Contingent neutrality in competing viral populations

Abstract

The replicative fitness of a genetically marked (MARM-C) population of vesicular stomatitis virus was examined in competition assays in BHK-21 cells. In standard fitness assays involving up to eight competition passages of the mixed populations, MARM-C competes equally with the wild type (wt), but very prolonged competitions always led to the wt gaining dominance over MARM-C in a very slowed, nonlinear manner (J. Quer et al., J. Mol. Biol. 264:465-471, 1996). In the present study we show that a number of quite unrelated environmental perturbations, which decreased virus replication during competitions, all led to an accelerated dominance of the wt over MARM-C. These perturbations were (i) the presence of added (or endogenously generated) defective interfering particles, (ii) the presence of the chemical mutagen 5-fluorouracil (5-FU), or (iii) an increase in temperature to 40.5 degrees C. Thus, the "neutral fitness" of the MARM-C population is contingent. We have determined the entire genomic consensus sequence of MARM-C and have identified only six mutations. Clearly, some or all of these mutations allowed the MARM-C quasispecies population to compete equally with wt in a defined constant host environment, but the period of neutrality was shortened when the environment was perturbed during competitions. Interestingly, when four passages of each population were carried out independently in the presence of 5-FU (but in the absence of competition), no significant differences were detected in the fitness changes of wt and MARM-C, nor was there a difference in their subsequent abilities to compete with each other in a standard fitness assay. We propose a model for this contingent neutrality. The conditions employed to generate the MARM-C quasispecies population selected a small number of mutations in the consensus sequence. It appears that the MARM-C quasispecies population has moved into a segment of sequence space in which the average fitness value is neutral but, under environmental stress, beneficial mutations cannot be generated rapidly enough to compete with those being generated concurrently by competing wt virus quasispecies populations.

FIG. 1

Competition between wt and MARM-C VSV in serial undiluted passages. Results of 12 parallel competition experiments are shown in two panels for clarity. Procedures for infection of BHK-21 cells and determination of the proportion of wt and MARM-C genomes are detailed in Materials and Methods.

FIG. 2

Competition between wt and MARM-C VSVs in the absence (open symbols) or presence (solid symbols) of exogenous DIPs. DIPs were prepared as indicated in Materials and Methods. At each passage an amount of purified DIPs, giving a reduction of 98% in VSV production in a standard assay with VSV-Indiana, was added. The procedures for infection are detailed in Materials and Methods.

FIG. 3

Competition between wt and MARM-C VSVs in the absence (open symbols) or the presence (solid symbols) of 5-FU. The conditions of treatment with 5-FU and the procedures are detailed in Materials and Methods.

FIG. 4

Competition passages between wt and MARM-C VSVs at 37°C (open symbols) or at 40.5°C (solid symbols). The conditions for culture at high temperature are detailed in Materials and Methods.