The SARS-CoV-2 subgenome landscape and its novel regulatory features

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a global pandemic. CoVs are known to generate negative subgenomes (subgenomic RNAs [sgRNAs]) through transcription-regulating sequence (TRS)-dependent template switching, but the global dynamic landscapes of coronaviral subgenomes and regulatory rules remain unclear. Here, using next-generation sequencing (NGS) short-read and Nanopore long-read poly(A) RNA sequencing in two cell types at multiple time points after infection with SARS-CoV-2, we identified hundreds of template switches and constructed the dynamic landscapes of SARS-CoV-2 subgenomes. Interestingly, template switching could occur in a bidirectional manner, with diverse SARS-CoV-2 subgenomes generated from successive template-switching events. The majority of template switches result from RNA-RNA interactions, including seed and compensatory modes, with terminal pairing status as a key determinant. Two TRS-independent template switch modes are also responsible for subgenome biogenesis. Our findings reveal the subgenome landscape of SARS-CoV-2 and its regulatory features, providing a molecular basis for understanding subgenome biogenesis and developing novel anti-viral strategies. [Display omitted] •Dynamic subgenome landscapes of SARS-CoV-2 in two host cells are constructed•Bidirectional and successive template switching diversify sgRNA biogenesis•Several key determinants governing template switching efficacy are discovered•Canonical TRS-independent RNA-RNA interaction mediates template switches Wang et al. construct dynamic landscapes of SARS-CoV-2 subgenomic RNAs (sgRNAs) by using integrated poly(A) RNA sequencing at various time points after infection. Computational analyses reveal novel modes of viral sgRNA biogenesis and decode regulatory features, including several key determinants governing the efficacy of template switching in coronaviral RNA transcription.