Fig. 1.

SARS-CoV-2 RdRp stimulates the expression of CoV-Gluc reporter. (A) Schematic diagram of the Gluc reporter system. The expression cassette of Gluc is in sense strand, which is flanked by the 5′ and 3′ untranslated regions (UTRs) of SARS-COV-2. The negative-sense vRNA is first synthesized by SARS-CoV-2 RdRp, followed by transcription into plus-strand RNA (mRNA) to express Gluc. (B) HEK293T cells (4 × 10⁵) were co-transfected with the indicated plasmids (0.5 μg of each plasmid). At 48 h post-transfection, cells were collected for Western blot. Western blots were probed with anti-Flag antibody to detect nsp12 (upper panel), nsp7 and nsp8 (middle panel). β-actin was detected (lower panel) as the loading control. The supernatants of the transfected cells were subjected to Gluc activity assay. Results shown are the average of three independent experiments. (C) Levels of both plus-strand and minus-strand CoV-Gluc RNA were determined by quantitative RT-PCR from HEK293T cells that were transfected with the indicated plasmid DNA. Results shown are the average of three independent experiments. (D) The D760A RdRp mutant disables CoV-Gluc activity. HEK293T cells were transfected with the indicated plasmid DNA. Expression of RdRp, nsp7, and nsp8 was examined by Western blotting. Levels of Gluc in the culture supernatants were measured, and the level from cells expressing CoV-Gluc and wild type RdRp was arbitrarily set at 1. Results shown are the average of three independent transfection experiments.