Structure of the full SARS-CoV-2 RNA genome in infected cells

Purpose: SARS-CoV-2 is a betacoronavirus responsible for the COVID-19 pandemic. Currently, limited understanding of the molecular and structural biology of the virus hampers the development of antiviral drugs. Methods: Vero cells were infected with SARS-CoV-2 and treated with dimethyl sulfate (DMS). RNA was extracted, reverse-transcribed using TGIRT-III, PCR-amplified, and sequenced using an Illumina iSeq100. Reads were trimmed with TrimGalore and mapped to the SARS-CoV-2 genome with Bowtie2. Mutations were quantified with DREEM, and the frequencies were used as folding constraints in RNAstructure. The output structures were visualized with VARNA. Results: A data-driven secondary structure of the full RNA genome was generated, including structures previously lacking experimental validation, such as the TRSs. An alternative structure was identified for the frameshift element that differs drastically from previous in vitro models. The canonical frameshift element pseudoknot was not detected; in its place was an alternative stem partially overlapping with the canonical pseudoknot. Two alternative structures containing this stem were identified. Conclusions: The genome-wide structure of SARS-CoV-2 provides a basis for developing therapeutics targeted to key regulatory structures. In particular, the model suggests that frameshifting is caused by a novel 75 nt stem rather than the canonical pseudoknot, which will guide the search for therapeutics that disrupt frameshifting.. Overall design: Secondary structure of SARS-CoV-2 RNA genome in two cultures of infected Vero cells.