Slow fitness recovery in a codon-modified viral genome

Abstract

Extensive synonymous codon modification of viral genomes appears to be an effective way of attenuating strains for use as live vaccines. An assumption of this method is that codon changes have individually small effects, such that codon-attenuated viruses will be slow to evolve back to high fitness (and thus to high virulence). The major capsid gene of the bacterial virus T7 was modified to have varying levels of suboptimal synonymous codons in different constructs, and fitnesses declined linearly with the number of changes. Adaptation of the most extreme design, with 182 codon changes, resulted in a slow fitness recovery by standards of previous experimental evolution with this virus, although fitness effects of substitutions were higher than expected from the average effect of an engineered codon modification. Molecular evolution during recovery was modest, and changes evolved both within the modified gene and outside it. Some changes within the modified gene evolved in parallel across replicates, but with no obvious explanation. Overall, the study supports the premise that codon-modified viruses recover fitness slowly, although the evolution is substantially more rapid than expected from the design principle.

FIG. 1.

Fitness declines approximately linearly with the level of codon deoptimization. The wild type is represented by the leftmost point, at 68% preferred codons in the major capsid gene, 10A. Four engineered constructs span 0.5–0.1 preferred codons in the gene. Fitness is measured in doublings per hour, a geometric measure (from left to right, mean fitnesses are 43.2, 40.4, 39.1, 36.1, and 35.7). The difference in fitness between the wild-type and most extreme engineered construct, T7-10A_0.1, is 7.5.

FIG. 2.

Fitness of the initial T7-10A_0.1 (leftmost, 35.7) and of four lines evolved from it (from S1 to R+, 36.2, 36.2, 38.7, and 43.2, respectively). The rightmost bar is for the phage T7-10A_0.1R+ (labeled R+), created as a T7-10A_0.1 recombined with a wild-type gene 10 plasmid and adapted for 6 h; it is used as the wild type in this paper and is also the leftmost point in figure 1.