long-term competitions against wild-type (wt) VSV.27,28 Because of its history of repeated bottleneck passages, this outcome was proposed to have resulted
0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
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Low Adaptability of Bottlenecked VSV Strains
from the accumulation of mutations per Muller’s ratchet, leading to lower adaptability. The term adaptability was used to describe the potential of a population to improve in fitness.28 In order to test the validity of this hypothesis, several populations were generated by repeated plaque-to-plaque passages, followed by additional large population passages when fitness losses were observed. These neutral populations were competed against wt virus and their fate followed through a period of up to 79 passages. The results showed that, as previously seen for MARM C, bottlenecked populations were unable to outcompete wt VSV, indicating impaired adaptability.
Results Bottlenecked populations were generated by 20 plaque-to-plaque passages of MARM U as described,16 and amplified at low multiplicity of infection (m.o.i.) for a single passage to generate a large stock of each strain. A total of 18 replicas of this passage regime were done, and fitness of the resulting populations was determined by direct competition against wt. Most of the strains showed fitness decrease, in agreement with previous work.16,19 Six of the populations were selected to continue the study (Table 1). Mutant population MRy showed fitness increase. Mutant MRb maintained a neutral fitness value relative to wt and was not passaged further. Mutants MRd, MRm, MRq and MRr showed different degrees of fitness decrease, and were then allowed to recover by repeated passages of large populations (2 £ 105 PFU) at low m.o.i., which has been shown to result in exponential fitness increases of strains having similar initial fitness.29 Fitness was tested during these large population passages until neutrality was regained (i.e. a relative fitness of 1). As described below, MRr showed no fitness recovery after six large population passages (Table 1). MARM U is the surrogate wt and has the same
genomic sequence, with the exception of a U ! G mutation at nucleotide 3853 (Ala ! Asp) that confers the resistance to I1 monoclonal antibody (mAb). Short-term competitions, of one to six passages, showed that MARM U and wt have the same fitness (Table 1), as reported.21 The fitness assays were continued, until one of the populations lost the competition. A MARM population, including MARM U, was considered as a loser when its frequency fell below 5% of its initial value. The rationale for stopping these competitions before genuine extinction is threefold. First, mutant frequencies are between 1023 and 1024 for the I1 epitope, due to several mutations conferring resistance to I1 mAb.30 Thus, our method cannot truly measure mutant extinction, since there will always be a small fraction of naturally occurring MARM clones. A second reason is the existence of memory in viral quasispecies, as reported by Domingo’s group.31,32 A third argument is the potential effects of complementation in maintaining low-fitness mutants, particularly when rare.33,34 The competitions between MARM U and wt ended with three MARM U wins and five MARM U losses, so the null hypothesis (i.e. random behavior) could not be rejected (Table 2). Two representative competitions, one leading to MARM U dominance and one resulting in MARM U loss are shown in Figure 1. Thus, fitness gains could occur equally in MARM U and wt populations, indicating no differences in adaptability. MRr, one of the low fitness populations, was unable to recover fitness after six large population passages (Table 2). This is in contrast with other bottlenecked strains that were able to do so after the same number of passages,19,29,35 including those described here (Table 2). This is the first description of a low fitness VSV strain unable to recover fitness under these conditions, suggesting decreased adaptability compared to wt or other VSV strains. MRy was one of the two populations among the 18 replicas that showed a high fitness phenotype
Table 1. Origin and fitness of VSV strains Strain Wild-type MARM U MRb Mry MRd Rd MRm Rm MRq Rq MRr MRr þ 6 a
Historya
Fitnessb
Replication in BHK-21 cells MARM clone of wt MARM U plus 20 plaque-to-plaque passages MARM U plus 20 plaque-to-plaque passages MARM U plus 20 plaque-to-plaque passages MRd plus two amplification passages MARM U plus 20 plaque-to-plaque passages MRm plus six amplification passages MARM U plus 20 plaque-to-plaque passages MRq plus four amplification passages MARM U plus 20 plaque-to-plaque passages MRr plus six amplification passages
1.0 1.00 ^ 0.05 1.01 ^ 0.05 1.23 ^ 0.03 0.82 ^ 0.07 0.99 ^ 0.04 0.15 ^ 0.07 1.00 ^ 0.07 0.6 ^ 0.1 1.0 ^ 0.1 0.43 ^ 0.08 0.45 ^ 0.3
pc
0.05 0.0005 0.027 0.48
