SARS-COV-2 leader enables the virus to escape repression by the NSP1 protein, but not its R124A and RK124AA mutants. (A) Schematic representation of genomic SARS-CoV-2 RNA (gRNA) and subgenomic RNAs (sgRNAs). Red line: viral leader; gray oval: transcriptional regulatory sequences (TRS); gray rectangles: open reading frames (ORFs). Please note that not all TRSs and ORFs are shown. (B) Schematic representation of NSP1 protein, including analyzed mutants R124A, RK124AA, KH164AA with partial alignment of mutated regions. The numbers correspond to the amino acid positions. (C) Repression of RL mRNA by SARS-CoV-2 NSP1 and its point mutants. HEK293T cells were cotransfected with Renilla luciferase (RL) plasmid and increasing amounts of plasmids, encoding flag-tagged SARS-CoV-2 NSP1, or indicated NSP1 point mutants. As negative control, the vector encoding flag alone instead of flag-NSP1 plasmid was used. Open bars: vector, green: WT NSP1, purple: RK124AA NSP1, blue: R124A NSP1, gray: KH164AA NSP1. Values are presented as a percentage of luciferase produced in the presence of the vector. Values represent means ±SD from at least three experiments. P-values ([*] <0.1, [**] <0.01, [***] <0.001), calculated with a two-sample t-test comparing vector and NSP1 samples, are shown above the relevant bars. Expression of flag-NSP1 fusion protein and its point mutants was estimated by western blotting with anti-flag antibodies and shown below the reporter assay. Note that KH164AA mutant migrates slower than WT. ActB was used as a loading control. (D) Repression of RL mRNA by SARS-CoV NSP1 and its point mutants. The experiment was performed as in C, but SARS-CoV NSP1 instead of SARS-CoV-2 NSP1 was used. (E,F) Reporter bearing SARS-CoV-2 leader (CoV-2-RL) escapes repression by WT NSP1, but is repressed by NSP1 R124A and NSP1 RK124AA mutants. Panel E shows the effects of NSP1 from SARS-CoV-2 and panel F—NSP1 from SARS-CoV. CoV-2-RL reporter bears the viral leader, TRS and the beginning of viral ORF9 (encoding nucleocapsid protein) fused with the RL coding sequence. See Materials and Methods for more details.
