Figure 1.

Schematic diagram of the cell-based SARS-CoV-2 RdRp activity assay system. A SARS-CoV-2 RdRp reporter assay system consists of a bicistronic reporter plasmid and nsp12 gene expression plasmid. A p(+)FLuc-(−)UTR-NLuc reporter plasmid contained the sense-oriented (+) firefly luciferase gene, (+)FLuc under the CMV promoter and the antisense-oriented 3′-UTR of SARS-CoV-2, Nano-Glo luciferase sequence and 5′-UTR of SARS-CoV-2, (−)UTR-NLuc, which were flanked with HDV ribozyme self-cleavage sequences. The host RNA polymerase transcribed the (+)FLuc-(−)UTR-NLuc RNA, which was then processed at the HDV ribozyme self-cleavage sequence. The cleaved (−)3′-UTR-(−)NLuc-(−)5′-UTR RNA sequences were replicated by the SARS-CoV RdRp protein. Then, the replicated sense-oriented NLuc RNA was translated. Therefore, NLuc activity indicated the activity of SARS-CoV-2 RdRp, whereas FLuc activity acted as an internal control.