All the clonal strains were amplified once at low m.o.i. to produce a working stock. Fitness values as calculated in short-term competitions (one to five passages). Comparison between a bottlenecked strain and its recovered counterpart. p indicates the probability that the fitness values of each pair is the same (one-tailed t test). b c
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Low Adaptability of Bottlenecked VSV Strains
Table 2. Outcome of long-term competitions between VSV wt and strains with and without a history of genetic bottleneck Strain
Number of assays
Number lost
Mutants without a history of genetic bottleneck MARM U 8 5
pa 0.21875
Mutants with a history of genetic bottleneck Mutants without a period of recovery MRb 8 Mry 8
8 8
0.0039 0.0039
Mutants with a period of recovery 8 MARM Cb Rd 6 Rm 6 Rq 8
8 6 6 8
0.0039 0.0156 0.0156 0.0039
a Probability of a strain losing all the competitions at random, calculated based on binomial distributions. b Data from Quer et al.30,31
in single-passage competitions against wt virus (Table 1). However, at the second competition passage MRy frequency started decreasing, and this trend continued until extinction (Figure 2). This result could be interpreted as wt VSV starting at a low point of a high fitness peak, while MRy would start from a higher point, but at a lower fitness peak (Figure 3). To test this model wt and MRy were allowed to replicate separately for a single passage at low m.o.i. and the corresponding progenies were competed against each other. As expected, the initial frequency increase of MRy ceased to be observed and the frequency decrease took place at the same rate as in competitions between the parental wt and MRy strains (Figure 2). In a third experiment MRy was passaged three times and competed against original wt. The result was indistinguishable from that displayed by the original MRy strain (Figure 2). Four neutral VSV populations with a history of repeated genetic bottleneck were competed against wt. These included a strain without overall fitness loss after the repeated genetic bottleneck (MRb) and three strains that did lose fitness but were allowed to recover (Rd, Rm, Rq). A fifth strain has been previously described (MARM C).27,28 Long-
term competitions led to variable periods of coexistence followed by eventual extinction of the mutant strains in every replica (Figure 4). The period of co-existence was only reproducible for Rq, it was limited to a single competition passage, and frequency decrease took place at a similar rate in all the replicas. The time at which MRb, Rd and Rm started decaying ranged between seven and 60 passages, and the rates of frequency decrease were also variable.
Discussion The present results show that VSV strains with a history of genetic bottleneck consistently lose in competition with wild-type virus. All 44 competitions carried out with six different bottleneck strains resulted in mutant losses. In addition, MRr þ 6 was unable to regain fitness after six large-population passages. The possible outcomes of long-term competitions can be treated as a binomial distribution under the null hypothesis that competing populations behave randomly. Therefore, the null hypothesis cannot be rejected for MARM U, but the probability of six out of six or eight out of eight competitions leading to the same outcome by chance events is 0.03125 and 0.0078125, respectively, and thus these results are statistically significant. A non-random behavior can be a priori translated into overall wins or overall losses, decreasing the probabilities of the actual results (all losses) by half in each case (Table 2). Lack of fitness loss in MRb does not imply no mutations accumulated, and non-random behavior of MRb during long-term competitions against wt has two possible (and not mutually exclusive) explanations. First, there could be accumulation of neutral mutations; and second, there could be accumulation of deleterious mutations accompanied by beneficial mutations, resulting in no net fitness change. Beneficial mutations can be fixed during genetic bottleneck, though with much lower probability than that for deleterious or even neutral mutations. One potential explanation for the present results is that the bottlenecked populations have a fitness
Figure 1. Changes in normalized MARMU:wt ratios during longterm competitions in BHK-21 cells. Two representative replicas are shown. These competitions were initiated at an approximately 1:1 ratio and were done in parallel. The horizontal line represents no change compared to the initial ratio. One of the competitions resulted in MARM U overcoming wt (W), and the other led to wt dominance (X).
64
Low Adaptability of Bottlenecked VSV Strains
Figure 2. Representative results of changes of MARM:wt ratios during long-term competitions between MRy and wt (W), between MRy and wt after a single amplification passage of each population (B), and between MRy after three amplification passages and wt (K) in BHK-21 cells. The standard error of the passage 1 value is smaller than the size of the symbol. The horizontal line represents no change compared to the initial ratio.
