Sex and the Evolution of Intrahost Competition in RNA Virus φ6

Sex allows beneficial mutations that occur in separate lineages to be fixed in the same genome. For this reason, the Fisher-Muller model predicts that adaptation to the environment is more rapid in a large sexual population than in an equally large asexual population. Sexual reproduction occurs in populations of the RNA virus φ6 when multiple bacteriophages coinfect the same host cell. Here, we tested the model’s predictions by determining whether sex favors more rapid adaptation of φ6 to a bacterial host, Pseudomonas phaseolicola. Replicate populations of φ6 were allowed to evolve in either the presence or absence of sex for 250 generations. All experimental populations showed a significant increase in fitness relative to the ancestor, but sex did not increase the rate of adaptation. Rather, we found that the sexual and asexual treatments also differ because intense intrahost competition between viruses occurs during coinfection. Results showed that the derived sexual viruses were selectively favored only when coinfection is common, indicating that within-host competition detracts from the ability of viruses to exploit the host. Thus, sex was not advantageous because the cost created by intrahost competition was too strong. Our findings indicate that high levels of coinfection exceed an optimum where sex may be beneficial to populations of φ6, and suggest that genetic conflicts can evolve in RNA viruses. IF sex is defined as the exchange of genetic material the role of promoting linkage equilibrium (Felsenstein 1974). In the first case, the environment is variable between organisms (Michod and Levin 1988), then sexual reproduction is found to be extremely wideand sex brings together novel and genetic combinations favored by positive selection. In the second case, the spread in nature. This is surprising because sex has certain costs associated with it. For example, the producenvironment may be constant, but the genome is changing because the rate of deleterious mutations is high. tion of males leads to a twofold cost of sex (Williams 1975; Maynard Smith 1978; Seger and Hamilton Sex brings together parts of genomes that have not been destroyed by mutations, and selection then acts to purify 1988). Another consequence of sex is that it tends to them through the removal of deleterious mutations. break apart well-adapted combinations of genes (coA model of positive selection developed by Fisher adapted gene complexes). Thus, whenever favorable (1930) and Muller (1932; see also Maynard Smith combinations of genes are brought together into single 1988) proposes that adaptation is more rapid in a large individuals via mutation, recombination, and/or synfinite population of sexual organisms than in an equally gamy, sex has the potential to immediately tear them large, but asexual, population evolving in the same enviapart at its next occurrence (Shields 1988). For these ronment. Suppose that two favorable mutations, A and reasons one would expect asexuality to be selectively B, can arise in separate individuals in the same populafavored; therefore, the prevalence of sex in natural poption and that each mutation can increase under seleculations of organisms remains an intriguing question tion. In an asexual population, these mutations can at in evolutionary biology (Michod and Levin 1988; best compete with each other until one or the other Hurst and Peck 1996). spreads to fixation [see Gerrish and Lenski (1998) Two general hypotheses have been suggested for the for theoretical treatment of this process]. Thus, an AB evolution of sex. Positive selection models propose that individual can arise only if the two mutations appear sex may be advantageous because it generates beneficial sequentially in a single evolving lineage (e.g., A occurs variation in novel or changing environments. On the and increases to fixation, but B can be fixed only if it other hand, purifying selection models argue that sex occurs in an individual that is already A). In contrast, may have evolved because it reduces or prevents the if A and B occur in different individuals in a sexual buildup of deleterious mutations (mutational load). population, genetic exchange allows the two mutations Both hypotheses are similar in that they ascribe to sex to be combined in a single descendant. For this reason, the Fisher-Muller hypothesis predicts that sex has the potential to accelerate the pace of adaptive evolution. Corresponding author: Paul E.Turner, Department of Biology, UniverNote that this argument applies only in large finite popsity of Maryland, College Park, MD 20742. E-mail: [email protected] ulations. An infinite population is so large that the geGenetics 150: 523–532 (October 1998) 524 P. E. Turner and L. Chao netic combination AB can arise spontaneously, whereas, small, medium, and large, respectively. Because a single phage contains all three segments (Day and Mindich in a small population, the advantage is offset because any beneficial mutation that occurs is likely to be fixed 1980), a lone phage infecting a host cell can reproduce, but reproduction is then asexual. In contrast, genetic (or lost) before the next one appears. Sexual reproduction in RNA viruses is analogous to exchange (sex) occurs when multiple φ6 viruses coinfect the same host cell and generate reassortant (hybrid) that in eukaryotes (Chao 1994). When two or more viruses coinfect the same host cell, hybrid progeny are progeny (Mindich et al. 1976). RNA virus φ6 provides a powerful system to explore produced