Journal: Med (New York, N.y.)

Article Title: Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases

doi: 10.1016/j.medj.2021.08.001

Figure Lengend Snippet: Antibodies to coronaviral spikes in pediatric and adolescent JIA, JDM, JSLE, and MIS-C patients and age-matched controls (A) Mirror plots of the specific MFI increase of HEK293T cells expressing HCoV-OC43 spike (top) or SARS-CoV-2 spike (bottom) caused by individual sera. Each bar is an individual healthy control or patient. Samples are plotted according to the signal of antibodies to HCoV-OC43 spike and in the same position in the mirror plots. Antibody levels to SARS-CoV-2 spike in MIS-C patients are plotted on a different scale from the rest. (B) Mirror plots of the specific MFI increase of HEK293T cells expressing ERV3-1 (top) or HERV-K113 (bottom) envelope glycoproteins caused by individual sera. Samples are plotted in the same order as in (A).

Article Snippet: pcCMV3- HCoV-OC43_spike , SinoBiological , Cat# VG40607-CF.

Techniques: Expressing

Journal: Med (New York, N.y.)

Article Title: Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases

doi: 10.1016/j.medj.2021.08.001

Figure Lengend Snippet: Prevalence of IgG antibodies to OC43 and SARS-CoV-2 spikes in JIA, JDM, and JSLE patients.

Article Snippet: pcCMV3- HCoV-OC43_spike , SinoBiological , Cat# VG40607-CF.

Techniques:

Journal: Med (New York, N.y.)

Article Title: Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases

doi: 10.1016/j.medj.2021.08.001

Figure Lengend Snippet: Antibodies to coronaviral spikes in pediatric and adolescent JIA, JDM, JSLE, and MIS-C patients and controls of different age Specific MFI increases of HEK293T cells expressing HCoV-OC43 spike (A and C) or SARS-CoV-2 spike (B and D) caused by individual sera from the indicated age and disease group. Each symbol is an individual healthy control or patient. Antibody levels to SARS-CoV-2 spike in MIS-C patients are plotted on a different scale from the rest. Red and blue numbers within the plots denote the p values of statistically significant increases and decreases, respectively, when comparing each disease group with the respective healthy control of the same age group. The older control group was also compared with the younger control group.

Article Snippet: pcCMV3- HCoV-OC43_spike , SinoBiological , Cat# VG40607-CF.

Techniques: Expressing

Journal: Med (New York, N.y.)

Article Title: Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases

doi: 10.1016/j.medj.2021.08.001

Figure Lengend Snippet: SARS-CoV-2 neutralizing antibodies in pediatric and adolescent JIA, JDM, JSLE, and MIS-C patients (A) SARS-CoV-2-neutralizing antibody titers in the indicated age and disease group. Only patients with SARS-CoV-2 spike-binding antibodies detectable by flow cytometry were included. JIA, JDM, and JSLE patients of both age groups combined were compared with MIS-C patients by ANOVA on ranks tests. (B) Correlation of SARS-CoV-2-neutralizing antibody titers with levels of flow-cytometry-detectable antibodies to SARS-CoV-2 spike (left) or HCoV-OC43 spike (right). In (A) and (B), each symbol is an individual patient. In (B), one JSLE patient was removed from the regression analysis as an outlier for the HCoV-OC43 spike antibodies, based on the Kurtosis coefficient of the group.

Article Snippet: pcCMV3- HCoV-OC43_spike , SinoBiological , Cat# VG40607-CF.

Techniques: Binding Assay, Flow Cytometry

Journal: Med (New York, N.y.)

Article Title: Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases

doi: 10.1016/j.medj.2021.08.001

Figure Lengend Snippet: Antibodies to coronaviral nucleoproteins in pediatric and adolescent JIA, JDM, JSLE, and MIS-C patients and age-matched controls (A–C) Mirror plots of the specific MFI increase of HEK293T cells expressing HCoV-OC43 nucleoprotein (top) or SARS-CoV-2 nucleoprotein (bottom) caused by individual sera. Each bar is an individual child or adolescent healthy control or JIA, JDM, or JSLE patient (A), MIS-C patient (B), or adult healthy control (C). Samples are plotted according to the signal of antibodies to the HCoV-OC43 nucleoprotein and in the same position in the mirror plots. (D) Correlation of the proportion of total HCoV-OC43 nucleoprotein-binding antibodies represented by IgM (top) or IgG (bottom) classes, and the age of the donor or patient. Each symbol is an individual sample.

Article Snippet: pcCMV3- HCoV-OC43_spike , SinoBiological , Cat# VG40607-CF.

Techniques: Expressing, Binding Assay

Journal: Med (New York, N.y.)

Article Title: Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases

doi: 10.1016/j.medj.2021.08.001

Figure Lengend Snippet: Antibodies to coronaviral nucleoproteins in pediatric and adolescent JIA, JDM, JSLE, and MIS-C patients and controls of different ages Specific MFI increase of HEK293T cells expressing HCoV-OC43 nucleoprotein (A and C) or SARS-CoV-2 nucleoprotein (B and D) caused by individual sera from the indicated age and disease group. Each symbol is an individual healthy control or patient. Red and blue numbers within the plots denote the p values of statistically significant increases and decreases, respectively, when comparing each disease group with the respective healthy control of the same age group. The older control group was also compared with the younger control group.

Article Snippet: pcCMV3- HCoV-OC43_spike , SinoBiological , Cat# VG40607-CF.

Techniques: Expressing

Journal: Med (New York, N.y.)

Article Title: Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases

doi: 10.1016/j.medj.2021.08.001

Figure Lengend Snippet: Ratios of the levels of antibodies to coronaviral spikes and nucleoproteins in pediatric and adolescent JIA, JDM, and JSLE patients and age-matched controls (A) The log2-transformed ratios of total antibodies to the HCoV-OC43 spike to total antibodies to the HCoV-OC43 nucleoprotein (S: N) are plotted for the indicated age and disease group. Each symbol is an individual sample. Numbers within the plots denote the p values of statistically significant increases, when comparing each disease group with the respective healthy control of the same age group. (B) Heatmap of ranked S: N ratios in the same samples, with each column representing a patient or control. The sample annotations for disease; age; disease activity; and treatment with steroids, biologics, or disease-modifying anti-rheumatic drugs (DMARDs) are also indicated.