disadvantage smaller than the error of the fitness assay. If that were the case, one would expect a linear decline in the relative abundance of the bottlenecked competitor until extinction is reached. None of the strains described here behaved linearly, but instead there was a period of stable coexistence followed by a phase of more or less drastic frequency decline (Figures 2 and 4). A second argument is provided by MARM G, a VSV mutant with initial high fitness, that could be nonetheless outcompeted by wt.36 The same behavior was seen with MRy, the relative abundance of which actually increased during the first competition passage only to plunge during subsequent replication. Also, in some replicas with several bottleneck strains the critical period of decline was immediately preceded by a period of frequency increase, a type of trajectory that had already been described for MARM C (see Figure 1, series C, by Quer et al.27). A second possible explanation of these results is that the wt population has had the opportunity to achieve high levels of heterogeneity, while the mutant populations are still relatively homogeneous. Several facts point against the argument that low diversity is due to lack of opportunity to generate it. First, all the populations, including the
wt, are originally clonal. MARM U is a clone with one amplification passage, the same as MRb and MRy, and it is capable of outcompeting wt. In another study, MARM H and MARM D are strictly clonal strains, and were able to outcompete wt.36 In contrast, Rm had undergone a total of seven large population passages (including the first amplification passage), and still was unable to prevail in any of the competitions against wt. Many of the competitions, particularly involving MRb, Rd and Rm, showed stable mutant frequencies for many passages, providing additional opportunities for genetic diversification. The representative example depicted in Figure 4, shows co-existence of wt and Rm for 28 passages. MRr þ 6 was unable to recover fitness significantly in six recovery passages, so impairment was evident even in the absence of competing wt. Studies on plant alphaviruses further support this argument. Two recent reports, showed that the quasispecies cloud size in a variety of virus –plant pairs achieved its maximum after a single passage.37,38 Limitations in the ability to gain fitness are better explained by differences in the potential of the bottlenecked genomes to accumulate mutations with a net beneficial effect on fitness. One possible
Figure 3. Bidimensional fitness landscape showing the position of the VSV strains described here. The horizontal line represents neutrality. The open circle indicates the position of wt and MARM U; the filled circles represent the bottlenecked strains and recovered strains. The continuous line representing the fitness landscape is theoretical, and the different mutants may be at the same or at different fitness peaks, but the positions of peaks and valleys relative to the data points are designed to reflect the relative likelihood of favorable, neutral or deleterious mutations for each population (see Discussion).
Low Adaptability of Bottlenecked VSV Strains
65
Figure 4. Representative results of changes in MARM:wt ratios during long-term competitions between wt and MRb (X), Rd (B), Rm (þ) and Rq (V) in BHK-21 cells. For each strain the replica was chosen randomly. The horizontal line represents no change compared to the initial ratio.
mechanism, as discussed above, is by limiting the amount of variability generated because of lower mutation rates. It seems unlikely that mutations leading to higher polymerase fidelity occurred and were fixed in all mutants, since Muller’s ratchet is fundamentally a random process. Assuming similar overall mutation rates, differences may occur in the beneficial mutation rate and the deleterious mutation rate, that is, the quantity and/or quality of mutations that can be generated. A higher deleterious mutation rate translates into low genome robustness. Differences in genome robustness are more likely to operate when both populations are at the top of a fitness peak,39 a situation that does not apply to these competing VSV populations. Thus, the best explanation for the results is that mutant populations have a lower beneficial mutation rate than wt. The ability of a genome to generate beneficial mutations is determined by its sequence. The actual mutations are, of course, random, but a genome’s position on the fitness landscape biases the likely effects of such mutations.40 Muller’s ratchet provides a mechanism to generate diverse starting points for the generation and accumulation of variation, and recovery by compensation would likely promote further deviation from wt sequence and its corresponding fitness peak. Compensation has an important contribution to fitness recovery in phage f6, foot-and-mouth disease virus, and human immunodeficiency virus type 1.41 – 43 These results are in better agreement with the Wrightian model of rugged landscapes44 than with Fisher’s model,45 although the latter cannot be ruled out. Some of the mutants may be at the top of fitness peaks, for instance, MRy (see Figure 3). Since the neutral strains were able of stable co-existence with wt for a variable period of time, and wt is increasing in fitness during this time, it is reasonable to assume that the MARM populations are increasing in fitness as well, as predicted by the Red Queen hypothesis.46 At least MARM C has been confirmed to be gaining fitness during competition.27,29 However, this
fitness gain could not keep pace with the fitness increase of wt. Lack of fitness recovery in MRr þ 6 suggests that MRr is far from any substantial fitness peaks (Figure 3). This cannot be interpreted at this point as an inability to evolve, since MRr could potentially accumulate neutral or deleterious mutations. Additional large population passages may eventually lead to fitness recovery, but it is clear that this population could not gain fitness as well as other strains. It is possible that within larger samples of bottlenecked strains some may be found in fitness peaks higher than that where wt VSV is located. Such populations, with better adaptability than wt, would behave as overall winners in long-term competitions. However, no such strain was identified here, or even any strain with adaptability similar to that of wt. The results, with seven out of seven bottlenecked strains showing compromised adaptability, agree with a scenario where wt is at an optimal fitness peak, and Muller’s ratchet has an overall negative effect by placing the mutant strains in suboptimal locations of the fitness landscape. This process should be intensified if compensation prevails during recovery. Figure 3 shows the representation of a bidimensional fitness landscape with the location of mutant strains most consistent with the experimental data. It is also possible that adaptability differences would disappear if larger population sizes could be used. This may allow sampling of additional beneficial mutations in bottlenecked strains at each competition passage. The strains presently described will be useful for understanding the process of fitness recovery in negative strand viruses, particularly with the availability of reverse genetics for VSV.47,48 In addition we can learn about the basis of adaptability. What type of mutations lead to differences in adaptability? Are these mutations selectively neutral? Analyses of the mutations accumulated during competitions can discriminate between a quantitative and a qualitative model of low beneficial
66
mutation rates (or show the validity of both). These questions are currently under investigation and will help to improve our knowledge of the rules governing the evolution of RNA viruses.
