SARS-COV-2 leader alleviates NSP1-mediated silencing via its stem–loop 1 (SL1) structure and enhances NSP1 expression. (A) SL1 in SARS-COV-2 leader is both necessary and sufficient to escape NSP1-mediated repression. HEK293T cells were cotransfected with the indicated SARS-COV-2 leader reporters and NSP1-encoding constructs, either WT or indicated mutants. CoV-2-RL reporter is the same as in Figure 1D–E (contains a full-length leader, TRS [gray oval] and the beginning of viral ORF9 fused with RL coding sequence), CoV-2-ΔSL1-RL lacks stem–loop 1 (SL1), and CoV-2-SL1-RL and CoV-SL1-RL carry SL1 alone, originating from SARS-CoV-2 and SARS-CoV, correspondingly. The experiment was performed, and data presented as in Figure 1C. Values are shown as a percentage of luciferase produced in the presence of the vector for each reporter. Values represent means ± SD from at least three experiments. P-values are calculated and presented as in Figure 1. (B) NSP1 encoded by mRNA carrying the viral leader enhances its own expression. Constructs encoding SARS-CoV-2 NSP1 or its point mutants and carrying 5′UTR of genomic SARS-CoV-2 RNA (gCoV-2-flag-NSP1) were transfected in HEK293T cells, and cell lysates were analyzed by western blotting with anti-flag antibody. Beta-actin was used as a loading control.
