The mutational landscape of SARS-CoV-2 provides new insight into viral evolution and fitness.

Although vaccines and treatments have strengthened our ability to combat the COVID-19 pandemic, new variants of SARS-CoV-2 continue to emerge in human populations. Because the evolution of SARS-CoV-2 is driven by mutation, a better understanding of its mutation rate and spectrum could improve our ability to forecast the trajectory of the pandemic. Here, we use circular RNA consensus sequencing (CirSeq) to determine the mutation rate of six SARS-CoV-2 variants and perform a short-term evolution experiment to determine the impact of these mutations on viral fitness. Our analyses indicate that the SARS-CoV-2 genome mutates at a rate of ∼1.5 × 10/base per viral passage and that the spectrum is dominated by C → U transitions. Moreover, we find that the mutation rate is significantly reduced in regions that form base-pairing interactions and that mutations that affect these secondary structures are especially harmful to viral fitness. In this work, we show that the biased mutation spectrum of SARS-CoV-2 is likely a result of frequent cytidine deamination and that the secondary structure of the virus plays an important role in this process, providing new insight into the parameters that guide viral evolution and highlighting fundamental weaknesses of the virus that may be exploited for therapeutic purposes.