Materials and Methods Cells and viruses Host cells were Baby Hamster Kidney (BHK-21) cells from John Holland’s laboratory grown in MEM supplemented with 7% (v/v) heat-inactivated bovine calf serum and 0.06% (w/v) proteose peptone no. 3 to a density of 0.8 – 1 £ 105 cells/cm2. I1 monoclonal antibody hybridoma cells were a kind gift from Douglas Lyles and they generate an antibody that targets the I1 epitope of the VSV G glycoprotein.49 Mutations that confer resistance to I1 are selectively neutral in a variety of environments, providing a good marker that allows the differentiation of wt from MARM populations during competitions. Virus stocks were prepared from the wt and MARM U strains described by Novella et al.21 by a single low m.o.i. amplification passage on BHK-21 cells. Large volumes of stocks were produced, aliquoted, and kept at 2 86 8C in order to avoid additional manipulation of these strains. MARM U was employed as the parental strain to generate all the strains described here (Table 1). Virus competitions Fitness determinations were done as originally described by Holland and co-workers.50 We arbitrarily gave wt a fitness of 1.0, and we compared the replicative ability of the different MARM strains in competition. In short, mixtures of wt and MARM at approximately 1:1 ratio were used to infect a BHK-21 monolayer in T-25 flasks with a population size of 2 £ 105 PFU (m.o.i. 0.1). Virus progeny after 20 – 24 hours was harvested and used to infect a fresh monolayer under the same conditions. The original mixture and viral progeny after each competition passage were carefully titrated in the presence and absence of I1 mAb to quantify relative ratios of wt and MARM populations. Log transformed changes on those ratios were normalized by the initial ratio, and the slope of the linear fit is the fitness value. Additional details of these methods can be found elsewhere.20,50 Fitness assays were done at least in duplicate for competitions carried out for two or more passages, and at least six times for single-passage assays. The competitions can be continued until one population undergoes extinction, providing the means to observe differences in adaptability.
Acknowledgements I am grateful to Eric Miller, Selene Za´rate, Robert Blumenthal, Roger Herr, and Ramo´n Sua´rez Valdivieso for invaluable help. Bonnie Ebendick provided outstanding technical assistance. Work in my laboratory is supported by NIAID (NIH) grant R01-AI45686.
Low Adaptability of Bottlenecked VSV Strains
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Edited by J. Karn (Received 23 September 2003; received in revised form 24 November 2003; accepted 2 December 2003)
J. Mol. Biol. (2004) 336, 61–67
Negative Effect of Genetic Bottlenecks on the Adaptability of Vesicular Stomatitis Virus I. S. Novella* Department of Microbiology and Immunology, Medical College of Ohio, 3055 Arlington Avenue, Toledo, OH 43614-5806, USA
Muller’s ratchet is a principle of evolutionary genetics describing mutant accumulation in populations that are repeatedly subjected to genetic bottleneck. The immediate effect of Muller’s ratchet, overall loss of fitness, has been confirmed in several viral systems belonging to different groups. This report shows that in addition to fitness loss, genetic bottlenecks also have longer-term effects, namely changes in the capacity of viral populations to adapt. Thus, vesicular stomatitis virus strains with a history of genetic bottleneck have lower adaptability than strains maintained at relatively large population sizes. This lower adaptability is illustrated by their reduced ability to regain fitness and by their inability to outcompete wildtype populations in situations where the initial fitness of the bottlenecked mutant is the same or even higher than the initial fitness of the wild-type. q 2003 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: quasispecies; VSV; experimental evolution; genetic bottleneck; Muller’s ratchet
Introduction RNA viruses are excellent systems to study evolutionary processes.1,2 With mutation rates of 1023 –1025 substitutions per nucleotide and round of replication (or an average of about one mutation per genome copied3), RNA viruses generate highly polymorphic populations known and quasispecies. Quasispecies theory was originally developed by Manfred Eigen and colleagues,4 – 8 and it can be viewed as a special case of the mutation– selection balance model for extremely high mutation rates.9 One of the most important features of quasispecies populations is the dominance of group selection, in that selection acts at the level of the whole populations rather than of individual virions. Since the original publication of the theoretical models, several groups have provided experimental support for quasispecies as a good descriptor of RNA virus populations,10 – 15 although some controversy can still be found in the literature.10 – 13 One of the genetic principles that has been studied most thoroughly with RNA viruses is Muller’s ratchet.14 Muller’s ratchet describes the accumulation of deleterious mutations and overall fitness loss that occurs after repeated genetic Abbreviations used: VSV, vesicular stomatitis virus; wt, wild-type; m.o.i., multiplicity of infection. E-mail address of the corresponding author: [email protected]
