s ar s cov 2  (ATCC)

Journal: Nucleic acids research

Article Title: The differential effect of SARS-CoV-2 NSP1 on mRNA translation and stability reveals new insights linking ribosome recruitment, codon usage, and virus evolution.

doi: 10.1093/nar/gkaf261

Figure Lengend Snippet: Figure 2. Influence of mRNA codon usage bias on sensitivity to NSP1. ( A ) Correlation between tAIg of reporter mRNAs and their corresponding sensitivities to NSP1 WT or M2. Data were scored as described in Fig. 1 . The R 2 and P -values are indicated. ( B ) Effect of RL CDS recoding on sensitivity to NSP1 WT and M2. The CAI, tAIg, and CSCg values for parental (RL) and RL-1 to three variants are indicated. Right panel shows a relative quantification of RL and RL-3 mRNA le v els in HEK293T cells expressing NSP1 WT or M2. ( C ) Correlation between TE, tAIg, and sensitivity to NSP1 WT and M2. Upper left panel shows TE calculated for the indicated mRNAs by polysome profiling followed of RT-qPCR. The resulting heat map of the relative quantification is shown. Correlation between TE and tAIg (upper right panel) or TE and sensitivity to mRNA-induced degradation by NSP1 WT or M2 (bottom panel). Data were scored as described above and the resulting R 2 is indicated. Bottom right panel shows the correlation between TE of human mRNAs and sensitivity to NSP1-induced mRNA degradation in Calu3 cells infected with SARS-CoV-2 WT compared with that infected by a mutant virus defective in NSP1 ( NSP1). Data were taken from GSE200422 [ 27 ] and analyzed as described in the ‘Materials and methods’ section; the resulting Spearman correlation coefficient (r) is indicated. ( D ) Effect of SARS-CoV-2 infection on expression of RL-3, FFL, and EGFP in VeroE6 cells. Cells were infected, transfected with a combination of the three plasmids and analyzed at 16 and 24 hpi for luciferase activity (upper panel) or by western blot against NSP1, RPS6, and EGFP. In parallel wells, cells were co-transfected with pNSP1 and a combination of RL-3, FFL, and EGFP plasmids and analyzed 24 h later (top panels). A comparative analysis of NSP1 levels accumulated in infected and transfected cells is shown (bottom panel). Blots were also probed with anti-eIF2 α as loading control. The 1 / 2 and 1 / 4 dilutions of cell extracts are also indicated.

Article Snippet: For this, nsp1 coding sequences were amplified by polymerase chain reaction (PCR) from pDONR207 S AR S-CoV-2 nsp1 (wild type (WT), Addgene #141255), pDONR207 S AR S-CoV-2 nsp1 Delta RC (M1, K164A / H165A, Addgene #164522), and pDONR207 S AR S-CoV-2 nsp1 R124A / K125A (M2, Addgene #164523) plasmids using the primers 5-EX-NSP1 and 3-NB-NSP1( Supplementary Table S3 ) and cloned into pEGFP-N1 digested with EcoR I and Not I enzymes.

Techniques: Quantitative Proteomics, Expressing, Quantitative RT-PCR, Infection, Mutagenesis, Virus, Transfection, Luciferase, Activity Assay, Western Blot, Control

Journal: Nucleic acids research

Article Title: The differential effect of SARS-CoV-2 NSP1 on mRNA translation and stability reveals new insights linking ribosome recruitment, codon usage, and virus evolution.

doi: 10.1093/nar/gkaf261

Figure Lengend Snippet: Figure 3. Influence of 5 ′ UTR length and secondary str uct ure on sensitivity to NSP1. ( A ) RL plasmids with 5 ′ UTRs ranging from 8 to 650 nt were co-transfected with 82-FFL plasmid in HEK293T cells and the resulting normalized activities were expressed as a percentage relative to the 82-RL construct (-NSP1). The sensitivity to NSP1 WT expression (+NSP1) for every construct is expressed as described in previous figures. ( B ) Effect of NSP1 WT and M2 on 82-RL and TISU-RL expression (left panel) or 94-EGFP and TISU-EGFP expression assayed by western blot (middle panel). The effect of increasing amount of pNSP1 WT or M2 plasmids on TISU-EGFP expression is also shown. ND, not detected. Right panel shows mRNA level quantification of 94-EGFP and TISU-EGFP in cells expressing NSP1 WT or M2. Significance was scored as described before. ( C ) Influence of stable stem-loops (SLs) placed immediately before the AUG of FFL on the sensitivity to NSP1s. The placement and st abilit y ( G ◦) of SLs are shown (left panel). Effect of SLs on FFL expression relative to 82-FFL (lower left panel) and sensitivity to NSP1 WT and M2 (top middle panel). SL50-FFL expression relief by NSP1 WT was also confirmed by western blot using anti-Luc antibodies (bottom middle panel). Transfections also included an EGFP expressing plasmid and the resulting western blot anti-EGFP is shown (bottom bottom panel). The right panels show relative quantification of 82-FFL and SL50-FFL mRNA le v els in control and cells co-e xpressing NSP1 WT or M2.

