SARS-CoV-2 point mutations are over-represented in terminal loops of RNA stem-loop structures that can be resolved by Nsp13 helicase in a unique manner with respect to nucleotide dependence.

To improve health outcomes for COVID-19 (coronavirus disease 2019) patients, the factors that influence coronavirus genome variation need to be ascertained. The SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) genome is rich in predicted RNA secondary structures, particularly stem-loops (SLs) formed by intramolecular base pairing within palindromic sequences. We analyzed the NCBI Virus collection of SARS-CoV-2 genome sequences from COVID-19 individuals to map variants relative to SL structural elements. Point mutations in the SARS-CoV-2 genome, with a C-to-U transition bias, were over-represented in unpaired nucleotides and, more specifically, within the terminal loops of RNA SL structures. As the sole helicase encoded by SARS-CoV-2, Nsp13 may operate in the timely resolution of secondary RNA structures to facilitate SARS-CoV-2 RNA copying or processing. We characterized Nsp13 to resolve SARS-CoV-2 sequence-derived unimolecular RNA SL substrates and determined that it does so in a functionally cooperative manner. In addition to ATP, Nsp13 resolves the unimolecular RNA SL structure in the absence of nucleotide, in contrast to the strict ATP requirement for a bimolecular RNA forked duplex. We suggest a model in which a series of binary and ternary complex interactions of Nsp13 with nucleotide and/or RNA SL pose mechanistic implications for RNA SL resolution.