bottleneck, and its operation has been demonstrated in several viral species.15 – 18 Vesicular stomatitis virus (VSV) was the first mammalian virus in which Muller’s ratchet was demonstrated,16 and work from John Holland’s laboratory has provided a better understanding of this process.19 – 22 VSV is one of the preferred models to study RNA virus evolution.23 VSV is the prototype of the Rhabdoviridae family,24 which also includes rabies virus. It has a non-segmented, negative-stranded, 11 kb genome that contains five open reading frames (ORFs). Three polypeptides (N, P and L) are required for RNA synthesis and two (M and G) have structural roles. The M and P ORFs also code for additional products that are less well characterized.25,26 VSV is an arbovirus with a very wide host range in both groups of its hosts: mammals and insects. Replication in mammalian cells is normally cytolytic, and leads to complete cell killing with titers of 109 –1010 plaque-forming units (PFU)/ml within 12 –48 hours, depending on the viral strain, size of infecting population and cell type. In contrast, insects and insect cells become persistently infected with little or no obvious cell damage, and titers are significantly lower. Two reports have described a VSV strain, called MARM C, having an initially neutral relative fitness but which consistently loses in long-term competitions against wild-type (wt) VSV.27,28 Because of its history of repeated bottleneck passages, this outcome was proposed to have resulted
0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
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Low Adaptability of Bottlenecked VSV Strains
from the accumulation of mutations per Muller’s ratchet, leading to lower adaptability. The term adaptability was used to describe the potential of a population to improve in fitness.28 In order to test the validity of this hypothesis, several populations were generated by repeated plaque-to-plaque passages, followed by additional large population passages when fitness losses were observed. These neutral populations were competed against wt virus and their fate followed through a period of up to 79 passages. The results showed that, as previously seen for MARM C, bottlenecked populations were unable to outcompete wt VSV, indicating impaired adaptability.
Results Bottlenecked populations were generated by 20 plaque-to-plaque passages of MARM U as described,16 and amplified at low multiplicity of infection (m.o.i.) for a single passage to generate a large stock of each strain. A total of 18 replicas of this passage regime were done, and fitness of the resulting populations was determined by direct competition against wt. Most of the strains showed fitness decrease, in agreement with previous work.16,19 Six of the populations were selected to continue the study (Table 1). Mutant population MRy showed fitness increase. Mutant MRb maintained a neutral fitness value relative to wt and was not passaged further. Mutants MRd, MRm, MRq and MRr showed different degrees of fitness decrease, and were then allowed to recover by repeated passages of large populations (2 £ 105 PFU) at low m.o.i., which has been shown to result in exponential fitness increases of strains having similar initial fitness.29 Fitness was tested during these large population passages until neutrality was regained (i.e. a relative fitness of 1). As described below, MRr showed no fitness recovery after six large population passages (Table 1). MARM U is the surrogate wt and has the same
genomic sequence, with the exception of a U ! G mutation at nucleotide 3853 (Ala ! Asp) that confers the resistance to I1 monoclonal antibody (mAb). Short-term competitions, of one to six passages, showed that MARM U and wt have the same fitness (Table 1), as reported.21 The fitness assays were continued, until one of the populations lost the competition. A MARM population, including MARM U, was considered as a loser when its frequency fell below 5% of its initial value. The rationale for stopping these competitions before genuine extinction is threefold. First, mutant frequencies are between 1023 and 1024 for the I1 epitope, due to several mutations conferring resistance to I1 mAb.30 Thus, our method cannot truly measure mutant extinction, since there will always be a small fraction of naturally occurring MARM clones. A second reason is the existence of memory in viral quasispecies, as reported by Domingo’s group.31,32 A third argument is the potential effects of complementation in maintaining low-fitness mutants, particularly when rare.33,34 The competitions between MARM U and wt ended with three MARM U wins and five MARM U losses, so the null hypothesis (i.e. random behavior) could not be rejected (Table 2). Two representative competitions, one leading to MARM U dominance and one resulting in MARM U loss are shown in Figure 1. Thus, fitness gains could occur equally in MARM U and wt populations, indicating no differences in adaptability. MRr, one of the low fitness populations, was unable to recover fitness after six large population passages (Table 2). This is in contrast with other bottlenecked strains that were able to do so after the same number of passages,19,29,35 including those described here (Table 2). This is the first description of a low fitness VSV strain unable to recover fitness under these conditions, suggesting decreased adaptability compared to wt or other VSV strains. MRy was one of the two populations among the 18 replicas that showed a high fitness phenotype