through genetic exchange between the parent genomes. In some RNA viruses, the exchange is by the evolution and advantage of sex (Chao 1990; Chao et al. 1992, 1997). Its characteristics include short generrecombination ( Jarvis and Kirkegaard 1991), but in others it is achieved by segmenting the virus into several ation times and extremely high rates of spontaneous mutation: on the order of 1023 to 1025 errors per nucleosmaller RNA molecules, and hybrid progeny are reassortants containing segments descending from the varitide replication (Chao 1988). These features allow φ6 to be easily propagated in the laboratory for hundreds ous coinfecting parents. Examples of the latter include certain viruses that infect humans (e.g., Hantavirus and of generations, permitting evolutionary processes to be studied in detail. influenza; Ramig 1991). Because recombination between segments is rare or nonexistent in segmented Experimental overview: Sex in viruses is easily manipulated by controlling the multiplicity of infection (moi), RNA viruses (Horiuchi 1975; Mindich et al. 1976; Holland et al. 1982), this suggests that reassortment evolved or ratio of viruses to bacterial cells. We chose to examine the effect of sex at moi’s of 0.002 and 5. At both moi’s, as an alternative to recombination for the purpose of promoting sex (Pressing and Reanney 1984; Chao and assuming Poisson sampling (Sokal and Rohlf 1981), the proportion of cells infected with 0, 1, and 1988). If so, sex in RNA viruses and eukaryotes may be independent evolutionary events, and segmentation in $2 phages is, respectively, P(0) 5 e2moi, P(1) 5 (e2moi 3 moi) / 1, and P($2) 5 1 2 P(0) 2 P(1). Thus, only these viruses becomes a particularly instructive model for testing theories for the evolution of sex as a general P($2) / (1 2 P(0)) or 0.1% of all infected cells contain two or more viruses at an moi of 0.002, and reproduction phenomenon. Previous experiments have shown that fitness of RNA is primarily asexual. By the same logic, at an moi of 5 coinfection by two or more viruses is common and 97% virus φ6 decreases when viral lineages are subjected to a succession of population bottlenecks (Chao 1990; of cells should experience multiple infections. A single clone of bacteriophage φ6 was divided into Chao et al. 1992). The fitness decline results because the intensified genetic drift produced by small bottlenecks three sexual (moi 5 5) and three asexual (moi 5 0.002) populations, and then allowed to evolve through propaleads to the buildup of deleterious mutations, a phenomenon termed Muller’s ratchet (Muller 1964). Sex gation on the bacterial host P. phaseolicola. Presence or absence of sex in experimental populations was imposed in φ6 may be advantageous in combating Muller’s ratchet because segment reassortment presumably refor 50 consecutive days, which is equivalent to 250 generations of viral evolution. Throughout the study, a daily creates (from mutated individuals) progeny with no or fewer mutations (Chao et al. 1992, 1997). These comsample from each population of evolving viruses was stored in the freezer for later study. At the end of the bined results suggest that the conditions favoring the evolution of sex through Muller’s ratchet may be easily 50-day experiment, samples from each population (taken at discrete time intervals) were competed against satisfied in an RNA virus such as φ6. However, to assess the generality of these results, alternative hypotheses a common competitor of the ancestral genotype to measure changes in fitness. In this way, we determined for the evolution of sex must also be evaluated. Here we present results of experiments initiated to whether phage adaptation was more rapid in sexual populations than in asexual populations. examine whether the conditions favoring an advantage of sex by the Fisher-Muller hypothesis could be similarly The Fisher-Muller model predicts that sex allows more rapid evolution in a sexual population than in an asexsatisfied in φ6. Experimental system—RNA virus φ6: The RNA virus ual population of equal size. Equal size is an important criterion because, all else being equal, any population used in this study is the bacteriophage φ6. Although its natural bacterial host is unknown, φ6 can be grown in of large N should evolve faster than a population of small N. This is simply because beneficial mutations are the laboratory on Pseudomonas phaseolicola, the phytopathogen responsible for bean blight (Vidaver et al. expected to appear more often in a larger population (i.e., more individuals are present where these muta1973). Phage φ6 has a genome that is divided into three double-stranded RNA molecules (Semancik et al. 1973). tions occur at random). Thus, a crucial component of our experimental design was to eliminate differences Total genome size in φ6 is 13,379 nucleotides, and the three segments comprise 22, 30, and 48% of the genome in population size among the sexual and asexual treatments. We did so by controlling the number of viral (McGraw et al. 1986; Gottlieb et al. 1988; Mindich et al. 1988); thus, the relative segments are referred to as progeny harvested in each treatment population. When 525 Intrahost Competition in Viruses moi 5 5 (i.e., 1 3 1010 phage/ml to 2 3 109 bacteria/ml). one or more phages infect a cell, the resultant viral These mixtures were placed in a nonshaking incubator for 40 progeny form a visible plaque on the surface of the min to allow phage adsorption. Following adsorption, 500 bacterial lawn. Each plaque in the asexual treatment was phages from each mixture were plated on a