Epistasis-Driven Evolution of the SARS-CoV-2 Secondary Structure

Area of Science:

• Molecular Biology
• Evolutionary Biology
• Virology

Background:

• Epistasis describes how a mutation's effect depends on its genetic context.
• RNA secondary structure, stabilized by Watson-Crick (WC) base pairs, is crucial for function and a source of epistasis.
• Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) provides a model to study RNA structural evolution.

Purpose of the Study:

• Investigate epistasis in the SARS-CoV-2 RNA secondary structure.
• Identify conserved topological motifs and analyze mutations at WC base-paired and unpaired sites.
• Determine the impact of mutations on secondary structure integrity and evolutionary constraints.

Main Methods:

• Examined derived alleles and their frequencies at WC and unpaired (UP) sites within 15 conserved topological motifs of the SARS-CoV-2 genome.
• Compared mutation frequencies at WC sites that abolish base pairing versus those forming G·U wobbles.
• Assessed epistatic constraint levels at WC sites in relation to specific RNA structural motifs.

Main Results:

• Fewer derived alleles and lower frequencies were observed at WC sites compared to UP sites, suggesting deleterious effects of secondary structure modification.
• Mutations abolishing base pairing had lower frequencies than those forming G·U wobbles, indicating partial preservation of structure by weak interactions.
• Strongest epistatic constraints were found in a pseudoknot motif essential for programmed ribosomal frameshifting, while weakest constraints were in 3' UTR motifs regulating viral processes.

Conclusions:

• Epistasis significantly shapes the evolution of SARS-CoV-2 RNA secondary structure.
• WC base-paired sites are under stronger evolutionary constraint than unpaired sites.
• Specific structural motifs, like the frameshifting pseudoknot, are critical targets of natural selection.