Mapping genome-wide distribution of transcriptionally engaged SARS-CoV-2 polymerase at base-pair resolution using novel nascent RNA sequencing to study transcriptional regulation

The COVID-19 pandemic caused by SARS-CoV-2 has emerged as a global health crisis and risen the urgent need for the drug development. However, the development of effective antiviral against SARS-CoV-2 has been challenged by the gap between the knowledge of viral genome and the molecular mechanisms of transcription and replication. The RNA-dependent-RNA polymerase (RdRp) is the core enzyme in the Replication/Transcription Complex (RTC), which serves as a promising target for antiviral development against SARS-CoV-2. The absence of an effective RdRp inhibitor as antiviral has prompted the critical need to elucidate the molecular mechanism of RdRp in viral genome transcription. The key question is how RTC regulates the viral genome transcription through interfering with the transcription regulatory sequences (TRS). On a genomic level, the central puzzle on the SARS-CoV-2 lies in the highly complex transcriptional regulatory networks RTC exhibits when transcribing its genome. RTC interferes with transcription at the Transcription Regulating Sequences (TRS) upstream of most Open Reading Frames during RNA synthesis. Then, TRS signals the RTC to undergo discontinuous transcription of subgenomic RNAs, where RNA synthesis is halted and reinitiated at the next TRS. We propose to conduct nascent RNA sequencing to map RdRp along regulatory genomic loci to determine how these sequences affect RTC transcription. Insights from our study can reveal the highly interactive nature of TRS and RTC, which in turn can provide therapeutic targets by interfering with the ways TRS and other sequences regulate transcription through interacting with RTC.