Table 1. Origin and fitness of VSV strains Strain Wild-type MARM U MRb Mry MRd Rd MRm Rm MRq Rq MRr MRr þ 6 a
Historya
Fitnessb
Replication in BHK-21 cells MARM clone of wt MARM U plus 20 plaque-to-plaque passages MARM U plus 20 plaque-to-plaque passages MARM U plus 20 plaque-to-plaque passages MRd plus two amplification passages MARM U plus 20 plaque-to-plaque passages MRm plus six amplification passages MARM U plus 20 plaque-to-plaque passages MRq plus four amplification passages MARM U plus 20 plaque-to-plaque passages MRr plus six amplification passages
1.0 1.00 ^ 0.05 1.01 ^ 0.05 1.23 ^ 0.03 0.82 ^ 0.07 0.99 ^ 0.04 0.15 ^ 0.07 1.00 ^ 0.07 0.6 ^ 0.1 1.0 ^ 0.1 0.43 ^ 0.08 0.45 ^ 0.3
pc
0.05 0.0005 0.027 0.48
All the clonal strains were amplified once at low m.o.i. to produce a working stock. Fitness values as calculated in short-term competitions (one to five passages). Comparison between a bottlenecked strain and its recovered counterpart. p indicates the probability that the fitness values of each pair is the same (one-tailed t test). b c
63
Low Adaptability of Bottlenecked VSV Strains
Table 2. Outcome of long-term competitions between VSV wt and strains with and without a history of genetic bottleneck Strain
Number of assays
Number lost
Mutants without a history of genetic bottleneck MARM U 8 5
pa 0.21875
Mutants with a history of genetic bottleneck Mutants without a period of recovery MRb 8 Mry 8
8 8
0.0039 0.0039
Mutants with a period of recovery 8 MARM Cb Rd 6 Rm 6 Rq 8
8 6 6 8
0.0039 0.0156 0.0156 0.0039
a Probability of a strain losing all the competitions at random, calculated based on binomial distributions. b Data from Quer et al.30,31
in single-passage competitions against wt virus (Table 1). However, at the second competition passage MRy frequency started decreasing, and this trend continued until extinction (Figure 2). This result could be interpreted as wt VSV starting at a low point of a high fitness peak, while MRy would start from a higher point, but at a lower fitness peak (Figure 3). To test this model wt and MRy were allowed to replicate separately for a single passage at low m.o.i. and the corresponding progenies were competed against each other. As expected, the initial frequency increase of MRy ceased to be observed and the frequency decrease took place at the same rate as in competitions between the parental wt and MRy strains (Figure 2). In a third experiment MRy was passaged three times and competed against original wt. The result was indistinguishable from that displayed by the original MRy strain (Figure 2). Four neutral VSV populations with a history of repeated genetic bottleneck were competed against wt. These included a strain without overall fitness loss after the repeated genetic bottleneck (MRb) and three strains that did lose fitness but were allowed to recover (Rd, Rm, Rq). A fifth strain has been previously described (MARM C).27,28 Long-
term competitions led to variable periods of coexistence followed by eventual extinction of the mutant strains in every replica (Figure 4). The period of co-existence was only reproducible for Rq, it was limited to a single competition passage, and frequency decrease took place at a similar rate in all the replicas. The time at which MRb, Rd and Rm started decaying ranged between seven and 60 passages, and the rates of frequency decrease were also variable.
Discussion The present results show that VSV strains with a history of genetic bottleneck consistently lose in competition with wild-type virus. All 44 competitions carried out with six different bottleneck strains resulted in mutant losses. In addition, MRr þ 6 was unable to regain fitness after six large-population passages. The possible outcomes of long-term competitions can be treated as a binomial distribution under the null hypothesis that competing populations behave randomly. Therefore, the null hypothesis cannot be rejected for MARM U, but the probability of six out of six or eight out of eight competitions leading to the same outcome by chance events is 0.03125 and 0.0078125, respectively, and thus these results are statistically significant. A non-random behavior can be a priori translated into overall wins or overall losses, decreasing the probabilities of the actual results (all losses) by half in each case (Table 2). Lack of fitness loss in MRb does not imply no mutations accumulated, and non-random behavior of MRb during long-term competitions against wt has two possible (and not mutually exclusive) explanations. First, there could be accumulation of neutral mutations; and second, there could be accumulation of deleterious mutations accompanied by beneficial mutations, resulting in no net fitness change. Beneficial mutations can be fixed during genetic bottleneck, though with much lower probability than that for deleterious or even neutral mutations. One potential explanation for the present results is that the bottlenecked populations have a fitness
Figure 1. Changes in normalized MARMU:wt ratios during longterm competitions in BHK-21 cells. Two representative replicas are shown. These competitions were initiated at an approximately 1:1 ratio and were done in parallel. The horizontal line represents no change compared to the initial ratio. One of the competitions resulted in MARM U overcoming wt (W), and the other led to wt dominance (X).
