The mutational spectrum of SARS-CoV-2 genomic and antigenomic RNA

Abstract

The raw material for viral evolution is provided by intra-host mutations occurring during replication, transcription or post-transcription. Replication and transcription of Coronaviridae proceed through the synthesis of negative-sense 'antigenomes' acting as templates for positive-sense genomic and subgenomic RNA. Hence, mutations in the genomes of SARS-CoV-2 and other coronaviruses can occur during (and after) the synthesis of either negative-sense or positive-sense RNA, with potentially distinct patterns and consequences. We explored for the first time the mutational spectrum of SARS-CoV-2 (sub)genomic and anti(sub)genomic RNA. We use a high-quality deep sequencing dataset produced using a quantitative strand-aware sequencing method, controlled for artefacts and sequencing errors, and scrutinized for accurate detection of within-host diversity. The nucleotide differences between negative- and positive-sense strand consensus vary between patients and do not show dependence on age or sex. Similarities and differences in mutational patterns between within-host minor variants on the two RNA strands suggested strand-specific mutations or editing by host deaminases and oxidative damage. We observe generally neutral and slight negative selection on the negative strand, contrasting with purifying selection in ORF1a, ORF1b and S genes of the positive strand of the genome.

Keywords: RNA editing; SARS-CoV-2; antigenomes; mutational spectrum.

Figure 1.

Example of the three types of mutations. The green-coloured bases show the original mutational changes; the magenta-coloured bases show the subsequent changes in base pairing. (Online version in colour.)

Figure 2.

Consensus > variant pair frequencies for SARS-CoV-2 positive and complemented negative strands; (a) shows mutational changes attributable to known mechanisms (i.e. ADARs, APOBECs, oxidative damage); (b) shows other mutational changes, with A > T, C > A, T > A, T > G frequencies (grey) on the negative strand not interpreted. (Online version in colour.)

Figure 3.

Genomic context of candidate sites for RNA editing on the positive (top row) and non-complemented negative (bottom row) strands. The site of change is labelled 0, with the base positions to the 5′ direction labelled −2 and −1, base positions to the 3′ direction labelled + 1 and + 2. The ‘ref’ columns show the genomic content of the SARS-CoV-2 reference sequence [GenBank ID: NC_045512.2]. (Online version in colour.)

Figure 4.

dN/dS of SARS-CoV-2 coding regions computed with differently filtered variants. Red circles are from positive-strand variant sites with no less than 1% MAF; red triangles are from positive-strand variant sites with no less than 3% MAF. Blue squares are from negative-strand variant sites with at least five reads supporting the minor variant, blue triangles are the same sites but with A > T, C > A, T > A, T > G sites masked. ‘Pos > = 3%’ dN/dS = Inf are not shown for ORF3a, ORF6 and ORF7b. No ‘Pos > = 3%’ variant sites were called within E; therefore, dN/dS is not shown. (Online version in colour.)

Figure 5.

SARS-CoV-2 polymorphic sites shared by both strands. The x-axis indicates the genomic positions of the sites, the y-axis ([pos] for positive strand, [neg] for negative strand) indicates the consensus base frequencies of the mismatching sites. 'O's mark sites with complementing consensus-variant pairs from the two strands (base frequencies not shown). 'X's mark consensus-variant pair mismatching sites, with positive consensus frequency on the top, negative consensus frequency on the bottom, connected by a vertical line. Each mismatching site is labelled with genomic position and positive consensus > variant in the top half of the plot and complemented negative consensus > variant (in the positive sense) in the lower half of the plot. (Online version in colour.)