Article Snippet: pcCMV3- HCoV-OC43_spike , SinoBiological , Cat# VG40607-CF.

Techniques: Transformation Assay, Activity Assay

Journal: Med (New York, N.y.)

Article Title: Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases

doi: 10.1016/j.medj.2021.08.001

Figure Lengend Snippet:

Article Snippet: pcCMV3- HCoV-OC43_spike , SinoBiological , Cat# VG40607-CF.

Techniques: Recombinant, Software

Journal: bioRxiv

Article Title: Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53

doi: 10.1101/2023.08.31.555625

Figure Lengend Snippet: A. RNA-seq volcano plot of SARS-CoV-2 infection versus mock infection in VeroE6 cells (Riva et al. ). FDR, false discovery rate. B. Gene set enrichment analysis of VeroE6 transcriptome changes upon SARS-CoV-2 infection and other cell models of SARS-CoV-2 infection (Blanco-Melo et al. ). C. Heatmap of relative expression levels (scaled counts via the variance stabilized transform [VST]) of differentially expressed (FDR < 0.05) genes upon VeroE6 SARS-CoV-2 infection by RNA-seq and differentially accessible transcription start sites (TSS’s) (FDR < 0.05 and |log2 Fold Change| > 0.5) by ATAC-seq. Legend, Z-scaled VST counts. D. ATAC-seq volcano plot demonstrating the changes in accessible chromatin loci upon HKU5-SARS-CoV-1-S infection versus mock infection in VeroE6 cells 48 hr post-infection. FDR, false discovery rate. E. ATAC-seq volcano plot demonstrating the changes in accessible chromatin loci upon MERS-CoV infection versus mock infection in VeroE6 cells 48 hr post-infection. FDR, false discovery rate. F. Principal component analysis of ATAC-seq profiles (principal components 3 and 4). G. Principal component analysis of ChromVAR transcription factor motif profiles from ATAC-seq datasets.

Article Snippet: Gene inserts from pDONR221-EGFP (Addgene #25899), pDONR223 SARS-CoV-2 spike (Addgene #149329), B.1.1.7 SARS-CoV-2 spike (Sino Biological, VG40771-CF), and B.1.617.2 SARS-CoV-2 spike (Sino Biological, VG40804-UT) sequences were subcloned into pInducer20 (Addgene #44012) via Gateway cloning. pLenti-HiBiT-Blast was generated by swapping out the dCas9-VP64-T2A insert of lenti-dCas9-VP64-Blast (Addgene #61425) by Q5 site-directed mutagenesis (NEB). pLenti-LgBiT-T2A-Blast was generated by subcloning the LgBiT coding sequence from the FRB-LgBiT plasmid (Promega N2016) into the lenti-dCas9-VP64-Blast plasmid.

Techniques: RNA Sequencing Assay, Infection, Expressing

Journal: bioRxiv

Article Title: Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53

doi: 10.1101/2023.08.31.555625

Figure Lengend Snippet: A. Experimental design and schematic of the iDAPT approach (made with Biorender). B. ATAC-seq volcano plot demonstrating the changes in accessible chromatin loci upon SARS-CoV-2 infection versus mock infection in VeroE6 cells 48 hr post-infection. FDR, false discovery rate. C. Principal component analysis of ATAC-seq profiles upon infection with SARS-CoV-2, HKU5-SARS-CoV-1-S, or MERS-CoV and mock infection. PC, principal component. D. iDAPT-MS volcano plot of SARS-CoV-2 infection (MOI 0.5) versus mock infection in VeroE6 cells 48 hr post-infection. Black circles, significant transcription factors (FDR < 0.05 and |log2 Fold Change| > 0.5). E. Principal component analysis of iDAPT-MS profiles upon infection with SARS-CoV-2, HKU5-SARS-CoV-1-S, or MERS-CoV and mock infection. F. Heatmap of relative protein abundance levels of differentially abundant (FDR < 0.05 and |log2 Fold Change| > 0.5) transcription factors from SARS-CoV-2 vs. mock infection iDAPT-MS analyses. Specificity is defined as the Pearson correlation between iDAPT-MS protein abundance and SARS-CoV-2 infection status. Sig, significant differential chromatin abundance by iDAPT-MS (FDR < 0.05 and |log2 Fold Change| > 0.5) upon infection relative to mock-infected cells. CRISPR, VeroE6 SARS-CoV-2 CRISPR/Cas9 screening results from Wei et al . Positive, Z-score > 2. Negative, Z-score < -2. G. TP53 sgRNA enrichment from CRISPR/Cas9 pooled screening in VeroE6 cells for the three coronaviruses assessed versus mock infection (Wei et al ). Z-scores are quantile-normalized for improved comparison between distributions. H. Top, Western blotting analysis of the indicated proteins after CRISPR/Cas9-based targeting of two TP53-targeting sgRNAs (TP53-1 and TP53-2) or negative control (NT, nontargeting). Bottom, the corresponding Vero E6 cells were infected with VSV pseudotyped viral particles (VSVpp) expressing VSV-G or different coronaviral spike proteins. Luciferase relative to the VSV-G-expressing VSVpp control was measured 24 h after infection. The mean ± s.e.m. are shown. The Welch two-sample t-test with Holm p-value correction was used to assess statistical significance relative to negative control: n.s., not significant. I. Western blotting analysis of the indicated proteins after infection with the corresponding coronaviruses (MOI 0.1, 24 h). Relative ratios of TP53/ACTB (normalized to mock) are annotated below TP53. J. Top, TP53 sgRNA enrichment across published CRISPR/Cas9 genetic screens stratified by TP53 mutational status of the parental cell line (Rebendenne et al ). The mean Z-score of each group is demarcated by a horizontal line. p-value, Welch two-sample t-test. Bottom, TP53 mutational status associated with each parental cell line and corresponding impact of TP53 point mutations on its transcriptional activity as determined in Ursu et al. .

