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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. [ABSTRACT FROM AUTHOR]

COVID-19 has spread worldwide since 2019 and is now a severe threat to public health. We previously identified the causative agent as a novel SARS-related coronavirus (SARS-CoV-2) that uses human angiotensin-converting enzyme 2 (hACE2) as the entry receptor. Here, we successfully developed a SARS-CoV-2 hACE2 transgenic mouse (HFH4-hACE2 in C3B6 mice) infection model. The infected mice generated typical interstitial pneumonia and pathology that were similar to those of COVID-19 patients. Viral quantification revealed the lungs as the major site of infection, although viral RNA could also be found in the eye, heart, and brain in some mice. Virus identical to SARS-CoV-2 in full-genome sequences was isolated from the infected lung and brain tissues. Last, we showed that pre-exposure to SARS-CoV-2 could protect mice from severe pneumonia. Our results show that the hACE2 mouse would be a valuable tool for testing potential vaccines and therapeutics. • SARS-CoV-2 could infect HFH4-hACE2 mice and cause death • SARS-CoV-2 infection localizes to lungs of mice and causes typical interstitial pneumonia • Pre-exposure to SARS-CoV-2 protects mice from lethal challenge A SARS-CoV-2 hACE2 transgenic mouse infection model recapitulates a number of infection symptoms and pathology in COVID-19 patients. Pre-exposure to SARS-CoV-2 was able to protect mice from severe pneumonia. [ABSTRACT FROM AUTHOR]