64
Low Adaptability of Bottlenecked VSV Strains
Figure 2. Representative results of changes of MARM:wt ratios during long-term competitions between MRy and wt (W), between MRy and wt after a single amplification passage of each population (B), and between MRy after three amplification passages and wt (K) in BHK-21 cells. The standard error of the passage 1 value is smaller than the size of the symbol. The horizontal line represents no change compared to the initial ratio.
disadvantage smaller than the error of the fitness assay. If that were the case, one would expect a linear decline in the relative abundance of the bottlenecked competitor until extinction is reached. None of the strains described here behaved linearly, but instead there was a period of stable coexistence followed by a phase of more or less drastic frequency decline (Figures 2 and 4). A second argument is provided by MARM G, a VSV mutant with initial high fitness, that could be nonetheless outcompeted by wt.36 The same behavior was seen with MRy, the relative abundance of which actually increased during the first competition passage only to plunge during subsequent replication. Also, in some replicas with several bottleneck strains the critical period of decline was immediately preceded by a period of frequency increase, a type of trajectory that had already been described for MARM C (see Figure 1, series C, by Quer et al.27). A second possible explanation of these results is that the wt population has had the opportunity to achieve high levels of heterogeneity, while the mutant populations are still relatively homogeneous. Several facts point against the argument that low diversity is due to lack of opportunity to generate it. First, all the populations, including the
wt, are originally clonal. MARM U is a clone with one amplification passage, the same as MRb and MRy, and it is capable of outcompeting wt. In another study, MARM H and MARM D are strictly clonal strains, and were able to outcompete wt.36 In contrast, Rm had undergone a total of seven large population passages (including the first amplification passage), and still was unable to prevail in any of the competitions against wt. Many of the competitions, particularly involving MRb, Rd and Rm, showed stable mutant frequencies for many passages, providing additional opportunities for genetic diversification. The representative example depicted in Figure 4, shows co-existence of wt and Rm for 28 passages. MRr þ 6 was unable to recover fitness significantly in six recovery passages, so impairment was evident even in the absence of competing wt. Studies on plant alphaviruses further support this argument. Two recent reports, showed that the quasispecies cloud size in a variety of virus –plant pairs achieved its maximum after a single passage.37,38 Limitations in the ability to gain fitness are better explained by differences in the potential of the bottlenecked genomes to accumulate mutations with a net beneficial effect on fitness. One possible
Figure 3. Bidimensional fitness landscape showing the position of the VSV strains described here. The horizontal line represents neutrality. The open circle indicates the position of wt and MARM U; the filled circles represent the bottlenecked strains and recovered strains. The continuous line representing the fitness landscape is theoretical, and the different mutants may be at the same or at different fitness peaks, but the positions of peaks and valleys relative to the data points are designed to reflect the relative likelihood of favorable, neutral or deleterious mutations for each population (see Discussion).
Low Adaptability of Bottlenecked VSV Strains
65
Figure 4. Representative results of changes in MARM:wt ratios during long-term competitions between wt and MRb (X), Rd (B), Rm (þ) and Rq (V) in BHK-21 cells. For each strain the replica was chosen randomly. The horizontal line represents no change compared to the initial ratio.