Article Snippet: Gene inserts from pDONR221-EGFP (Addgene #25899), pDONR223 SARS-CoV-2 spike (Addgene #149329), B.1.1.7 SARS-CoV-2 spike (Sino Biological, VG40771-CF), and B.1.617.2 SARS-CoV-2 spike (Sino Biological, VG40804-UT) sequences were subcloned into pInducer20 (Addgene #44012) via Gateway cloning. pLenti-HiBiT-Blast was generated by swapping out the dCas9-VP64-T2A insert of lenti-dCas9-VP64-Blast (Addgene #61425) by Q5 site-directed mutagenesis (NEB). pLenti-LgBiT-T2A-Blast was generated by subcloning the LgBiT coding sequence from the FRB-LgBiT plasmid (Promega N2016) into the lenti-dCas9-VP64-Blast plasmid.

Techniques: Infection, CRISPR, Comparison, Western Blot, Negative Control, Expressing, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53

doi: 10.1101/2023.08.31.555625

Figure Lengend Snippet: A. Representative Ponceau S and western blotting analysis of iDAPT-MS experiment. TP, transposase-peroxidase; T, transposase only. B. Gene set enrichment analysis (GSEA) of sequence-specific transcription factors of iDAPT-MS profiles comparing transposase-peroxidase (TP) enrichment versus transposase (T)- only enrichment. Log2 fold change values were used to rank proteins. NES, normalized enrichment score. p, GSEA p-value. C. Top, significant (FDR < 0.05 and |log2 Fold Change| > 0.5) transcription factor chromatin abundance changes from SARS-CoV-2 vs. mock iDAPT-MS, ordered by SARS-CoV-2 specificity. Middle, corresponding RNA abundance changes from RNA-seq. Bottom, corresponding ChromVAR motif enrichment changes from ATAC-seq. FDR, false discovery rate. D. Principal component analysis of iDAPT-MS profiles (principal components 3 and 4). E. iDAPT-MS volcano plot of HKU5-SARS-CoV-1-S infection (MOI 0.5) versus mock infection in VeroE6 cells 48 hr post-infection. Black circles, significant transcription factors (FDR < 0.05 and |log2 Fold Change| > 0.5). Black points, labeled transcription factors. Red point, TP53. F. iDAPT-MS volcano plot of MERS-CoV infection (MOI 0.5) versus mock infection in VeroE6 cells 48 hr post-infection. Black circles, significant transcription factors (FDR < 0.05 and |log2 Fold Change| > 0.5). Black points, labeled transcription factors. Red point, TP53. G. Gene set enrichment analysis of TP53 target genes (Fischer et al. ) in published RNA-seq (Riva et al. or Blanco-Melo et al. ) or single-cell RNA-seq (Delorey et al. ) datasets. Genes are ranked by log2 fold change. NES, normalized enrichment score. H. Top, heatmap of relative protein abundance levels of differentially abundant (FDR < 0.05 and |log2 Fold Change| > 0.5) proteins from iDAPT-MS analyses that contribute to the SARS-CoV-2 cytopathic effect (CRISPR/Cas9 pooled screening gene effect |Z| > 2) and not the MERS-CoV nor HKU5-SARS-CoV-1-S cytopathic effects (|Z| < 2). Specificity, Pearson correlation between iDAPT-MS abundances and SARS-CoV-2 infection (see Methods). Sig, significance (FDR < 0.05 and |log2 Fold Change| > 0.5) of infection condition relative to mock-infected cells. Middle, sgRNA enrichment across published CRISPR/Cas9 genetic screens (Rebendenne et al ). The mean Z-score of each gene is demarcated by a horizontal line. Bottom, significance of Z-score distribution per gene. The Welch one-sample t-test with Benjamini-Hochberg p-value correction was used to assess statistical significance. FDR, false discovery rate.

Article Snippet: Gene inserts from pDONR221-EGFP (Addgene #25899), pDONR223 SARS-CoV-2 spike (Addgene #149329), B.1.1.7 SARS-CoV-2 spike (Sino Biological, VG40771-CF), and B.1.617.2 SARS-CoV-2 spike (Sino Biological, VG40804-UT) sequences were subcloned into pInducer20 (Addgene #44012) via Gateway cloning. pLenti-HiBiT-Blast was generated by swapping out the dCas9-VP64-T2A insert of lenti-dCas9-VP64-Blast (Addgene #61425) by Q5 site-directed mutagenesis (NEB). pLenti-LgBiT-T2A-Blast was generated by subcloning the LgBiT coding sequence from the FRB-LgBiT plasmid (Promega N2016) into the lenti-dCas9-VP64-Blast plasmid.

Techniques: Western Blot, Sequencing, RNA Sequencing Assay, Infection, Labeling, CRISPR