Article Snippet: For this, nsp1 coding sequences were amplified by polymerase chain reaction (PCR) from pDONR207 S AR S-CoV-2 nsp1 (wild type (WT), Addgene #141255), pDONR207 S AR S-CoV-2 nsp1 Delta RC (M1, K164A / H165A, Addgene #164522), and pDONR207 S AR S-CoV-2 nsp1 R124A / K125A (M2, Addgene #164523) plasmids using the primers 5-EX-NSP1 and 3-NB-NSP1( Supplementary Table S3 ) and cloned into pEGFP-N1 digested with EcoR I and Not I enzymes.

Techniques: Transfection, Plasmid Preparation, Construct, Expressing, Western Blot, Quantitative Proteomics, Control

Journal: Nucleic acids research

Article Title: The differential effect of SARS-CoV-2 NSP1 on mRNA translation and stability reveals new insights linking ribosome recruitment, codon usage, and virus evolution.

doi: 10.1093/nar/gkaf261

Figure Lengend Snippet: Figure 4. The presence of Gs near the 5 ′ cap critically determines the sensitivity to NSP1. ( A ) Schematic diagram of G-less 5 ′ UTR and its variants including G, SL, or GA motifs at the indicated positions. The composition (% G) and st abilit y ( G ◦in kcal / mol) of the resulting sequences are indicated. The Gs introduced in the sequences are in bold. The first 22-nt sequence of 5 ′ L N of SARS-CoV-2 is also shown. ( B ) Left panel shows a comparative analysis of RL expression with G-less or 82 5 ′ UTRs in combination with optimal or suboptimal codon usage (RL versus RL-3) in HEK293T cells. Effect of NSP1 WT or M2 on the expression of G-less 5 ′ UTR in combination with optimal or suboptimal codon usage (middle and right panels). Significance was scored as described in previous figures. ( C ) Sensitivity of G-less and its variants to increasing amounts of NSP1 WT. Data are the average of two independent experiments. ( D ) Reduced sensitivity of G-less-RL expression to eIF4A inhibition by silvestrol (left panel) or RocA (right panel) compared to RL, FFL, or M1 and M4 variants of G-less-RL. Inhibitors were added 1 h after transfection and measurements were taken 16 h later. Data are the mean ± standard deviation from three independent experiments.

Article Snippet: For this, nsp1 coding sequences were amplified by polymerase chain reaction (PCR) from pDONR207 S AR S-CoV-2 nsp1 (wild type (WT), Addgene #141255), pDONR207 S AR S-CoV-2 nsp1 Delta RC (M1, K164A / H165A, Addgene #164522), and pDONR207 S AR S-CoV-2 nsp1 R124A / K125A (M2, Addgene #164523) plasmids using the primers 5-EX-NSP1 and 3-NB-NSP1( Supplementary Table S3 ) and cloned into pEGFP-N1 digested with EcoR I and Not I enzymes.

Techniques: Sequencing, Expressing, Inhibition, Transfection, Standard Deviation

Journal: Nucleic acids research

Article Title: The differential effect of SARS-CoV-2 NSP1 on mRNA translation and stability reveals new insights linking ribosome recruitment, codon usage, and virus evolution.

