We used vesicular stomatitis virus to test the effect of complementation on the relative fitness of a deleterious mutant, monoclonal antibody-resistant mutant (MARM) N, in competition with its wild-type ancestor. We carried out competitions of MARM N and wild-type populations at different multiplicities of infection (MOIs) and initial ratios of the wild type to the mutant and found that the fitness of MARM N relative to that of the wild type is very sensitive to changes in the MOI (i.e., the degree of complementation) but depends little, if at all, on the initial frequencies of MARM N and the wild type. Further, we developed a mathematical model under the assumption that during coinfection both viruses contribute to a common pool of protein products in the infected cell and that they both exploit this common pool equally. Under such conditions, the fitness of all virions that coinfect a cell is the average fitness in the absence of coinfection of that group of virions. In the absence of coinfection, complementation cannot take place and the relative fitness of each competitor is only determined by the selective value of its own products. We found good agreement between our experimental results and the model predictions, which suggests that the wild type and MARM N freely share all of their gene products under coinfection.RNA viruses form highly polymorphic populations called quasispecies (8,9,13). The origin of much of their genetic diversity is high mutational pressure. Most RNA viruses have mutation rates on the order of one miscopied base per genome and generation (10, 11). However, high mutational pressure is not necessarily the only factor promoting genetic diversity. Niche differentiation can allow stable polymorphisms in an infected host. For instance, during a polyclonal immune response, different subpopulations may have different sensitivities to individual antibodies; variation within a host can also be related to differences in the tropism of different viral subpopulations. Virus-virus interactions may also promote stable polymorphisms in an infected host. In cell culture, such interactions readily occur during replication inside a cell and are likely to be an important contributor to the maintenance of variation. When two or more virions coinfect the same cell, complementation can take place. Not every function can be complemented in trans. In positive-stranded viruses, translation and replication are coupled (25); therefore, many functions need to be provided in cis and cannot be rescued by complementation (33, 37). Other proteins can freely interact with heterologous genomes or replicons, even from different viral species (18,21). In the absence of compartmentalization or other limitations to the diffusion of viral products, soluble proteins can interact with any genome inside the cell, potentially changing the phenotype of the virions and masking targets for natural selection to operate. A remarkable example of this phenomenon is phenotypic mixing and hiding, particularly for monoclonal antibody (...

Abstract. RNA viruses are widely used to study evolution experimentally. Many standard protocols of virus propagation and competition are done at nominally low multiplicity of infection (m.o.i.), but lead during one passage to two or more rounds of infection, of which the later ones are at high m.o.i. Here, we develop a model of the competition between wild type (wt) and a mutant under a regime of alternating m.o.i. We assume that the mutant is deleterious when it infects cells on its own, but derives a selective advantage when rare and coinfecting with wt, because it can profit from superior protein products created by the wt. We find that, under these assumptions, replication at alternating low and high m.o.i. may lead to the stable coexistence of wt and mutant for a wide range of parameter settings. The predictions of our model are consistent with earlier observations of frequency-dependent selection in vesicular stomatitis virus and human immunodeficiency virus type 1. Our results suggest that frequency-dependent selection may be common in typical evolution experiments with viruses.Key words. Complementation, experimental evolution, frequency-dependent selection, quasispecies, vesicular stomatitis virus.Received November 10, 2003. Accepted November 18, 2003 RNA virus populations grown on cell culture in the laboratory are often considered single-niche systems, and are used to test basic evolutionary theories, such as Muller's ratchet, the evolution of recombination, or the potential costs of host radiation (Chao 1990;Duarte et al. 1992;Escarmís et al. 1996;Chao et al. 1997;Yuste et al. 1999;Turner and Elena 2000). In a single-niche system, mutation pressure is the only source of polymorphisms in the population, but polymorphisms can also be maintained by negative frequencydependent selection if several niches are available. Frequency-dependent selection in RNA viruses grown in vitro (Elena et al. 1997;Turner and Chao 1999;Yuste et al. 2002;Turner and Chao 2003) demonstrates that multiple niches can be available even in these simple laboratory systems.Frequency-dependent selection among viruses grown in vitro is typically caused by complementation, that is, withincell interactions between different virus strains. When several viruses coinfect the same cell, they share genetic material and protein products while they replicate. This type of interaction can for example lead to the accumulation of defective interfering particles (DIPs; Bangham and Kirkwood 1990; Szathmary 1992;Frank 2000), virus particles that cannot replicate by themselves because they lack essential genes. Defective interfering particles can coexist with nondefective virus particles because they complement their defective genomes with genes from the nondefective particles when both coinfect the same cell. Other effects caused by within-cell interactions are phenotypic mixing and hiding (Novick and Szilard 1951;Brenner 1957;Huang et al. 1974;Holland et al. 1989;Wilke and Novella 2003) or recombination and reassortment (King et al. 1982;Lai 1992...

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RNA viruses are widely used to study evolution experimentally. Many standard protocols of virus propagation and competition are done at nominally low multiplicity of infection (m.o.i.), but lead during one passage to two or more rounds of infection, of which the later ones are at high m.o.i. Here, we develop a model of the competition between wild type (wt) and a mutant under a regime of alternating m.o.i. We assume that the mutant is deleterious when it infects cells on its own, but derives a selective advantage when rare and coinfecting with wt, because it can profit from superior protein products created by the wt. We find that, under these assumptions, replication at alternating low and high m.o.i. may lead to the stable coexistence of wt and mutant for a wide range of parameter settings. The predictions of our model are consistent with earlier observations of frequency-dependent selection in vesicular stomatitis virus and human immunodeficiency virus type 1. Our results suggest that frequency-dependent selection may be common in typical evolution experiments with viruses.