P. phaseolicola produced through infection of one phage (on average), lawn for 24 hr incubation. The next day, the propagation cycle was completed when 100 of the resultant plaques from and every day we harvested 500 plaques to propagate each population were harvested to prepare a new phage lysate; each asexual population (N 5 500). In contrast, each because each plaque contained the progeny of five viruses plaque in the sexual treatment was produced through (on average), N equaled 500 in each S population. The propacoinfection of five phages (on average), and here we gation cycle was then repeated using the new lysate, and a harvested only 100 plaques for daily propagation (N 5 total of 50 cycles was conducted for each population. The P. phaseolicola hosts used in propagation were grown daily from 500). a frozen stock. This prevented evolution of phage resistance by the host bacteria and eliminated the possibility that bacteria and phage would coevolve. As each cycle represents approxiMATERIALS AND METHODS mately 5 generations of viral evolution, 250 generations occurred during the experiment. Following daily propagation, Phage and bacteria: All viruses were originally derived from a sample from each population’s lysate was stored in a 2208 a single clone of bacteriophage φ6, previously described by freezer for future study. Chao et al. (1992). We also obtained a spontaneous host-range During the first five cycles of the experiment (and periodimutant of φ6, referred to as φ6h. Host-range ability occurs cally thereafter), lysates were titered to gauge the exact conthrough a point mutation on the medium segment, and a centration of phage per milliliter. These data were used to previous study showed that the h marker imposed a 5% fitness ensure the accuracy ofmoi during the subsequent propagation cost (Chao et al. 1992). Preliminary experiments confirmed cycle. Because the titer of phage lysates was not highly variable that φ6h carries a 7% fitness cost under our experimental (data not shown), cycles propagated without titering were conditions (data not shown). All fitness measurements relabased on the mean titer in the initial five cycles (z2 3 1010 tive to φ6h reported below are adjusted to reflect the cost of phage/ml). the h marker. Asexual treatment: The same φ6 lysate described above was The P. phaseolicola host strain used in all experiments was used to found the three replicate populations in the asexual purchased from the American TypeCulture Collection (ATCC treatment, designated “A.” Each A population was mixed with No. 21781). An additional host strain, P. pseudocaligenes ERA, an overnight culture of P. phaseolicola at moi 5 0.002 (i.e., 4 3 was obtained from the laboratory of L. Mindich (Public Health 10 phage/ml to 2 3 10 bacteria/ml). Adsorption followed Research Institute, New York). φ6h forms clear plaques when by plating was identical to that described in the S treatment. plated on a mixed lawn containing both P. phaseolicola and The next day, the propagation cycle was completed when 500 P. pseudocaligenes. In contrast, non-host-range phages form turof the resultant plaques from each population were harvested bid plaques on a mixed lawn because they do not kill the to prepare a new phage lysate; because each plaque contained P. pseudocaligenes cells present. the progeny of only one phage (on average), N equaled 500 Culture conditions and media: All phages and bacteria were in each A population. As in the S treatment, later propagation grown, plated, incubated, and diluted at 258 in LC medium, cycles were based upon the mean titer of lysates in the first a modification of Luria broth (Mindich et al. 1976). Liquid five cycles (z1 3 10 phage/ml). Lysateswere stored in a 2208 LC medium allows a stationary-phase bacterial density of z4 3 freezer as previously described. Aside from possible phage 109 cells/ml for P. phaseolicola, and z5 3 1010 cells/ml for interactions during adsorption, the asexual treatment was deP. pseudocaligenes ERA. All bacterial cultures were inoculated signed to minimize interactions between viruses. Figure 1 deby a single bacterial colony placed into 10 ml LC medium in picts major features of the propagation cycle in each experia sterile flask. Culture flasks were grown for 24 hr in a shaking mental treatment. incubator at 258 and 120 rpm. During this 24-hr period, bactePaired-growth experiments: After the method of Chao rial cultures attained stationary-phase densities. All bacterial (1990), fitness was measured by comparing the growth rate stocks were stored in a 4:6 glycerol/LC (v/v) solution at 2208. of a test phage (or mixed population of phages) relative to that Agar concentrations in plates were 1.5 and 0.7% for bottom of the ancestral phage bearing an h marker (φ6h). Competitors and top LC agar, respectively. The volume of top agar was were mixed at a 1:1 volumetric ratio, and z400 viruses were 3 ml/plate, and that of bacterial lawns was 200 ml. Plates plated with top agar on a lawn containing 200 ml of overnight used in all evolution experiments contained lawns made from P. phaseolicola culture (z8 3 108 cells). Because no preadsorpovernight bacterial cultures of P. phaseolicola. P/E plates used tion occurred before plating, every virus in the lawn infected in some assays contained a mixture of P. phaseolicola and a cell alone. After 24 hr incubation, the resulting 400 plaques P. pseudocaligenes ERA at a 200:1 volumetric ratio; ordinary and were then harvested and filtered to produce a lysate. The ratio host-range phages produce turbid and clear plaques, respecof test