Journal: bioRxiv

Article Title: Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53

doi: 10.1101/2023.08.31.555625

Figure Lengend Snippet: A. Top, schematic of the SARS-CoV-2 viral genome with encoded proteins. Bottom, number of significant (FDR < 0.05) ATAC-seq peaks upon transfection of plasmid-encoded viral proteins relative to empty vector and EGFP (48 hr) in VeroE6 cells. Black triangles, peaks with increasing accessibility. Red circles, peaks with decreasing accessibility. B. Comparison of thresholded (FDR < 0.05) differential ATAC-seq peaks between SARS-CoV-2 vs. mock infection (24 hr, MOI 0.5) and plasmid-encoded transfection of SARS-CoV-2 nsp1 vs. controls in VeroE6 cells (48 hr). Blue line, best-fit trendline. R, Pearson correlation. p-value, Pearson correlation p-value. C. Comparison of thresholded (FDR < 0.05) differential ATAC-seq peaks between SARS-CoV-2 vs. mock infection (24 hr, MOI 0.5) and plasmid-encoded transfection of SARS-CoV-2 spike vs. controls in VeroE6 cells (48 hr). Blue line, best-fit trendline. R, Pearson correlation. p-value, Pearson correlation p-value. D. Venn diagram analysis of differential ATAC-seq peaks (FDR < 0.05, positive and negative combined) upon nsp1 or spike expression relative to controls. E. Western blotting analysis of VeroE6 cells 48 hr after transfection with plasmids encoding the indicated proteins. EV, empty vector. Relative ratios of TP53/ACTB (normalized to the corresponding EV) are annotated below TP53. F. Western blotting analysis of VeroE6 cells 48 hr after transfection with plasmids encoding the indicated FLAG-tagged proteins. EV, empty vector. Relative ratios of TP53/ACTB (normalized to EV) are annotated below TP53. G. Schematic of cell-cell fusion assay. A 1:1 mixture of VeroE6 cells stably transduced with either pLenti-HiBiT-Bsr or pLenti-LgBiT-P2A-Bsr is transfected with corresponding plasmids. After 48 hr, the cell-cell fusion effect is measured by NanoBiT luciferase complementation. Made with Biorender. H. Cell-cell fusion analysis of VeroE6 cells transfected with the corresponding spike proteins. S2P, “2P” mutant of the spike which is incapable of membrane fusion. The Welch two-sample t-test with Holm p-value correction was used to assess statistical significance relative to the S2P mutant as negative control: ***, p < 0.001; *, p < 0.05; n.s., not significant. I. Schematic of VSV-G acidification assay. Cells were transfected with plasmid encoding VSV-G. 24 h later, cells were treated with a 3 min pulse of DMEM buffered with 10 mM morpholineethanesulfonic acid (MES) at different pH levels. Cells were analyzed 24 h after acidification. J. Cell-cell fusion analysis of VeroE6 cells transfected with VSV-G plasmid and treated with buffers at different pH levels. The Welch two-sample t-test with Holm p-value correction was used to assess statistical significance relative to pH 7.5 as control: ***, p < 0.001; **, p < 0.01; n.s., not significant. K. Western blotting analysis of VeroE6 cells transfected with VSV-G plasmid and treated with buffers at different pH levels. Relative ratios of TP53/ACTB (normalized to pH 7.5) are annotated below TP53.

Article Snippet: Gene inserts from pDONR221-EGFP (Addgene #25899), pDONR223 SARS-CoV-2 spike (Addgene #149329), B.1.1.7 SARS-CoV-2 spike (Sino Biological, VG40771-CF), and B.1.617.2 SARS-CoV-2 spike (Sino Biological, VG40804-UT) sequences were subcloned into pInducer20 (Addgene #44012) via Gateway cloning. pLenti-HiBiT-Blast was generated by swapping out the dCas9-VP64-T2A insert of lenti-dCas9-VP64-Blast (Addgene #61425) by Q5 site-directed mutagenesis (NEB). pLenti-LgBiT-T2A-Blast was generated by subcloning the LgBiT coding sequence from the FRB-LgBiT plasmid (Promega N2016) into the lenti-dCas9-VP64-Blast plasmid.

Techniques: Transfection, Plasmid Preparation, Comparison, Infection, Expressing, Western Blot, Cell-Cell Fusion Assay, Stable Transfection, Transduction, Luciferase, Mutagenesis, Membrane, Negative Control

Journal: bioRxiv

Article Title: Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53

doi: 10.1101/2023.08.31.555625

Figure Lengend Snippet: A. Cell-cell fusion analysis of VeroE6 cells transfected with the corresponding spike proteins. S2P, “2P” mutant of the spike. The Welch two-sample t-test with Holm p-value correction was used to assess statistical significance relative to the S2P mutant as negative control: ***, p < 0.001. B. Western blotting analysis of VeroE6 cells 48 hr after transfection with plasmids encoding the indicated FLAG-tagged proteins. EV, empty vector. Relative ratios of TP53/ACTB (normalized to EV) are annotated below TP53. C. Western blotting analysis of A549ACE2 pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL doxycycline (Dox) for 48 hr. Relative ratios of TP53/ACTB (normalized to EGFP -Dox) are annotated below TP53. D. Representative brightfield images of A549ACE2 pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL Dox for 48 hr. Scale bar, 200 μm. E. Western blotting analysis of the indicated proteins 48 hr after 1 μg/mL Dox treatment of VeroE6 pInducer20 cells transduced with the indicated gene constructs. F. RNA-seq analysis of TP53 target gene upon ancestral, alpha, or delta spike expression versus EGFP expression. p-value, gene set enrichment analysis. Also see . G. Pearson correlation of ATAC-seq profiles upon ancestral, alpha, or delta spike expression versus EGFP expression compared to treatment with or without 1 μM nutlin-3a for 24 hr. p-value, Pearson correlation. Also see . H. iDAPT-MS volcano plots of ancestral, alpha, or delta spike expression versus EGFP expression in VeroE6 pInducer20 cells 48 hr after 1 μg/mL doxycycline induction. Black points, significant transcription factors (FDR < 0.05 and |log2 Fold Change| > 0.5). I. Western blotting analysis of the indicated proteins after infection with the corresponding SARS-CoV-2 viral variants and MOIs (24 h). Relative ratios of TP53/ACTB (normalized to mock) are annotated below TP53. J. Percentage of TP53-positive cells in COVID-19 patients with pre-Delta or Delta SARS-CoV-2 variants versus uninfected (negative) patient lung biopsies. Immunohistochemistry quantification was performed using QuPath. p-value, linear regression coefficient. K. Representative immunohistochemistry images of TP53 protein, SARS-CoV-2 spike, and CXCL8/IL8 protein epitopes in the lungs of Delta variant-infected COVID-19 patients. Scale bar, 100 μm.