mechanism, as discussed above, is by limiting the amount of variability generated because of lower mutation rates. It seems unlikely that mutations leading to higher polymerase fidelity occurred and were fixed in all mutants, since Muller’s ratchet is fundamentally a random process. Assuming similar overall mutation rates, differences may occur in the beneficial mutation rate and the deleterious mutation rate, that is, the quantity and/or quality of mutations that can be generated. A higher deleterious mutation rate translates into low genome robustness. Differences in genome robustness are more likely to operate when both populations are at the top of a fitness peak,39 a situation that does not apply to these competing VSV populations. Thus, the best explanation for the results is that mutant populations have a lower beneficial mutation rate than wt. The ability of a genome to generate beneficial mutations is determined by its sequence. The actual mutations are, of course, random, but a genome’s position on the fitness landscape biases the likely effects of such mutations.40 Muller’s ratchet provides a mechanism to generate diverse starting points for the generation and accumulation of variation, and recovery by compensation would likely promote further deviation from wt sequence and its corresponding fitness peak. Compensation has an important contribution to fitness recovery in phage f6, foot-and-mouth disease virus, and human immunodeficiency virus type 1.41 – 43 These results are in better agreement with the Wrightian model of rugged landscapes44 than with Fisher’s model,45 although the latter cannot be ruled out. Some of the mutants may be at the top of fitness peaks, for instance, MRy (see Figure 3). Since the neutral strains were able of stable co-existence with wt for a variable period of time, and wt is increasing in fitness during this time, it is reasonable to assume that the MARM populations are increasing in fitness as well, as predicted by the Red Queen hypothesis.46 At least MARM C has been confirmed to be gaining fitness during competition.27,29 However, this
fitness gain could not keep pace with the fitness increase of wt. Lack of fitness recovery in MRr þ 6 suggests that MRr is far from any substantial fitness peaks (Figure 3). This cannot be interpreted at this point as an inability to evolve, since MRr could potentially accumulate neutral or deleterious mutations. Additional large population passages may eventually lead to fitness recovery, but it is clear that this population could not gain fitness as well as other strains. It is possible that within larger samples of bottlenecked strains some may be found in fitness peaks higher than that where wt VSV is located. Such populations, with better adaptability than wt, would behave as overall winners in long-term competitions. However, no such strain was identified here, or even any strain with adaptability similar to that of wt. The results, with seven out of seven bottlenecked strains showing compromised adaptability, agree with a scenario where wt is at an optimal fitness peak, and Muller’s ratchet has an overall negative effect by placing the mutant strains in suboptimal locations of the fitness landscape. This process should be intensified if compensation prevails during recovery. Figure 3 shows the representation of a bidimensional fitness landscape with the location of mutant strains most consistent with the experimental data. It is also possible that adaptability differences would disappear if larger population sizes could be used. This may allow sampling of additional beneficial mutations in bottlenecked strains at each competition passage. The strains presently described will be useful for understanding the process of fitness recovery in negative strand viruses, particularly with the availability of reverse genetics for VSV.47,48 In addition we can learn about the basis of adaptability. What type of mutations lead to differences in adaptability? Are these mutations selectively neutral? Analyses of the mutations accumulated during competitions can discriminate between a quantitative and a qualitative model of low beneficial
66
mutation rates (or show the validity of both). These questions are currently under investigation and will help to improve our knowledge of the rules governing the evolution of RNA viruses.
Materials and Methods Cells and viruses Host cells were Baby Hamster Kidney (BHK-21) cells from John Holland’s laboratory grown in MEM supplemented with 7% (v/v) heat-inactivated bovine calf serum and 0.06% (w/v) proteose peptone no. 3 to a density of 0.8 – 1 £ 105 cells/cm2. I1 monoclonal antibody hybridoma cells were a kind gift from Douglas Lyles and they generate an antibody that targets the I1 epitope of the VSV G glycoprotein.49 Mutations that confer resistance to I1 are selectively neutral in a variety of environments, providing a good marker that allows the differentiation of wt from MARM populations during competitions. Virus stocks were prepared from the wt and MARM U strains described by Novella et al.21 by a single low m.o.i. amplification passage on BHK-21 cells. Large volumes of stocks were produced, aliquoted, and kept at 2 86 8C in order to avoid additional manipulation of these strains. MARM U was employed as the parental strain to generate all the strains described here (Table 1). Virus competitions Fitness determinations were done as originally described by Holland and co-workers.50 We arbitrarily gave wt a fitness of 1.0, and we compared the replicative ability of the different MARM strains in competition. In short, mixtures of wt and MARM at approximately 1:1 ratio were used to infect a BHK-21 monolayer in T-25 flasks with a population size of 2 £ 105 PFU (m.o.i. 0.1). Virus progeny after 20 – 24 hours was harvested and used to infect a fresh monolayer under the same conditions. The original mixture and viral progeny after each competition passage were carefully titrated in the presence and absence of I1 mAb to quantify relative ratios of wt and MARM populations. Log transformed changes on those ratios were normalized by the initial ratio, and the slope of the linear fit is the fitness value. Additional details of these methods can be found elsewhere.20,50 Fitness assays were done at least in duplicate for competitions carried out for two or more passages, and at least six times for single-passage assays. The competitions can be continued until one population undergoes extinction, providing the means to observe differences in adaptability.
Acknowledgements I am grateful to Eric Miller, Selene Za´rate, Robert Blumenthal, Roger Herr, and Ramo´n Sua´rez Valdivieso for invaluable help. Bonnie Ebendick provided outstanding technical assistance. Work in my laboratory is supported by NIAID (NIH) grant R01-AI45686.
Low Adaptability of Bottlenecked VSV Strains
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Edited by J. Karn (Received 23 September 2003; received in revised form 24 November 2003; accepted 2 December 2003)