The SARS-CoV-2 Subgenome Landscape and its Novel Regulatory Features

Summary COVID-19, caused by Coronavirus SARS-CoV-2, is now in global pandemic. Coronaviruses are known to generate negative subgenomes (sgRNAs) through Transcription-Regulating Sequence (TRS)-dependent template switch, but the global dynamic landscapes of coronaviral subgenomes and regulatory rules remain unclear. Here, using NGS short-read and Nanopore long-read poly(A) RNA sequencing in two cell types at multiple time points post-infection of SARS-CoV-2, we identified hundreds of template switches and constructed the dynamic landscapes of SARS-CoV-2 subgenomes. Interestingly, template switch could occur in 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. Moreover, two TRS-independent template switch modes are also responsible for subgenome biogenesis. Collectively, 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.