Article Snippet: Gene inserts from pDONR221-EGFP (Addgene #25899), pDONR223 SARS-CoV-2 spike (Addgene #149329), B.1.1.7 SARS-CoV-2 spike (Sino Biological, VG40771-CF), and B.1.617.2 SARS-CoV-2 spike (Sino Biological, VG40804-UT) sequences were subcloned into pInducer20 (Addgene #44012) via Gateway cloning. pLenti-HiBiT-Blast was generated by swapping out the dCas9-VP64-T2A insert of lenti-dCas9-VP64-Blast (Addgene #61425) by Q5 site-directed mutagenesis (NEB). pLenti-LgBiT-T2A-Blast was generated by subcloning the LgBiT coding sequence from the FRB-LgBiT plasmid (Promega N2016) into the lenti-dCas9-VP64-Blast plasmid.

Techniques: Transfection, Mutagenesis, Negative Control, Western Blot, Plasmid Preparation, Transduction, Construct, RNA Sequencing Assay, Expressing, Infection, Immunohistochemistry, Variant Assay

Journal: bioRxiv

Article Title: Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53

doi: 10.1101/2023.08.31.555625

Figure Lengend Snippet: A. Representative brightfield images of VeroE6 pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL doxycycline for 48 hr. Scale bar, 200 μm. B. RNA-seq volcano plots of ancestral, alpha, or delta spike expression versus EGFP expression in VeroE6 pInducer20 cells 48 hr after 1 μg/mL doxycycline induction. Black points, significant TP53 target genes. C. ATAC-seq volcano plots of ancestral, alpha, or delta spike expression versus EGFP expression in VeroE6 pInducer20 cells 48 hr after 1 μg/mL doxycycline induction. D. Western blotting analysis of VeroE6 cells treated with either DMSO or 1 μM nutlin-3a for 24 hr. E. ATAC-seq volcano plot of 1 μM nutlin-3a versus DMSO treatment in VeroE6 cells for 24 hr. FDR, false discovery rate. F. Comparison of thresholded (FDR < 0.05) differential ATAC-seq peaks between nutlin-3a treatment (24 hr, 1 μM) and doxycycline-induced expression (48 hr, 1 μg/mL) of SARS-CoV-2 ancestral, alpha, or delta spike relative to expression of EGFP in VeroE6 pInducer20 cells. Blue line, best-fit trendline. R, Pearson correlation. p-value, Pearson correlation test. G. Heatmap of relative expression levels (scaled counts via the variance stabilized transform [VST]) of differentially expressed (FDR < 0.05) TP53 target genes from VeroE6 pInducer20 EGFP, ancestral (Spike), Alpha (B.1.1.7), or Delta (B.1.617.2) spike sequences after 48 hr of 1 μg/mL doxycycline induction. Legend, Z-scaled VST counts.

Article Snippet: Gene inserts from pDONR221-EGFP (Addgene #25899), pDONR223 SARS-CoV-2 spike (Addgene #149329), B.1.1.7 SARS-CoV-2 spike (Sino Biological, VG40771-CF), and B.1.617.2 SARS-CoV-2 spike (Sino Biological, VG40804-UT) sequences were subcloned into pInducer20 (Addgene #44012) via Gateway cloning. pLenti-HiBiT-Blast was generated by swapping out the dCas9-VP64-T2A insert of lenti-dCas9-VP64-Blast (Addgene #61425) by Q5 site-directed mutagenesis (NEB). pLenti-LgBiT-T2A-Blast was generated by subcloning the LgBiT coding sequence from the FRB-LgBiT plasmid (Promega N2016) into the lenti-dCas9-VP64-Blast plasmid.

Techniques: Transduction, Construct, RNA Sequencing Assay, Expressing, Western Blot, Comparison

Journal: bioRxiv

Article Title: Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53

doi: 10.1101/2023.08.31.555625

Figure Lengend Snippet: A. Top, western blotting analysis of A549ACE2 cells infected with VSVpp-SARS-CoV-2-S (VSVpp-S) displaying the indicated SARS-CoV-2 spike sequences for 24 hr. A549ACE2 pInducer20 cells transduced with the Delta spike sequence (pInd20-Delta) and treated with or without 1 μg/mL doxycycline (Dox) for 24 hr are shown for comparison. Relative ratios of TP53/ACTB (normalized to Delta +Dox) are annotated below TP53. Bottom, representative brightfield images of A549ACE2 cells infected with the corresponding VSVpp-SARS-CoV-2-S viruses for 24 hr. Scale bar, 200 μm. B. Top, western blotting analysis of 1:1 mixtures of A549 and A549ACE2 pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL Dox for 48 hr. Relative ratios of TP53/ACTB (normalized to Delta +Dox) are annotated below TP53. Bottom, representative brightfield images of A549/A549ACE2 pInducer20 cell mixtures as above treated with or without 1 μg/mL Dox for 48 hr. Scale bar, 200 μm. C. Top, western blotting analysis of A549ACE2 pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL Dox and with the corresponding amount of neutralizing antibody (NAb, μg/mL, Sino Biological #40592-R001) for 24 hr. Relative ratios of TP53/ACTB (normalized to Delta +Dox) are annotated below TP53. Bottom, representative brightfield images of A549ACE2 pInducer20-Delta spike cells treated with or without 1 μg/mL Dox and with the corresponding amount of neutralizing antibody (μg/mL) for 24 hr. Scale bar, 200 μm. D. Top, western blotting analysis of A549ACE2 pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL Dox and with the corresponding amount of CMK (μM) for 24 hr. Relative ratios of TP53/ACTB (normalized to Delta +Dox) are annotated below TP53. Bottom, representative brightfield images of A549ACE2 pInducer20-Delta spike cells treated with or without 1 μg/mL Dox and with the corresponding amount of CMK (μM) for 24 hr. Scale bar, 200 μm. E. Top, western blotting analysis of VeroE6 cells 48 hr after transfection with plasmids encoding the indicated FLAG-tagged proteins. Relative ratios of TP53/ACTB (normalized to EV) are annotated below TP53. Bottom, cell-cell fusion analysis of VeroE6 cells transfected with the corresponding spike proteins. S-2P/D-2P, “2P” mutant of the spike which is incapable of membrane fusion. The Welch two-sample t-test with Holm p-value correction was used to assess statistical significance relative to the S-2P mutant as negative control: ***, p < 0.001. F. Top, western blotting analysis of A549ACE2 pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL Dox and with the corresponding amount of 25-hydroxycholesterol (μM) for 24 hr. Relative ratios of TP53/ACTB (normalized to Delta +Dox) are annotated below TP53. Bottom, representative brightfield images of A549ACE2 pInducer20-Delta spike cells treated with or without 1 μg/mL Dox and with the corresponding amount of 25-hydroxycholesterol (μM) for 24 hr. Scale bar, 200 μm.