doi: 10.1093/nar/gkaf261

Figure Lengend Snippet: Figure 5. NSP1 promotes slotting of PIC to mRNA. ( A ) Effect of progressive shortening of 5 ′ UTR G-less on sensitivity to NSP1 WT in HEK293T cells. Co-transfections also included FFL plasmid to normalize for FFL activity. Data are expressed as log 2 fold-change expression. Data points correspond to the mean from at least three independent experiments that were adjusted to a sigmoid function (R 2 = 0.92); shading represents the 95% confidence interv al. T he resulting estimated blind spot of 1 2–1 5 nt is sho wn. A model of 48S-PIC sho wing the position of mRNA residues along the mRNA channel of 40S is also represented (right panel). ( B ) Comparative analysis of the expression of G-less-RL and Slot-RL constructs (left panel). Comparative analysis of the sensitivity of 82-RL and Slot-RL to NSP1s (right panel). Significance was scored as described in previous figures. ( C ) Effect of upstream out-of-frame AUGs (uAUG) on expression of G-less-RL and sensitivity to NSP1. The uAUG was inserted where indicated and the resulting uORFs partially o v erlap the 5 ′ of the main RL ORF (line). T he presence of eIF4A-binding motif (G A) 6 is indicated. HEK293T cells w ere cotransfected with the indicated constructs together with pFFL and the resulting expression was normalized for FFL activity and expressed as % of G-less-RL. Data represent the mean of four independent experiments that were adjusted to a sigmoid function (R 2 = 0.89); shading represents the 95% confidence interval (left panel). Middle and right panels show the effect of increasing amounts of pNSP1 WT plasmid on the expression of the indicated construct derived from pG-less-RL (open circles) or pG-M4-RL (black circles).

Article Snippet: For this, nsp1 coding sequences were amplified by polymerase chain reaction (PCR) from pDONR207 S AR S-CoV-2 nsp1 (wild type (WT), Addgene #141255), pDONR207 S AR S-CoV-2 nsp1 Delta RC (M1, K164A / H165A, Addgene #164522), and pDONR207 S AR S-CoV-2 nsp1 R124A / K125A (M2, Addgene #164523) plasmids using the primers 5-EX-NSP1 and 3-NB-NSP1( Supplementary Table S3 ) and cloned into pEGFP-N1 digested with EcoR I and Not I enzymes.

Techniques: Transfection, Plasmid Preparation, Activity Assay, Expressing, Construct, Binding Assay, Derivative Assay

Journal: Nucleic acids research

Article Title: The differential effect of SARS-CoV-2 NSP1 on mRNA translation and stability reveals new insights linking ribosome recruitment, codon usage, and virus evolution.

doi: 10.1093/nar/gkaf261

Figure Lengend Snippet: Figure 6. A combination of 5 ′ UTR and codon usage bias promotes resistance of N mRNA SARS-CoV-2 to NSP1. ( A ) Plot of tAIg and GC3 (%) distributions among human and coronavirus (hCoV) mRNAs. Values corresponding to human ACTB and SARS-CoV-2 mRNA N and its variant N1 are indicated, along with the Pearson correlation. ( B ) A representative western blot analysis of N and EGFP accumulated in HEK293T cells co-expressing NSP1 M1, WT or M2 (upper panel) and the corresponding quantification (bottom left panel). R elativ e quantification of mRNA le v els of N variants in cells co-expressing NSP1 WT (bottom right panel). Significance was scored as described in previous figures. The combination of 5 ′ UTR (95 or 5 ′ L) with CDS N is indicated at the top of each panel. The tAIg values for N and N1 are 0.21 and 0.248, respectively. ( C ) TE of 5 ′ L-N mRNA in HEK293T cells compared to some other reporters included in the co-transfection experiment (upper panel). Comparative analysis of the expression level and sensitivity to NSP1 among N variants with different combinations of 5 ′ UTR and codon usage. ( D ) Analysis of the expression of 5 ′ L-RL, G-less-RL and G-less_67-RL compared to 82-RL (left panel). The sensitivities of 5 ′ L-RL expression (middle panel) and its variant (5 ′ L(Gs)-RL, right panel) to NSP1 WT are shown.

Article Snippet: For this, nsp1 coding sequences were amplified by polymerase chain reaction (PCR) from pDONR207 S AR S-CoV-2 nsp1 (wild type (WT), Addgene #141255), pDONR207 S AR S-CoV-2 nsp1 Delta RC (M1, K164A / H165A, Addgene #164522), and pDONR207 S AR S-CoV-2 nsp1 R124A / K125A (M2, Addgene #164523) plasmids using the primers 5-EX-NSP1 and 3-NB-NSP1( Supplementary Table S3 ) and cloned into pEGFP-N1 digested with EcoR I and Not I enzymes.

