"Fertile" Mutations in SARS-CoV-2 RNA More Frequently Occurred in Hairpin Loops That Determine Virus Evolution

RNA hairpins may constitute a foundation of genetic evolution both in viruses and other organisms. Stem-loops theoretically comprise a stable part, the double-stranded stem, and a single-stranded loop allowing evolution. Here we tested for SARS-CoV-2 if "fertile" mutations were in loops while mutations in stems were poorly tolerated and rarely found in consensus genomes. We combined information on the frequencies of mutations, either "fertile" (present in ≥ 50 genomes) or "non-fertile" (neutral, weakly deleterious or lethal) in 61,397 SARS-CoV-2 genomes, and on whether these mutations occurred at positions where nucleotides were predicted to be either paired or unpaired. The proportion of positions harboring "fertile" mutations was significantly higher in loops than in stems for the whole genome (11.6% vs. 7.6%; p < 0.001, Yates-corrected chi-square test). This was also the case in the RNA-dependent RNA polymerase gene (10.0% vs. 4.9%; p = 0.0003) or in the spike gene (12.3% vs. 8.9%; p = 0.0049). All four most frequent mutations in our set of genomes were located in loops. Thus, apart from some observations in "accessory" genes, evolution in SARS-CoV-2 predominantly occurred in loops while mutations in stems were relatively "non-fertile." These stems could be potential antiviral targets, possibly through their disruption by RNA interference.