Abstract
Because synonymous mutations do not change the amino acid sequence of a protein, they are generally considered to be selectively neutral. Empiric data suggest, however, that a significant fraction of viral mutational fitness effects may be attributable to synonymous mutation. Bias in synonymous codon usage in viruses may result from selection for translational efficiency, mutational bias, base pairing requirements in RNA structures, or even selection against specific dinucleotides by innate immune effectors. Experimental analyses of codon usage and genome evolution have been facilitated by advances in synthetic biology, which now make it feasible to generate viral genomes that contain large numbers of synonymous mutations. The generally pleiotropic effects of synonymous mutation on viral fitness have, at times, made it difficult to define the mechanistic basis for the observed attenuation of these heavily mutated viruses. We have addressed this problem by developing a bioinformatic tool for the generation and analysis of viral sequences with large-scale synonymous mutation. A variety of permutation strategies are applied to shuffle codons within an open reading frame. After measuring the dinucleotide frequency, codon usage, codon pair bias, and free energy of RNA folding for each permuted genome, we used z-score normalization and a least squares regression model to quantify their overall distance from the starting sequence. Using this approach, the user can easily identify a large number of synonymously mutated sequences with varying similarity to a wild-type genome across a range of nucleic-acid-based determinants of viral fitness. We believe that this tool will be useful in designing genomes for subsequent experimental studies of the fitness impacts of synonymous mutation.