phage to φ6h in the starting mixture (R0) and in the tively, on P/E plates. harvested lysate (R1) was estimated by plaques formed on Phage lysates were prepared by plating plaque-purified phage with top agar and a P. phaseolicola lawn. After 24 hr, P/E plates, where the ratio of the two phages was based on the h marker. Thus, fitness was assayed on a P. phaseolicola plaques in the top agar were resuspended in 3 ml of LC broth and centrifuged at 3000 rpm for 10 min. Supernatant lawn, but the starting and final ratios were assayed on a mixed lawn of hosts. The number of plaques per paired-growth plate containing the phage lysate was filtered (0.22 mm, Durapore; Millipore, Bedford, MA) to remove bacteria. Phage lysates and mixed lawn plate was maximized at 400 because this minimized plaque overlap and, hence, interaction between were stored at 2208 in a 4:6 glycerol/LC (v/v) solution. Sexual treatment: A single clone of φ6 was used to prepare phages. Fitness (W) is defined as W 5 R1/R0. If W 5 1, then the test phage has the same fitness as the reference phage a phage lysate as described above. At the start of the experiment this lysate was used to found three replicate populations (φ6h); if W , 1, it has a lower fitness and accordingly for W . 1. For increased sensitivity when fitness differences were in the sexual treatment, designated “S.” Each S population was then mixed with an overnight culture of P. phaseolicola at small, our protocol was repeated, in which case W t 5 Rt/R0, 526 P. E. Turner and L. Chao Figure 1.—Summary of the propagation schemes for the asexual and sexual treatment groups. Most aspects of propagation were identical in the two treatments. Phage (d) adsorbed to bacterial cells (h) at a given multiplicity-of-infection (moi), and this mixture was used to seed a bacterial lawn. During overnight growth, the viral progeny formed visible plaques (s). These plaques were harvested, and the bacteria were removed by filtration to create a new lysate. (A) The asexual treatment contained moi 5 0.002, ensuring that each plaque produced was the result of a single infection, (B) whereas, the sexual treatment contained moi 5 5, ensuring that each plaque was the result of coinfection by five viruses (on average). To control for differences in population size between Figure 2.—Fitness improvement in terms of host exploitathe two groups, one-fifth as many plaques were harvested in tion (paired-growth) for each population in the two treatthe sexual treatment as in the asexual treatment. There were ments. (A–C) Populations A1, A2, and A3, respectively, from three replicate populations in each group, and all of the poputhe asexual treatment; (D–F) populations S1, S2, and S3 from lations were propagated for 50 days. See text for details. the sexual treatment. Each point represents mean fitness (6SE) relative to the common competitor (φ6h) based on 3 replicate assays, except for the ancestor that is based on 10 where t is the number of repetitions, and Rt is the ratio after assays. See text for statistical analyses. t repetitions (Chao 1990). Modified fitness assays: Fitness was also estimated in the two evolutionary environments: moi 5 5 and moi 5 0.002. (n 5 3) for each population. As shown in Figure 2, the The above fitness assay was modified so that two competitors S and A treatment populations underwent very different were mixed at an equal volumetric ratio, but were allowed to adsorb to P. phaseolicola for 40 min at a given moi. Following fitness gains during the experiment. Population A3 adsorption, the phages were plated with top agar on a showed a final fitness improvement of approximately P. phaseolicola lawn. To ensure that modified fitness assays twice that in any other experimental population (Figure matched the treatment conditions as closely as possible, the 2C). More importantly, final mean values for fitness in total number of phage per plate equaled that in the experithe A populations exceeded those in the S populations, ment proper (z500 plaques per plate). W in the modified fitness assays was calculated as described above. and a nonparametric test showed that this ranking of final fitness values in the two treatments was statistically significant (one-tailed Mann-Whitney rank test with RESULTS Us 5 9, n1 5 n2 5 3, P 5 0.05). We then calculated the grand mean fitness for the three replicate populations Fitness improvement in experimental populations: To address the Fisher-Muller model, we sought to deterat each time point. The A populations experienced a positive linear improvement in fitness over time (linear mine whether the S and A treatment populations differed in their rates of fitness improvement. Pairedregression: slope 5 0.0047, t 5 16.494, d.f. 5 4, P , 0.001). In contrast, the fitness trajectory in the S populagrowth experiments (Chao 1990) measure fitness as the ability for an infecting virus to exploit its host in tions was concave; these populations appeared to quickly reach a selective plateau that was followed by a the absence of interaction with competing viruses (see materials and methods). We estimated fitness at 50 fitness decline. The regression model that best fit the experimental observations in the S treatment was a negageneration intervals for each population in the S and A treatments relative to the common competitor of the tive quadratic (F(2,3) 5 20.768, d.f. 5 3, P 5 0.017). Our results clearly indicated that the A populations experiancestral genotype, φ6h. Fitness assays were replicated 527 Intrahost Competition in Viruses enced a more rapid increase in fitness than