Article Snippet: Gene inserts from pDONR221-EGFP (Addgene #25899), pDONR223 SARS-CoV-2 spike (Addgene #149329), B.1.1.7 SARS-CoV-2 spike (Sino Biological, VG40771-CF), and B.1.617.2 SARS-CoV-2 spike (Sino Biological, VG40804-UT) sequences were subcloned into pInducer20 (Addgene #44012) via Gateway cloning. pLenti-HiBiT-Blast was generated by swapping out the dCas9-VP64-T2A insert of lenti-dCas9-VP64-Blast (Addgene #61425) by Q5 site-directed mutagenesis (NEB). pLenti-LgBiT-T2A-Blast was generated by subcloning the LgBiT coding sequence from the FRB-LgBiT plasmid (Promega N2016) into the lenti-dCas9-VP64-Blast plasmid.

Techniques: Western Blot, Infection, Transduction, Sequencing, Comparison, Construct, Transfection, Mutagenesis, Membrane, Negative Control

Journal: bioRxiv

Article Title: Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53

doi: 10.1101/2023.08.31.555625

Figure Lengend Snippet: A. Representative brightfield images of A549ACE2 TP53ko pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL Dox for 48 hr. Scale bar, 200 μm. B. Top, Western blotting analysis of whole cell lysates of A549ACE2 or A549ACE2 TP53ko pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL Dox for 48 hr. Relative ratios of TP53/ACTB (normalized to EGFP -Dox) are annotated below TP53. Bottom, Western blotting analysis of supernatants (SUP) of the corresponding cell constructs and treatments. C. MSigDB pathway enrichment analysis of differential RNA-seq profiles upon Dox-induced expression (48 hr, 1 μg/mL) of SARS-CoV-2 ancestral or delta spike relative to expression of EGFP in either A549ACE2 or A549ACE2 TP53ko pInducer20 cells. D. Cell viability as assessed by CellTiter-Glo in the indicated A549ACE2 or A549ACE2 TP53ko pInducer20 cells treated with or without 1 μg/mL Dox for 72 hr (mean ± s.e.m.). The Welch two-sample t-test with Holm p-value correction was used to assess statistical significance: ***, p < 0.001; **, p < 0.01. E. RNA levels of CDKN1A by RT-qPCR in the indicated A549ACE2 or A549ACE2 TP53ko pInducer20 cells treated with or without 1 μg/mL doxycycline (dox) for 48 hr (mean ± s.e.m.). The Welch two-sample t-test with Benjamini-Hochberg p-value correction was used to assess statistical significance relative to the EGFP -Dox condition: ***, p < 0.001; **, p < 0.01; *, p < 0.05; n.s., not significant. F. Senescence-associated beta-galactosidase activity in the indicated A549ACE2 or A549ACE2 TP53ko pInducer20 cells treated with or without 1 μg/mL Dox for 48 hr (n = 14, mean ± s.e.m.). The Welch two-sample t-test with Holm p-value correction was used to assess statistical significance: **, p < 0.01; n.s., not significant. G. Left, ATAC-seq volcano plots of ancestral or delta spike versus EGFP expression in A549ACE2 pInducer20 cells 48 hr after 1 μg/mL doxycycline induction. Right, number of significant (FDR < 0.05) ATAC-seq peaks upon doxycycline-induced expression (48 hr, 1 μg/mL) of SARS-CoV-2 ancestral or delta spike relative to expression of EGFP in either A549ACE2 or A549ACE2 TP53 KO pInducer20 cells. Black triangles, peaks with increasing accessibility. Red circles, peaks with decreasing accessibility. H. Left, heatmap of relative levels of differentially expressed microRNAs (FDR < 0.05 and baseMean > 10) from ancestral or delta spike versus EGFP expression in A549ACE2 or A549ACE2 TP53ko pInducer20 cells 48 hr after 1 μg/mL doxycycline induction. Right, number of significant (FDR < 0.05) differentially expressed microRNAs upon doxycycline-induced expression (48 hr, 1 μg/mL) of ancestral or delta spike versus EGFP expression in A549ACE2 or A549ACE2 TP53ko pInducer20 cells. Black triangles, significantly upregulated microRNAs. Red circles, significantly downregulated microRNAs. I. Heatmap of relative levels of cytokines in supernatants of A549ACE2 or A549ACE2 TP53ko pInducer20 cells treated with 1 μg/mL doxycycline for 48 hr. The Welch two-sample t-test with Benjamini-Hochberg p-value correction was used to assess statistical significance. Sig, FDR < 0.05. J. Representative brightfield images of HNEpC-ACE2 pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL Dox for 48 hr. Scale bar, 200 μm. K. Western blotting analysis of supernatants of HNEpC-ACE2 pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL Dox for 48 hr.

Article Snippet: Gene inserts from pDONR221-EGFP (Addgene #25899), pDONR223 SARS-CoV-2 spike (Addgene #149329), B.1.1.7 SARS-CoV-2 spike (Sino Biological, VG40771-CF), and B.1.617.2 SARS-CoV-2 spike (Sino Biological, VG40804-UT) sequences were subcloned into pInducer20 (Addgene #44012) via Gateway cloning. pLenti-HiBiT-Blast was generated by swapping out the dCas9-VP64-T2A insert of lenti-dCas9-VP64-Blast (Addgene #61425) by Q5 site-directed mutagenesis (NEB). pLenti-LgBiT-T2A-Blast was generated by subcloning the LgBiT coding sequence from the FRB-LgBiT plasmid (Promega N2016) into the lenti-dCas9-VP64-Blast plasmid.