Techniques: Variant Assay, Western Blot, Expressing, Cotransfection

Journal: Nucleic acids research

Article Title: The differential effect of SARS-CoV-2 NSP1 on mRNA translation and stability reveals new insights linking ribosome recruitment, codon usage, and virus evolution.

doi: 10.1093/nar/gkaf261

Figure Lengend Snippet: Figure 2. Influence of mRNA codon usage bias on sensitivity to NSP1. ( A ) Correlation between tAIg of reporter mRNAs and their corresponding sensitivities to NSP1 WT or M2. Data were scored as described in Fig. 1 . The R 2 and P -values are indicated. ( B ) Effect of RL CDS recoding on sensitivity to NSP1 WT and M2. The CAI, tAIg, and CSCg values for parental (RL) and RL-1 to three variants are indicated. Right panel shows a relative quantification of RL and RL-3 mRNA le v els in HEK293T cells expressing NSP1 WT or M2. ( C ) Correlation between TE, tAIg, and sensitivity to NSP1 WT and M2. Upper left panel shows TE calculated for the indicated mRNAs by polysome profiling followed of RT-qPCR. The resulting heat map of the relative quantification is shown. Correlation between TE and tAIg (upper right panel) or TE and sensitivity to mRNA-induced degradation by NSP1 WT or M2 (bottom panel). Data were scored as described above and the resulting R 2 is indicated. Bottom right panel shows the correlation between TE of human mRNAs and sensitivity to NSP1-induced mRNA degradation in Calu3 cells infected with SARS-CoV-2 WT compared with that infected by a mutant virus defective in NSP1 ( NSP1). Data were taken from GSE200422 [ 27 ] and analyzed as described in the ‘Materials and methods’ section; the resulting Spearman correlation coefficient (r) is indicated. ( D ) Effect of SARS-CoV-2 infection on expression of RL-3, FFL, and EGFP in VeroE6 cells. Cells were infected, transfected with a combination of the three plasmids and analyzed at 16 and 24 hpi for luciferase activity (upper panel) or by western blot against NSP1, RPS6, and EGFP. In parallel wells, cells were co-transfected with pNSP1 and a combination of RL-3, FFL, and EGFP plasmids and analyzed 24 h later (top panels). A comparative analysis of NSP1 levels accumulated in infected and transfected cells is shown (bottom panel). Blots were also probed with anti-eIF2 α as loading control. The 1 / 2 and 1 / 4 dilutions of cell extracts are also indicated.

Article Snippet: For this, nsp1 coding sequences were amplified by polymerase chain reaction (PCR) from pDONR207 S AR S-CoV-2 nsp1 (wild type (WT), Addgene #141255), pDONR207 S AR S-CoV-2 nsp1 Delta RC (M1, K164A / H165A, Addgene #164522), and pDONR207 S AR S-CoV-2 nsp1 R124A / K125A (M2, Addgene #164523) plasmids using the primers 5-EX-NSP1 and 3-NB-NSP1( Supplementary Table S3 ) and cloned into pEGFP-N1 digested with EcoR I and Not I enzymes.

Techniques: Quantitative Proteomics, Expressing, Quantitative RT-PCR, Infection, Mutagenesis, Virus, Transfection, Luciferase, Activity Assay, Western Blot, Control

Journal: Nucleic acids research

Article Title: The differential effect of SARS-CoV-2 NSP1 on mRNA translation and stability reveals new insights linking ribosome recruitment, codon usage, and virus evolution.

doi: 10.1093/nar/gkaf261

Figure Lengend Snippet: Figure 3. Influence of 5 ′ UTR length and secondary str uct ure on sensitivity to NSP1. ( A ) RL plasmids with 5 ′ UTRs ranging from 8 to 650 nt were co-transfected with 82-FFL plasmid in HEK293T cells and the resulting normalized activities were expressed as a percentage relative to the 82-RL construct (-NSP1). The sensitivity to NSP1 WT expression (+NSP1) for every construct is expressed as described in previous figures. ( B ) Effect of NSP1 WT and M2 on 82-RL and TISU-RL expression (left panel) or 94-EGFP and TISU-EGFP expression assayed by western blot (middle panel). The effect of increasing amount of pNSP1 WT or M2 plasmids on TISU-EGFP expression is also shown. ND, not detected. Right panel shows mRNA level quantification of 94-EGFP and TISU-EGFP in cells expressing NSP1 WT or M2. Significance was scored as described before. ( C ) Influence of stable stem-loops (SLs) placed immediately before the AUG of FFL on the sensitivity to NSP1s. The placement and st abilit y ( G ◦) of SLs are shown (left panel). Effect of SLs on FFL expression relative to 82-FFL (lower left panel) and sensitivity to NSP1 WT and M2 (top middle panel). SL50-FFL expression relief by NSP1 WT was also confirmed by western blot using anti-Luc antibodies (bottom middle panel). Transfections also included an EGFP expressing plasmid and the resulting western blot anti-EGFP is shown (bottom bottom panel). The right panels show relative quantification of 82-FFL and SL50-FFL mRNA le v els in control and cells co-e xpressing NSP1 WT or M2.