the S populations, suggesting that sex is costly in this experimental system. The sexual and asexual treatments in this study also differ because intrahost competition between viruses occurs during coinfection. One possible explanation for our unexpected results is that sexual viruses evolved traits favoring within-host competition, rather than traits that improve host exploitation (as measured by pairedgrowth assays). To explore this hypothesis, we sought to determine whether the S and A treatment populations experienced fitness trajectories in their respective environments that differed from the paired-growth results (Figure 2). Rate of adaptation to treatment conditions: We measured the fitness relative to φ6h for each population at 50 generation intervals using a fitness assay that was modified to match the population’s evolutionary environment (i.e., moi 5 5 or moi 5 0.002; see materials and methods). Fitness assays were replicated (n 5 2) for each population, and the grand mean fitness of the three populations in each treatment group was calculated at each time point. The results are presented in Figure 3; for comparison, Figure 3 includes the grand mean data for paired-growth assays described above. Regression analysis shows that the A populations (Figure 3A) experienced a positive linear improvement in fitFigure 3.—Fitness improvement under treatment condiness in both their own environment (slope 5 0.0043, tions compared to fitness measured through paired-growth t 5 10.418, d.f. 5 4, P , 0.001), and in terms of paired assays. (A) Fitness improvement for the asexual populations measured at moi 5 0.002 (n), compared to their pairedgrowth (see above). This general result held for each growth trajectory (m). Both fitness trajectories are positive A population analyzed separately (data not shown), and and linear, and do not differ statistically (see text). (B) Fitness the high variance observed in Figure 3A was due to improvement for the sexual populations measured at moi 5 inflated fitness values for population A3. We compared 5 (s), and their paired-growth trajectory (d). Data show that the two regression lines in Figure 3A for equality of fitness improvement in sexual populations is positive and linear only when coinfection is common, conditions similar to slopes using a small-sample two-tailed t-test for parallelthose in their evolutionary environment. Each point repreism (Kleinbaum and Kupper 1978). This test shows that sents the grand mean (6SE) of three populations, except for the regression coefficients are not significantly different the ancestor that is based on 10 assays. See text for statistical at the a 5 0.05 level (T 5 0.802, t0.05[8] 5 2.306, d.f. 5 analyses. 8, P . 0.4). We concluded that the A populations showed an equally rapid rate of improvement in their own environment as that predicted by changes in pairedthe sexual phages are evolved to be strong intrahost competitors, but are poorly adapted to conditions where growth. This result was not unexpected because both assay environments provide little opportunity for intercoinfection is uncommon. For completeness, we measured the fitness (n 5 2) action among competing phages. In marked contrast, we observed that the S populaat 50 generation intervals for each population relative to φ6h in the unevolved treatment environment (i.e., tions (Figure 3B) showed a very different fitness trajectory in their own environment when compared to moi 5 5 or moi 5 0.002). The grand mean fitness of the three replicate populations in each treatment group changes in paired-growth. Regression analysis indicates that these populations experienced a positive linear imwas calculated at each time point. Regression analysis shows that the fitness improvement of asexual phages provement in fitness at moi 5 5 (slope 5 0.0040, t 5 5.395, d.f. 5 4, P 5 0.006), unlike the fitness results at moi 5 5 was very rapid (slope 5 0.0060, t 5 3.931, d.f. 5 4, P 5 0.017). In fact, their rate of improvement from paired-growth assays (see above). This result held when each population was analyzed separately (data not was identical to that shown at moi 5 0.002 (t -test for parallelism: T 5 1.069, t0.05[8] 5 2.306, d.f. 5 8, P . 0.2) shown). Thus, the S populations showed a rapid rate of improvement in their own environment, but these and relative to changes in paired growth (T 5 0.833, t0.05[8] 5 2.306, d.f. 5 8, P . 0.4). At first, it may seem adaptive changes did not translate to rapid improvement in terms of paired-growth. We concluded that surprising that the A populations do equally well in 528 P. E. Turner and L. Chao TABLE 1 allows for coinfection, but do poorly when intrahost interactions are minimized. It is possible to explore this Final mean fitness relative to the ancestor (φ6h) for each hypothesis further by allowing single genotypes of sexexperimental population in three environments ual phages and asexual phages to compete directly in environments where levels of phage interaction differ. Treatment environment A switch to fitness assays involving head-to-head compePopulation Paired growth moi 5 0.002 moi 5 5 tition between evolved phages is desirable for two reaA1 1.471 1.778 2.334 sons. First, all fitness results reported thus far involved A2 1.645 1.581 1.824 mixed populations of evolved viruses. Thus, the obA3 3.203 2.965 4.472 served tradeoff must be a property of the majority of S1 1.276 1.366 1.438 genotypes present, but competitions involving a pure S2 1.250 1.615 2.278 clone of the majority genotype should serve only to S3 1.095 1.197 2.243 magnify the apparent