Techniques: Transduction, Construct, Western Blot, RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Activity Assay

Journal: bioRxiv

Article Title: Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53

doi: 10.1101/2023.08.31.555625

Figure Lengend Snippet: A. MSigDB pathway enrichment analysis of differential RNA-seq profiles upon doxycycline- induced expression (48 hr, 1 μg/mL) of SARS-CoV-2 ancestral, alpha, or delta spike relative to expression of EGFP in VeroE6 pInducer20 cells. B. Cell viability as assessed by CellTiter-Glo of the indicated A549 pInducer20 cells treated with or without 1 μg/mL doxycycline (dox) for 72 hr (mean ± s.e.m.). The Welch two-sample t-test with Holm p-value correction was used to assess statistical significance: **, p < 0.01; n.s., not significant. C. Senescence-associated beta-galactosidase activity in the indicated A549 pInducer20 cells treated with or without 1 μg/mL doxycycline (dox) for 48 hr (mean ± s.e.m.). The Welch two-sample t-test with Holm p-value correction was used to assess statistical significance: *, p < 0.05; n.s., not significant. D. Western blotting analysis of A549ACE2 pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL doxycycline (Dox) and with the corresponding amount of KU-55933 (μM) for 24 hr. Relative ratios of TP53/ACTB (normalized to Delta +Dox) are annotated below TP53. E. Western blotting analysis of A549ACE2 pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL doxycycline (Dox) and with the corresponding amount of berzosertib (μM) for 24 hr. Relative ratios of TP53/ACTB (normalized to Delta +Dox) are annotated below TP53. F. Enrichment of GOBP MAPK Cascade genes in RNA-seq profiles upon expression of SARS-CoV-2 delta spike versus EGFP in either VeroE6 or A549ACE2 pInducer20 cells. Genes are ranked by log2 fold change. NES, normalized enrichment score. p-value, gene set enrichment analysis. G. Top, Western blotting analysis of A549ACE2 pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL doxycycline (Dox) and with 5 μM of SCH772984 or temuterkib for 24 hr. Relative ratios of TP53/ACTB (normalized to Delta +Dox) are annotated below TP53. Bottom, representative brightfield images of A549ACE2 pInducer20-Delta spike cells treated with or without 1 μg/mL doxycycline and with 5 μM of SCH772984 or temuterkib for 24 hr. Scale bar, 200 μm.

Article Snippet: Gene inserts from pDONR221-EGFP (Addgene #25899), pDONR223 SARS-CoV-2 spike (Addgene #149329), B.1.1.7 SARS-CoV-2 spike (Sino Biological, VG40771-CF), and B.1.617.2 SARS-CoV-2 spike (Sino Biological, VG40804-UT) sequences were subcloned into pInducer20 (Addgene #44012) via Gateway cloning. pLenti-HiBiT-Blast was generated by swapping out the dCas9-VP64-T2A insert of lenti-dCas9-VP64-Blast (Addgene #61425) by Q5 site-directed mutagenesis (NEB). pLenti-LgBiT-T2A-Blast was generated by subcloning the LgBiT coding sequence from the FRB-LgBiT plasmid (Promega N2016) into the lenti-dCas9-VP64-Blast plasmid.

Techniques: RNA Sequencing Assay, Expressing, Activity Assay, Western Blot, Transduction, Construct

Journal: bioRxiv

Article Title: Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53

doi: 10.1101/2023.08.31.555625

Figure Lengend Snippet: A. Evolution of SARS-CoV-2 variants in the U.S.A. and the U.K. over the course of the COVID-19 pandemic. Gray, other SARS-CoV-2 variants. Data were last accessed from covariants.org on 4/3/2023 . B. Top, annotated SARS-CoV-2 spike subdomains. Bottom, sequence analysis of SARS-CoV-2 variant spike sequences. Colored boxes, differing sequences from ancestral spike at the given amino acid position. NTD, N-terminal domain; RBD, receptor-binding domain; PBCS, polybasic cleavage site; FP, fusion peptide; HR1/HR2, heptapeptide repeat sequences 1 and 2; TA, transmembrane anchor; CTD, C-terminal domain. C. Western blotting analysis of VeroE6 cells 48 hr after transfection with plasmids encoding the indicated FLAG-tagged proteins. EV, empty vector. Relative ratios of TP53/ACTB (normalized to EV) are annotated below TP53. D. Cell-cell fusion analysis of VeroE6 cells transfected with the corresponding spike proteins. EV, empty vector. The Welch two-sample t-test with Holm p-value correction was used to assess statistical significance relative to EV as negative control: ***, p < 0.001; **, p < 0.01; *, p < 0.05; n.s., not significant. E. Scatterplot of TP53 protein level and fusion effect by SARS-CoV-2 spike variant. R, Pearson correlation. p-value, Pearson correlation test. F. Proposed model of findings.

Article Snippet: Gene inserts from pDONR221-EGFP (Addgene #25899), pDONR223 SARS-CoV-2 spike (Addgene #149329), B.1.1.7 SARS-CoV-2 spike (Sino Biological, VG40771-CF), and B.1.617.2 SARS-CoV-2 spike (Sino Biological, VG40804-UT) sequences were subcloned into pInducer20 (Addgene #44012) via Gateway cloning. pLenti-HiBiT-Blast was generated by swapping out the dCas9-VP64-T2A insert of lenti-dCas9-VP64-Blast (Addgene #61425) by Q5 site-directed mutagenesis (NEB). pLenti-LgBiT-T2A-Blast was generated by subcloning the LgBiT coding sequence from the FRB-LgBiT plasmid (Promega N2016) into the lenti-dCas9-VP64-Blast plasmid.

