The key features of SARS-CoV-2 leader and NSP1 required for viral escape of NSP1-mediated repression

FIGURE 1.. 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, [***]…

FIGURE 2.. 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…

FIGURE 3.. Positions C19 and C20 in the loop of SARS-CoV-2 SL1 are required to escape NSP1-mediated repression. (A) Structures of SARS-CoV-2, bat CoV, SARS-CoV, and MERS-CoV SL1, predicted by mfold (Zuker 2003). The elements of SL1 are labeled: loop, two sections of stem 1 (stem1a and stem 1b), separated by bulge. (B) Mutagenesis of the loop shows the requirement of C19 and C20 for the derepressor function of SL1 in reporter assay. HEK293T cells were cotransfected with SL1-RL reporter or indicated SL1 mutants and the constructs encoding SARS-CoV-2 NSP1, either WT or indicated mutants. The introduced mutations (18U > A, 19C > G, 20C > G, 21C > G, [19C > G; 20C > G], [19C > G; 20C > G; 21C > G], [18U > A; 19C > G; 20C > G; 21C > G]) and resulting sequences of the loop are shown above the plots. The mutated residues are shown in red. The results are 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.

FIGURE 4.. Position C15 in the stem of SARS-CoV-2 SL1 is necessary to escape NSP1-mediated repression. (A) Sequence of stem 1b (see Fig. 2A) is important for derepressor function of SL1. The role of mutations, disrupting folding or modifying parameters of the stem and bulge in SL1, was tested in the NSP1-reporter assay, as described in Figure 3B. 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 as in Figure 1. Mutations and resulting predicted structures (mfold, Zuker 2003) are shown above the plots, with mutated residues in red: 7_11del GGUUUinsCCAAA (i.e., replacement of nucleotides 7 to 11 [GGUUU] by CCAAA) unfolding stem 1a, [17_11delGGUUUinsCCAAA; 29_33delAAACCinsUUUGG] restoring stem 1a, [7_11del GGUUUinsCCAAA; 14_17delACCUinsUGGA] unfolding the stem 1, [7_11del GGUUUinsCCAAA; 14_17delACCUinsUGGA; 22_25delAGGUinsUCCA; 29_33delAAACCinsUUUGG] restoring stem 1, [12_16insUAUAU; 34_38AUAUA] elongating stem 1 by 5 bp, [7_11delCCAAA; 29-33del UUUGG] shortening stem 1 by 5 bp, 27_28delACinsU removing bulge. (B) Position 15C in stem 1b is required for SL1…

FIGURE 5.. ASO targeting SL1 efficiently and specifically down-regulates viral RNA. (A) Sequences of ASOs used in the experiment. The sequence of ASO4 is from the study by Vora et al. (2021). (B) SL1-ASO down-regulates CoV-2 SL1-RL in a wide range of concentrations. HEK293T cells were transfected with the indicated SARS-COV-2 reporters and increasing amounts of ASOs targeting SL1. Scrambled ASO was used as negative control. CoV-2-SL1-RL and CoV-2-ΔSL1-RL reporters are as described in Figure 2A. Values are shown as a percentage of luciferase produced in the presence of the corresponding concentration of scrambled ASO for each reporter. 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 scrambled and SL1-targeting ASO samples, are shown above the relevant bars.

FIGURE 6.. NSP1 interacts with ribosomal proteins, ribosome biogenesis factors and viral response factors. (A) Scheme for NSP1 BioID. NSP1 is fused with a promiscuous biotin ligase (birA), leading to labeling of proteins within a ∼10 nm radius. Labeled proteins are isolated on streptavidin beads and quantified by LC-MS/MS. Unfunctional mutants of NSP1 and biotin ligase alone (mock) serve as negative controls. (B) Volcano plots showing proteins enriched in SARS-CoV-2 NSP1-BioID. −log10 P-values (Y) are plotted against log2 fold change of LFQ (label-free quantification) values between NSP1-BioID and the indicated NSP1 mutant or BioID alone (mock) (X). Specific ribosomal protein (green), nuclear export factor 1 (NXF1, black), viral defense factors (referred to in the text and assigned to the GO category “defense response to virus,” red), and nucleases (orange) are shown. (C) Volcano plots showing changes in total proteome of 293T cells upon expression of SARS-CoV-2 NSP1. −log10 P-values (Y) are plotted against log2 fold change of LFQ (label-free quantification) values between NSP1 and the indicated NSP1 mutant or tag alone (mock) (X). (D) Boxplot showing changes in expression of proteins…