tradeoff. Second, all previous Paired-growth and treatment environment values are competitions assayed fitness relative to φ6h. This asmeans, n 5 3 and 2, respectively. sumes that fitness is completely transitive in our system. Although most microbial studies show the magnitude of one derived genotype’s advantage relative to another environments that do and do not allow interactions can be accurately predicted from each one’s advantage between competing viruses. However, we emphasize relative to the ancestor (e.g., Lenski et al. 1991; Travthat these fitness results are relative to φ6h. Because it isano et al. 1995), nontransitivity and other complex is unknown whether this ancestral virus had ever experiselection dynamics have been demonstrated in some enced an environment similar to our sexual treatment, experiments (e.g., Chao and Levin 1981; Paquin and no prediction can be made regarding its performance. A Adams 1983; Turner et al. 1996; Souza et al. 1997). valid set of predictions could be made if derived asexual Thus, fitness assays involving direct competitions bephages were competed against derived sexual phages tween clonal isolates would serve to eliminate any quesin the two evolutionary environments. However, we extions regarding nontransitivity in this study. plore this scenario below and will reserve further comTradeoff between intrahost competition and host exment until that set of data is presented. ploitation in sexual phages: We sought evidence of A very different result was obtained in S populations. whether the tradeoff shown by sexual phages would Regression analysis shows that the performance of sexmanifest in direct competitions between sexual phages ual phages at moi 5 0.002 was positive and linear, but and asexual phages. To explore this question, we rannot significant (slope 5 0.0014, t 5 2.568, d.f. 5 4, domly chose a single phage clone from one population P 5 0.062). This rate of improvement at moi 5 0.002 in each treatment at a time-point where performance was less rapid than that shown by the sexual phages in at moi 5 5 exceeded that for paired growth (Figure 3). their evolved environment at moi 5 5 (small-sample φS2 is a single clone isolated at 200 generations from two-tailed t -test for parallelism with T 5 2.784, t0.05[8] 5 population S2, whereas φA1 is that from population A1. 2.306, d.f. 5 8, P , 0.05). Thus, the performance of We obtained a spontaneous host-range mutant of φA1, sexual phages coincided very well with the degree of referred to as φA1h. Paired-growth fitness (6SE) of φA1 competitive interactions that occurred between viruses. relative to φA1h was found to be 1.062 6 0.070 (n 5 That is, sexual phages did very well at moi 5 5, worse 7); all fitness results reported below are adjusted to at moi 5 0.002 (where intrahost competition is rare, account for the 6% fitness cost of the h marker. We but phages may interact during adsorption), and very then competed φS2 against φA1h at moi 5 5 (n 5 5) poorly in the complete absence of competitive interacand at moi 5 0.002 (n 5 5). Results showed that mean tions (paired growth). This relative ranking is emphafitness of φS2 relative to φA1h was 1.424 (60.052 SE) at sized in Table 1, where we list the final mean fitness at moi 5 5, but 0.772 (60.028 SE) at moi 5 0.002. A 250 generations for each experimental population in t -test clearly indicates that the fitness of φS2 is depenall environments. A one-way ANOVA confirms that the dent upon the amount of intrahost competition allowed effect of assay environment on mean fitness is significant (ts 5 11.025, d.f. 5 8, P , 0.001). These results provide for the sexual phages (MSE 5 0.093, d.f. 5 2, Fs 5 5.337, firm evidence that phages evolved in a sexual environP 5 0.047), but not for the asexual phages (MSE 5 ment are selectively favored only when coinfection is 1.148, d.f. 5 2, Fs 5 0.516, P 5 0.621). These data further common and, hence, the level of intrahost competition suggest that the cost created by intrahost competition is intense. was so strong that it masked any advantage of sex in the S populations. DISCUSSION A tradeoff between intrahost competition and host exploitation explains the results shown in Figure 3B. We examined the Fisher-Muller model for the evolutionary maintenance of sex (Fisher 1930; Muller The sexual phages do very well in an environment that 529 Intrahost Competition in Viruses 1932) using a bacteria-phage model system. This theory evidence that intense selection to compete for limited host resources lessens the ability of viruses to exploit predicts that adaptation to the environment is more rapid in a sexual population, than in an equally large the host. Although it was previously suggested that within-host competition may lead to the evolution of asexual population. Sex occurs in RNA virus φ6 when multiple viruses coinfect the same bacterial cell. The novel viral traits (Lewontin 1970), to our knowledge the present study is the first empirical evidence for this presence or absence of sex in viral populations can be easily manipulated in this system by controlling moi. A idea. Further experiments are needed to elucidate the nasingle clone of φ6 was used to found three S and three A populations. These experimental populations were ture of the tradeoff shown here. In the meantime, we discuss three potential mechanisms that may be involved then propagated on a P. phaseolicola host for 250 generations of viral evolution. At the end of the study, we in the observed tradeoff. These include the evolution of defective viral genotypes that parasitize ordinary