BPS2026 – Transcription dynamics of SARS-CoV-2 polymerase in RNA synthesis and sub-genomic template switching

COVID-19 has become an emergency global health threat since 2019. SARS-CoV-2 is the pathogen of COVID-19, which belongs to the coronavirus family. With the viral rapid evolution, more than 10,000 variants have been developed in the past years. In the life cycle of SARS-CoV-2, transcription plays an important role that controls the proliferation and assembly of the virus. NSP12 is the RNA-dependent RNA polymerase (RdRP) catalytic subunit of SARS-CoV-2, which interacts with other subunits NSP7 and NSP8 to form the basic transcription complex. The transcription complex undergoes both full genomic and sub-genomic transcription with the help of RNA secondary structures and transcription factors. However, the mechanism of mutations transcription and sub-genomic template switching were less-studied. We utilized both biochemical methods and single molecule optical tweezers on NSP12 (P323L), a mutation located in the interface region of NSP12 and maintained till the current main variant strains. We proved that NSP12 (P323L) could enhance transcription efficiency via reducing the pause duration time compared with the NSP12 wild-type, which agreed with the patients who were infected by viruses containing the P323L who showed a higher viral loading from published data. Furthermore, NSP12 (P323L) also had higher tolerance against pauses induced by remdesivir and nucleotide incorporation. Besides, we also performed the single molecule optical tweezers on sub-genomic template switching model of SARS-CoV-2. We demonstrated that N protein, transcription regulation sequence, TRS-L and TRS-B were essential to form the basic switching model. Furthermore, a triple-strand structure was necessary for RdRP switching between TRS-L and TRS-B. Finally, we observed the switching complex forming via single molecule optical tweezers imaging. This study sheds light on the mechanism of SARS-CoV-2 transcription dynamics in single-molecule level and provided the potential targets of drug development.