Fig. 4: Dominant co-mutation attenuates viral replication.

a Predicted RNA structure of the SARS-CoV-2 5′UTR. RNA structure of the 400-nt 5′UTR was predicted by “RNAstructure” (http://rna.urmc.rochester.edu/RNAstructureWeb). The start codon for nsp1 is gray, the TRS-L is orange, and the mutated nucleotides are red. The bottom panel shows the alignment of the 5′UTR of SARS-CoV-2 with 5′UTRs of related viruses, with c241 highlighted. b Structure of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) or RNA complex. The structure of SARS-CoV-2 RdRp/RNA complex (PDB, 6X2G) was visualized by Chimera (UCSF). The P323 mutation is highlighted in red, with the alignment of the amino acid sequences of SARS-CoV-2 and related viruses near the P323 position. c Schematic of the in vitro ligation system for SARS-CoV-2 replicon. Four plasmids encompassing the viral genome were digested by BsaI to release the four fragments. After gel purification, the fragments were ligated by T4 ligase. The ligation products were purified and used as template for RNA in vitro transcription. sGluc, secreted Gaussia luciferase; 2A, foot-and-mouth disease virus (FMDV) 2A peptide; BSD, blasticidin. d Huh7 cells were co-transfected with in vitro transcribed replicon RNA (wild type (WT) or the indicated mutants) and an mRNA encoding the SARS-CoV-2 N protein. The luciferase activity in the supernatants was measured at the time points indicated. Medium was changed at 8 h post-transfection. Data are shown as mean ± SEM (n = 8). SAA, the NSP12 polymerase active-site mutant. Unpaired Student’s t-test was performed between the mutants and WT and between the mutants as indicated. *P < 0.05, **P < 0.01, ***P < 0.001.