FX174 Attenuation by Whole Genome Codon Deoptimization

James T. Van Leuven; Martina M. Ederer; Katelyn Burleigh; LuAnn Scott; Randall A. Hughes; Vlad Codrea; Andrew D. Ellington; Holly Wichman; Craig Miller

It is therefore important to understand how many and what types of synonymous mutations can be made to viral genomes without completely ablating their ability to replicate in host cells. What viral genes should be attenuated? How many attenuating mutations should be made to the genome? What synonymous features should be targeted for deoptimization? In most studies to date, a limited number of deoptimized constructs (usually structural proteins) were tested. We showed that fitness decreases can be obtained by deoptimizing many of the ΦX174 genes, indicating that nonstructural genes may also be good targets for attenuation. One approach to avoid evolutionary reversion might be recoding multiple genes or entire viral genomes, balancing optimization and deoptimization to maintain sufficient virulence while increasing the genetic distance to wild type. This strategy could prevent recovery by mutation or by recombination with wild-type viruses. However, our work suggests that the effects of recoding will not be uniform across a genome. We found that the attenuating effects of recoding and the nature of epistatic interactions from combining fragments differ dramatically between genes.