viruses, compared the rate of fitness improvement, relative to a common competitor of the ancestral genotype, for the evolution of genetic conflicts, and the impact of hard and soft selection on viral adaptation. The various populations in the two treatments. Our study can be summarized by two major results. mechanisms are not mutually exclusive. Defective interfering particles: All viruses require livFirst, all experimental populations showed a significant increase in fitness relative to a common competitor of ing host cells to replicate. Certain viral genotypes are defective because they require helper activity from anthe ancestral genotype. However, we found no evidence that sex increased the rate of adaptation in terms of other virus genome or virus gene(s) to undergo replication (Holland 1991). In fact, defective viruses of this paired growth (competitive fitness in the absence of phage interactions). Rather, sexual populations of vitype have long been documented in association with many human and animal viruses (e.g., Henle and Henle ruses adapted at a rate much slower than that of their asexual counterparts and even showed a fitness decline 1943; Bellet and Cooper 1959; see Holland 1991 for review). Huang and Baltimore (1970) coined the term by the end of the study (Figure 2). To explain our findings, we hypothesized that viral “defective interfering (DI) particles” as a name for these viruses that lack essential RNA or DNA and that interfere evolution was in response to a key difference between the two treatment environments: the level of intrahost specifically with helper phage by replicating at their expense. Essentially, DI particles are intracellular paracompetition experienced by viruses. When a virus is alone in infecting a host cell, its reproduction is strictly sites that rely on functional proteins synthesized by coinfecting viruses. DI particles also have an intracellular asexual, but selection is primarily for a virus that best exploits the host cell. Paired-growth (Chao 1990) meareplicative advantage over ordinary phage. For example, when DI and standard particles of RNA poliovirus cosures fitness as the ability for an infecting virus to exploit its host in the absence of interactions with competing infect the same host cell, the viral progeny is enriched by about 5% for particles containing DI RNAs (Cole viruses (see materials and methods). In contrast, sexual or coinfecting viruses may be selected for host exand Baltimore 1973). DI particles vary in their mechanism of intrahost advantage. Because they typically conploitation and for within-host competition of limited host resources. In the latter case, adaptation to intrahost tain fewer genes, the smaller size of DI particles may be sufficient for them to gain a replicative advantage (e.g., competition may detract from the ability of the virus to exploit its host, and this within-host selection may then DI particles of influenza A; Holland 1991). In other cases, DI particles have evolved more elaborate mechacreate a cost for coinfection. Lewontin (1970) suggested that when multiple viral genotypes coinfect the nisms to parasitize helper phages. For instance, some DI particles have retained replication initiation sites but same host cell, strong intrahost competition may lead to the evolution of novel viral traits. Thus, we predicted have lost transcription initiation sites at the 39 terminus, allowing a replicative advantage over virus genomes that that fitness trajectories from paired-growth assays should be more representative of fitness dynamics shown by must engage in transcription (Perrault and Semler 1970; Kailash et al. 1983). asexual phages in their evolutionary environment than for those shown by sexual phages in their selective enviThe evolution or isolation of DI particles in association with RNA virus φ6 has never been documented. ronment. To explore this hypothesis, we measured the fitness of populations in their respective treatment enviHowever, the derived sexual viruses in this study appear similar to DI particles because they experience a selecronments and compared these results to fitness changes observed in paired-growth assays. tive advantage only when intrahost competition is allowed (Figure 3B). One explanation is that a sexual Our second major result is that our observations can be explained by a systematic tradeoff between intrahost environment allows these viruses to interfere, directly or indirectly, with the replication of other coinfecting competition and host exploitation in sexual phages. That is, the derived sexual viruses are selectively favored viral genotypes. The derived sexual phages cannot be DI particles per se because they are able to undergo only when coinfection and—hence—intrahost interactions are common (Table 1, Figure 3B). This is firm normal asexual reproduction without the aid of helper 530 P. E. Turner and L. Chao viruses (see results). Furthermore, it has been argued the available positions (Wallace 1970). Thus, W can change depending on whether soft selection is at work. that evolution and maintenance of DI particles would require an moi that is orders of magnitude higher than In φ6, intrahost competition is an environment where soft selection can act. The limited number of available that imposed in our sexual treatment (Chao 1988). Still, the possibility exists that mechanisms similar to “positions” is the burst size produced as a result of infection (z200–400 viruses). Whereas two competing vithose employed by DI particles may allow the sexual phages in this study to gain a selective advantage