Techniques: Sequencing, Variant Assay, Binding Assay, Western Blot, Transfection, Plasmid Preparation, Negative Control

Journal: bioRxiv

Article Title: Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray

doi: 10.1101/2020.03.24.006544

Figure Lengend Snippet: Content of coronavirus antigen microarray.

Article Snippet: Influenza , H1N1 , A/Beijing/22808/2009 , HA1+HA2 , ADD64203.1 , HEK293 , Sino Biological , N-(AA1-529)-His-C , 40035-V08H.

Techniques: Microarray

Journal: bioRxiv

Article Title: Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray

doi: 10.1101/2020.03.24.006544

Figure Lengend Snippet: Non-coronavirus respiratory virus antigens on microarray.

Article Snippet: Influenza , H1N1 , A/Beijing/22808/2009 , HA1+HA2 , ADD64203.1 , HEK293 , Sino Biological , N-(AA1-529)-His-C , 40035-V08H.

Techniques: Microarray, Expressing, Construct

Journal: Nature Communications

Article Title: Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography

doi: 10.1038/s41467-021-27611-y

Figure Lengend Snippet: a ELISA screen for identification of potential SARS-CoV-2 RBD binders. Negative control wells containing expression media, and positive control wells containing VNAR E06 (anti-serum albumin) and rabbit monoclonal CR3022-RB (anti-SARS-CoV-2 Spike) are indicated. b Primary screen for identification of neutralizing VNAR domains. Concentration-dependent neutralization of pseudotyped SARS-CoV-2 (black) or SARS-CoV-1 (red) in HEK293T cells transiently expressing ACE2. Data represents mean ± s.e.m. relative luminescence units (RLUs) from n = 3 independent biological experiments. c Left , rank-ordered IC 50 values for neutralizing VNARs from panel ( b ). VNARs with IC 50 < 10 nM (dashed line) were selected for further characterization. Upper right, depiction of primary sequences of selected VNARs, relative length of complementarity determining regions (CDR1, CDR3) and location of cysteine residues (teal) are shown. d Phylogenetic tree of selected virus taxa, divergent lineages of betacoronaviruses are shown. Glycoproteins encoded by the indicated viruses (*) were used to generate pseudoviruses. e Heatmap summarizing IC 50 values for neutralization of the indicated pseudovirus with the indicated VNAR antibody. Values are derived from experiments described in ( f ). f Secondary validation of selected neutralizing VNAR domains. Concentration-dependent neutralization of viral particles pseudotyped with glycoproteins natively encoded by either SARS-CoV-2 (black), SARS-CoV-1 (red), WIV1-CoV (blue). MERS-CoV (green), or VSV (purple) in Calu-3 cells. Cell viability was also assessed in the presence of increasing concentrations of VNARs (yellow). Data represents mean ± s.e.m. RLU values from n = 3 independent biological experiments. g Concentration-dependent neutralization of replication-competent authentic SARS-CoV-2, strain USA_WA1/2020 in Vero E6 cells. Data represents mean ± s.e.m. RFU values from n = 3 independent biological experiments.

Article Snippet: Plasmids used for generating pseudovirus stocks were sourced as follows: plasmid encoding an Env -defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.R-E − ) , was obtained through the NIH AIDS Reagent Program; plasmids encoding SARS-CoV-1 and SARS-CoV-2 Spike were from Fang Li (Addgene plasmid #145031 and #145032, respectively) ; WIV1-CoV Spike was from Alejandro Balazs (Addgene plasmid #164439) ; MERS-CoV Spike was from Sino Biological (VG40069-NF); and VSV-G was from Bob Weinberg (Addgene plasmid #8454).

Techniques: Enzyme-linked Immunosorbent Assay, Negative Control, Expressing, Positive Control, Concentration Assay, Neutralization, Derivative Assay

Journal: Nature Communications

Article Title: Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography

doi: 10.1038/s41467-021-27611-y

Figure Lengend Snippet: a Sequence alignment between SARS-CoV-1, SARS-CoV-2, and the MERS RBDs. Residues different from SARS-CoV-2 are highlighted in red and the interaction interface for VNAR-3B4 is marked with a blue box. Bolded letters indicate residues critical for the interaction between the RBD and 3B4 with arrows indicating the residues that form a backbone beta-sheet. The sequence alignment is numbered above according to SARS-CoV-2. b Surface representations of SARS-CoV-1 (pink, above) and MERS (green, below) with variant residues colored red. The ACE2 binding interface is highlighted in purple for SARS-CoV-1 and the DPP4 binding interface in orange for MERS. The homology-modeled interaction interface for 3B4 is colored blue for both structures. c Zoomed in view of the modeled interaction interface between 3B4 (blue) and SARS-CoV-1 (pink, above) and MERS (green, below), with 3B4 colored blue in both pictures. Interacting residues are highlighted as in Fig. , showing the backbone interactions in black dashes and sidechain to backbone or sidechain to sidechain interactions shown as yellow dashes. Insets show the orientation of the zoomed in view. d Overlays of the 3B4 interface from modeled RBDs and their matching RBDs obtained by x-ray crystallography. The panel above shows the modeled SARS-CoV-1 RBD, colored pink, aligned with the crystal structure of SARS-CoV-1 RBD bound to ACE2 (PDB id: 2AJF), colored magenta. The panel below shows the modeled MERS RBD, colored light green, aligned with the crystal structure of MERS RBD bound to DPP4 (PDB id: 4L72), colored dark green.

Article Snippet: Plasmids used for generating pseudovirus stocks were sourced as follows: plasmid encoding an Env -defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.R-E − ) , was obtained through the NIH AIDS Reagent Program; plasmids encoding SARS-CoV-1 and SARS-CoV-2 Spike were from Fang Li (Addgene plasmid #145031 and #145032, respectively) ; WIV1-CoV Spike was from Alejandro Balazs (Addgene plasmid #164439) ; MERS-CoV Spike was from Sino Biological (VG40069-NF); and VSV-G was from Bob Weinberg (Addgene plasmid #8454).

Techniques: Sequencing, Variant Assay, Binding Assay