# Evidence for Strong Mutation Bias toward, and Selection against, U Content in SARS-CoV-2: Implications for Vaccine Design

## TL;DR

This study finds that SARS-CoV-2 has a mutation bias toward uracil (U) but is selected against it, which could inform better vaccine design strategies.

## Contribution

The paper identifies a strong mutation bias toward U and selection against it in SARS-CoV-2, offering new insights for vaccine attenuation.

## Key findings

- SARS-CoV-2 shows a strong mutation bias toward uracil (U) at synonymous sites.
- Observed UU content is only a quarter of what is expected under neutrality, indicating selection against U.
- CpG content varies systematically between SARS-CoV-2 genes, suggesting gene-specific selection.

## Abstract

Large-scale re-engineering of synonymous sites is a promising strategy to generate vaccines either through synthesis of attenuated viruses or via codon-optimized genes in DNA vaccines. Attenuation typically relies on deoptimization of codon pairs and maximization of CpG dinucleotide frequencies. So as to formulate evolutionarily informed attenuation strategies that aim to force nucleotide usage against the direction favored by selection, here, we examine available whole-genome sequences of SARS-CoV-2 to infer patterns of mutation and selection on synonymous sites. Analysis of mutational profiles indicates a strong mutation bias toward U. In turn, analysis of observed synonymous site composition implicates selection against U. Accounting for dinucleotide effects reinforces this conclusion, observed UU content being a quarter of that expected under neutrality. Possible mechanisms of selection against U mutations include selection for higher expression, for high mRNA stability or lower immunogenicity of viral genes. Consistent with gene-specific selection against CpG dinucleotides, we observe systematic differences of CpG content between SARS-CoV-2 genes. We propose an evolutionarily informed approach to attenuation that, unusually, seeks to increase usage of the already most common synonymous codons. Comparable analysis of H1N1 and Ebola finds that GC3 deviated from neutral equilibrium is not a universal feature, cautioning against generalization of results.