Article Snippet: For this, nsp1 coding sequences were amplified by polymerase chain reaction (PCR) from pDONR207 S AR S-CoV-2 nsp1 (wild type (WT), Addgene #141255), pDONR207 S AR S-CoV-2 nsp1 Delta RC (M1, K164A / H165A, Addgene #164522), and pDONR207 S AR S-CoV-2 nsp1 R124A / K125A (M2, Addgene #164523) plasmids using the primers 5-EX-NSP1 and 3-NB-NSP1( Supplementary Table S3 ) and cloned into pEGFP-N1 digested with EcoR I and Not I enzymes.

Techniques: Transfection, Plasmid Preparation, Construct, Expressing, Western Blot, Quantitative Proteomics, Control

Journal: Nucleic acids research

Article Title: The differential effect of SARS-CoV-2 NSP1 on mRNA translation and stability reveals new insights linking ribosome recruitment, codon usage, and virus evolution.

doi: 10.1093/nar/gkaf261

Figure Lengend Snippet: Figure 4. The presence of Gs near the 5 ′ cap critically determines the sensitivity to NSP1. ( A ) Schematic diagram of G-less 5 ′ UTR and its variants including G, SL, or GA motifs at the indicated positions. The composition (% G) and st abilit y ( G ◦in kcal / mol) of the resulting sequences are indicated. The Gs introduced in the sequences are in bold. The first 22-nt sequence of 5 ′ L N of SARS-CoV-2 is also shown. ( B ) Left panel shows a comparative analysis of RL expression with G-less or 82 5 ′ UTRs in combination with optimal or suboptimal codon usage (RL versus RL-3) in HEK293T cells. Effect of NSP1 WT or M2 on the expression of G-less 5 ′ UTR in combination with optimal or suboptimal codon usage (middle and right panels). Significance was scored as described in previous figures. ( C ) Sensitivity of G-less and its variants to increasing amounts of NSP1 WT. Data are the average of two independent experiments. ( D ) Reduced sensitivity of G-less-RL expression to eIF4A inhibition by silvestrol (left panel) or RocA (right panel) compared to RL, FFL, or M1 and M4 variants of G-less-RL. Inhibitors were added 1 h after transfection and measurements were taken 16 h later. Data are the mean ± standard deviation from three independent experiments.

Article Snippet: For this, nsp1 coding sequences were amplified by polymerase chain reaction (PCR) from pDONR207 S AR S-CoV-2 nsp1 (wild type (WT), Addgene #141255), pDONR207 S AR S-CoV-2 nsp1 Delta RC (M1, K164A / H165A, Addgene #164522), and pDONR207 S AR S-CoV-2 nsp1 R124A / K125A (M2, Addgene #164523) plasmids using the primers 5-EX-NSP1 and 3-NB-NSP1( Supplementary Table S3 ) and cloned into pEGFP-N1 digested with EcoR I and Not I enzymes.

Techniques: Sequencing, Expressing, Inhibition, Transfection, Standard Deviation

Journal: Nucleic acids research

Article Title: The differential effect of SARS-CoV-2 NSP1 on mRNA translation and stability reveals new insights linking ribosome recruitment, codon usage, and virus evolution.