during ruses may be equally capable of producing progeny through asexual reproduction, the outcome may be very intrahost competition. Genetic conflicts: Sex requires that a genome expose different when the two must compete for limited host resources (and hence, limited positions in the viral progitself to foreign genetic material, creating an opportunity for the evolution of genetic conflicts. Genetic coneny). Here, it is the better intrahost competitor that contributes more progeny to the next generation. flicts occur during genetic exchange when a particular gene (or genes) promotes its own spread at the expense Our paired-growth assay to measure fitness does not allow intrahost interactions between competing phage of other genes (Werren et al. 1988; Hurst et al. 1996). Well-described examples include the meiotic drive genotypes and it is here that hard selection should play a major role in the competitive outcome. On the other genes in Drosophila melanogaster and in mice (Lyttle 1993; Silver 1993). The advantage gained by DI partihand, coinfecting viruses must compete for limited host resources, and our sexual treatment is an environment cles during coinfection provides another example of genetic conflict (Chao 1994). DI particles experience where we would expect soft selection to be important. Thus, another way to visualize the genetic tradeoff apa higher replication or encapsidation rate inside a coinfected cell, but this parasitism has a negative effect on parent in the S populations is in terms of the relative contributions to phage adaptation of hard and soft selecthe coinfection group (the group of viruses coinfecting a cell). For example, the total yield of polioviruses protion. This concept is clearly illustrated when one compares the grand mean data shown in Figure 3B. For most duced by an infected cell is inversely proportional to the frequency of DI particles in the coinfection group of the experiment the sexual phages showed equivalent fitness improvement in the presence and absence of (Cole and Baltimore 1973). Although genic selection favors DI RNAs, selection at the level of coinfection phage interactions (sex), indicating that hard and soft selection contributed equally to their total fitness groups opposes them, much the same way that selection on individuals may prevent deleterious meiotically gained. However, it was during the last 50 to 100 generations of viral evolution that the effects of soft selection driven alleles from becoming fixed (Lewontin 1970). We believe that the sexual phages in this study provide became paramount in the sexual populations. Presumably, soft selection led to the evolution of viral traits yet another example for the evolution of genetic conflicts. These viruses gain an obvious selective advantage that improve intrahost competition, but these phage adaptations seemed to occur at the expense of other during coinfection. As in the case of DI RNAs, there may be an opposing selective force that would stop these traits molded by hard selection. Relevance of findings to previous work, and concludviruses from sweeping to fixation. If so, we can predict that the fitness advantage shown by sexual phages during remarks: Several recent studies have empirically tested whether sex leads to an increase in fitness (Birding coinfection should be dependent upon their initial frequency in competition and that an equilibrium may sell and Wills 1996; Da Silva and Bell 1996; Souza et al. 1997; Zeyl and Bell 1997). Souza et al. (1997) be reached, where sexual phages and asexual phages are able to stably coexist. This hypothesis is untested, tested a model similar to that of Fisher-Muller, but using populations of the bacterium Escherichia coli. The rate but provides an intriguing possibility for future study. Hard and soft selection: A final analogy may be drawn of fitness improvement in these asexual populations had slowed considerably from an initially rapid pace (Lenski between our study and the concept of adaptation through hard and soft selection. Wallace (1970) deet al. 1991). The authors sought to determine whether sexual recombination with novel genotypes would reacfined hard selection as selection resulting from conditions that an organism must meet to function as a breedcelerate the rate of adaptation in these populations. Although sexual recombination yielded dramatic ining individual. As an extreme example, consider a single-locus diploid system with dominant allele A and creases in genetic variation, this variability did not translate to more rapid adaptive evolution. This surprising recessive allele a. If the homozygous combination aa is always lethal, then the failure of aa individuals to result can be explained by changes in the selective environment brought on by the recombinant genotypes reproduce has little to do with conditions such as overcrowding in the population. In contrast, soft selection themselves (Turner et al. 1996). That is, complex ecological interactions (such as bacterial cross-feeding) does not involve conditions necessary for reproduction; rather, it involves a fixed number of available positions among recombinants led to unexpected environmental changes, leaving simple fitness estimates as an inadethat can be filled by any viable individual but, in reality, are filled by those individuals most fit to compete for quate method for evaluating the Fisher-Muller type 531 Intrahost Competition in Viruses tion of anticompetitor toxins in bacteria. Proc. Natl. Acad. Sci. model (Souza et al. 1997). 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