Properties and Mechanisms of Deletions, Insertions, and Substitutions in the Evolutionary History of SARS-CoV-2.

SARS-CoV-2 has accumulated many mutations since its emergence in late 2019. Nucleotide substitutions leading to amino acid replacements constitute the primary material for natural selection. Insertions, deletions, and substitutions appear to be critical for coronavirus's macro- and microevolution. Understanding the molecular mechanisms of mutations in the mutational hotspots (positions, loci with recurrent mutations, and nucleotide context) is important for disentangling roles of mutagenesis and selection. In the SARS-CoV-2 genome, deletions and insertions are frequently associated with repetitive sequences, whereas C>U substitutions are often surrounded by nucleotides resembling the APOBEC mutable motifs. We describe various approaches to mutation spectra analyses, including the context features of RNAs that are likely to be involved in the generation of recurrent mutations. We also discuss the interplay between mutations and natural selection as a complex evolutionary trend. The substantial variability and complexity of pipelines for the reconstruction of mutations and the huge number of genomic sequences are major problems for the analyses of mutations in the SARS-CoV-2 genome. As a solution, we advocate for the development of a centralized database of predicted mutations, which needs to be updated on a regular basis.

Blastocrithidia nonstop mitochondrial genome and its expression are remarkably insulated from nuclear codon reassignment.

The canonical stop codons of the nuclear genome of the trypanosomatid Blastocrithidia nonstop are recoded. Here, we investigated the effect of this recoding on the mitochondrial genome and gene expression. Trypanosomatids possess a single mitochondrion and protein-coding transcripts of this genome require RNA editing in order to generate open reading frames of many transcripts encoded as 'cryptogenes'. Small RNAs that can number in the hundreds direct editing and produce a mitochondrial transcriptome of unusual complexity. We find B. nonstop to have a typical trypanosomatid mitochondrial genetic code, which presumably requires the mitochondrion to disable utilization of the two nucleus-encoded suppressor tRNAs, which appear to be imported into the organelle. Alterations of the protein factors responsible for mRNA editing were also documented, but they have likely originated from sources other than B. nonstop nuclear genome recoding. The population of guide RNAs directing editing is minimal, yet virtually all genes for the plethora of known editing factors are still present. Most intriguingly, despite lacking complex I cryptogene guide RNAs, these cryptogene transcripts are stochastically edited to high levels.

The 29-nucleotide deletion in SARS-CoV: truncated versions of ORF8 are under purifying selection.

BACKGROUND: Accessory proteins have diverse roles in coronavirus pathobiology. One of them in SARS-CoV (the causative agent of the severe acute respiratory syndrome outbreak in 2002-2003) is encoded by the open reading frame 8 (ORF8). Among the most dramatic genomic changes observed in SARS-CoV isolated from patients during the peak of the pandemic in 2003 was the acquisition of a characteristic 29-nucleotide deletion in ORF8. This deletion cause splitting of ORF8 into two smaller ORFs, namely ORF8a and ORF8b. Functional consequences of this event are not entirely clear. RESULTS: Here, we performed evolutionary analyses of ORF8a and ORF8b genes and documented that in both cases the frequency of synonymous mutations was greater than that of nonsynonymous ones. These results suggest that ORF8a and ORF8b are under purifying selection, thus proteins translated from these ORFs are likely to be functionally important. Comparisons with several other SARS-CoV genes revealed that another accessory gene, ORF7a, has a similar ratio of nonsynonymous to synonymous mutations suggesting that ORF8a, ORF8b, and ORF7a are under similar selection pressure. CONCLUSIONS: Our results for SARS-CoV echo the known excess of deletions in the ORF7a-ORF7b-ORF8 complex of accessory genes in SARS-CoV-2. A high frequency of deletions in this gene complex might reflect recurrent searches in "functional space" of various accessory protein combinations that may eventually produce more advantageous configurations of accessory proteins similar to the fixed deletion in the SARS-CoV ORF8 gene.

Deletions across the SARS-CoV-2 Genome: Molecular Mechanisms and Putative Functional Consequences of Deletions in Accessory Genes.

The analysis of deletions may reveal evolutionary trends and provide new insight into the surprising variability and rapidly spreading capability that SARS-CoV-2 has shown since its emergence. To understand the factors governing genomic stability, it is important to define the molecular mechanisms of deletions in the viral genome. In this work, we performed a statistical analysis of deletions. Specifically, we analyzed correlations between deletions in the SARS-CoV-2 genome and repetitive elements and documented a significant association of deletions with runs of identical (poly-) nucleotides and direct repeats. Our analyses of deletions in the accessory genes of SARS-CoV-2 suggested that there may be a hypervariability in ORF7A and ORF8 that is not associated with repetitive elements. Such recurrent search in a "sequence space" of accessory genes (that might be driven by natural selection) did not yet cause increased viability of the SARS-CoV-2 variants. However, deletions in the accessory genes may ultimately produce new variants that are more successful compared to the viral strains with the conventional architecture of the SARS-CoV-2 accessory genes.

No evidence for widespread positive selection on double substitutions within codons in primates and yeasts.

Nucleotide substitutions in protein-coding genes can be divided into synonymous (S) and non-synonymous (N) ones that alter amino acids (including nonsense mutations causing stop codons). The S substitutions are expected to have little effect on function. The N substitutions almost always are affected by strong purifying selection that eliminates them from evolving populations. However, additional mutations of nearby bases can modulate the deleterious effect of single N substitutions and, thus, could be subjected to the positive selection. This effect has been demonstrated for mutations in the serine codons, stop codons and double N substitutions in prokaryotes. In all abovementioned cases, a novel technique was applied that allows elucidating the effects of selection on double substitutions considering mutational biases. Here, we applied the same technique to study double N substitutions in eukaryotic lineages of primates and yeast. We identified markedly fewer cases of purifying selection relative to prokaryotes and no evidence of codon double substitutions under positive selection. This is consistent with previous studies of serine codons in primates and yeast. In general, the obtained results strongly suggest that there are major differences between studied pro- and eukaryotes; double substitutions in primates and yeasts largely reflect mutational biases and are not hallmarks of selection. This is especially important in the context of detection of positive selection in codons because it has been suggested that multiple mutations in codons cause false inferences of lineage-specific site positive selection. It is likely that this concern is applicable to previously studied prokaryotes but not to primates and yeasts where markedly fewer double substitutions are affected by positive selection.