doi: 10.1093/nar/gkaf261

Figure Lengend Snippet: Figure 5. NSP1 promotes slotting of PIC to mRNA. ( A ) Effect of progressive shortening of 5 ′ UTR G-less on sensitivity to NSP1 WT in HEK293T cells. Co-transfections also included FFL plasmid to normalize for FFL activity. Data are expressed as log 2 fold-change expression. Data points correspond to the mean from at least three independent experiments that were adjusted to a sigmoid function (R 2 = 0.92); shading represents the 95% confidence interv al. T he resulting estimated blind spot of 1 2–1 5 nt is sho wn. A model of 48S-PIC sho wing the position of mRNA residues along the mRNA channel of 40S is also represented (right panel). ( B ) Comparative analysis of the expression of G-less-RL and Slot-RL constructs (left panel). Comparative analysis of the sensitivity of 82-RL and Slot-RL to NSP1s (right panel). Significance was scored as described in previous figures. ( C ) Effect of upstream out-of-frame AUGs (uAUG) on expression of G-less-RL and sensitivity to NSP1. The uAUG was inserted where indicated and the resulting uORFs partially o v erlap the 5 ′ of the main RL ORF (line). T he presence of eIF4A-binding motif (G A) 6 is indicated. HEK293T cells w ere cotransfected with the indicated constructs together with pFFL and the resulting expression was normalized for FFL activity and expressed as % of G-less-RL. Data represent the mean of four independent experiments that were adjusted to a sigmoid function (R 2 = 0.89); shading represents the 95% confidence interval (left panel). Middle and right panels show the effect of increasing amounts of pNSP1 WT plasmid on the expression of the indicated construct derived from pG-less-RL (open circles) or pG-M4-RL (black circles).

Article Snippet: For this, nsp1 coding sequences were amplified by polymerase chain reaction (PCR) from pDONR207 S AR S-CoV-2 nsp1 (wild type (WT), Addgene #141255), pDONR207 S AR S-CoV-2 nsp1 Delta RC (M1, K164A / H165A, Addgene #164522), and pDONR207 S AR S-CoV-2 nsp1 R124A / K125A (M2, Addgene #164523) plasmids using the primers 5-EX-NSP1 and 3-NB-NSP1( Supplementary Table S3 ) and cloned into pEGFP-N1 digested with EcoR I and Not I enzymes.

Techniques: Transfection, Plasmid Preparation, Activity Assay, Expressing, Construct, Binding Assay, Derivative Assay

Journal: Nucleic acids research

Article Title: The differential effect of SARS-CoV-2 NSP1 on mRNA translation and stability reveals new insights linking ribosome recruitment, codon usage, and virus evolution.

doi: 10.1093/nar/gkaf261

Figure Lengend Snippet: Figure 6. A combination of 5 ′ UTR and codon usage bias promotes resistance of N mRNA SARS-CoV-2 to NSP1. ( A ) Plot of tAIg and GC3 (%) distributions among human and coronavirus (hCoV) mRNAs. Values corresponding to human ACTB and SARS-CoV-2 mRNA N and its variant N1 are indicated, along with the Pearson correlation. ( B ) A representative western blot analysis of N and EGFP accumulated in HEK293T cells co-expressing NSP1 M1, WT or M2 (upper panel) and the corresponding quantification (bottom left panel). R elativ e quantification of mRNA le v els of N variants in cells co-expressing NSP1 WT (bottom right panel). Significance was scored as described in previous figures. The combination of 5 ′ UTR (95 or 5 ′ L) with CDS N is indicated at the top of each panel. The tAIg values for N and N1 are 0.21 and 0.248, respectively. ( C ) TE of 5 ′ L-N mRNA in HEK293T cells compared to some other reporters included in the co-transfection experiment (upper panel). Comparative analysis of the expression level and sensitivity to NSP1 among N variants with different combinations of 5 ′ UTR and codon usage. ( D ) Analysis of the expression of 5 ′ L-RL, G-less-RL and G-less_67-RL compared to 82-RL (left panel). The sensitivities of 5 ′ L-RL expression (middle panel) and its variant (5 ′ L(Gs)-RL, right panel) to NSP1 WT are shown.

Article Snippet: For this, nsp1 coding sequences were amplified by polymerase chain reaction (PCR) from pDONR207 S AR S-CoV-2 nsp1 (wild type (WT), Addgene #141255), pDONR207 S AR S-CoV-2 nsp1 Delta RC (M1, K164A / H165A, Addgene #164522), and pDONR207 S AR S-CoV-2 nsp1 R124A / K125A (M2, Addgene #164523) plasmids using the primers 5-EX-NSP1 and 3-NB-NSP1( Supplementary Table S3 ) and cloned into pEGFP-N1 digested with EcoR I and Not I enzymes.

Techniques: Variant Assay, Western Blot, Expressing, Cotransfection