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

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 de-optimisation 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 favoured 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 towards 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 includes 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.

Short sequence motif dynamics in the SARS-CoV-2 genome suggest a role for cytosine deamination in CpG reduction

“…CpG, CpA, CpC, CpU and GpA also showed a negative net gain but not as prominent as UpC, CpA or ApG. We find that the majority of dinucleotide losses were due to C>U changes, in agreement with a recent study by Rice and colleagues (Rice et al 2020). The remaining dinucleotides showed relative stability across the time period.…”

“…It was recently shown that synonymous substitutions disrupting CpG dinucleotides appear to contribute to the increased virulence of attenuated poliovirus (Stern et al 2017). We observe that more than half of the C>U substitutions disrupting CpG dinucleotides were synonymous, consistent with recent studies (Di Gioacchino et al 2020; Rice et al 2020) (Figure S6). In the future, the SARS-CoV-2 virus may level up its CpG frequency with its host, achieving so-called host-cell mimicry (Greenbaum et al 2008), but the observed CpG loss suggests that equilibrium between gains and losses have not yet been reached.…”

Impact of Early Pandemic Stage Mutations on Molecular Dynamics of SARS-CoV-2 Mpro

“…Current genomic sequence data suggests a generally low rate of mutation in SARS-CoV-2. 82 , 83 This being said, in the case of the M pro enzyme, a relatively higher rate of non-synonymous mutations has occurred at residue position 15 (G15D/S) in domain I, residue position 157 (V157I/L) in domain II, and at position 184 (P184L/S) within the interdomain linker region. On the 3D structure, it can be seen that these mutable areas occur away from the core areas of domains I and II, hence the probable lower selective pressure for these regions.…”

An evolutionary portrait of the progenitor SARS-CoV-2 and its dominant offshoots in COVID-19 pandemic

“…2a ; Supplementary Table 1 ). Out of a total of six initial mutations of proCoV2 (μ’s and α’s), the first five were synonymous U→C changes, whose functional significance is being debated 20 , 21 as they are the most common early nucleotide changes observed so far in the evolution of SARS-CoV-2 22 .…”

“…A McDonald-Kreitman test 38 rejects the similarity of molecular evolutionary patterns observed within the SARS-CoV-2 population (29KG dataset) and between human proCoV2 and the bat coronavirus. It is not prudent to automatically invoke the action of positive selection using such neutrality tests, because synonymous polymorphisms in SARS-CoV-2 genomes are affected by molecular mechanisms (e.g., RNA editing) 21 , 39 as well as negative selection 21 . Furthermore, even slightly deleterious alleles can become common when there is a population expansion 40 .…”