Journal: Antiviral Research

Article Title: Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibit the infection of SARS-CoV-2 variants and syncytium formation

doi: 10.1016/j.antiviral.2022.105428

Figure Lengend Snippet: Astersaponin I from Aster koraiensis inhibits SARS-CoV-2 infection. (A) The effects of various plant extracts on the entry of SARS-CoV-2 pseudovirus (pSARS-CoV-2) into ACE2 + and ACE2/TMPRSS2 + H1299 cells. (B, C) pSARS-CoV-2 entry and cell viability assays for the 70% EtOH extract of Aster koraiensis (B) and triterpenoid saponins isolated from A. koraiensis (C) in ACE2 + and ACE2/TMPRSS2 + H1299 cells. The data from pSARS-CoV-2 entry assay were representative of three independent experiments. The error bars indicate the SEM (n > 3). P values were determined by one-way ANOVA followed by Tukey's post hoc test. *P < 0.05; **P < 0.01, ****P < 0.0001.

Article Snippet: TMPRSS2 lentiviral expression vector, RRL.sin.cPPT.SFFV/TMPRSS2 (variant 1).IRES-neo.WPRE (MT130) was gifted by Caroline Goujon (Addgene plasmid # 145843).

Techniques: Infection, Isolation

Journal: Antiviral Research

Article Title: Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibit the infection of SARS-CoV-2 variants and syncytium formation

doi: 10.1016/j.antiviral.2022.105428

Figure Lengend Snippet: Astersaponin I isolated from Aster koraiensis blocks viral membrane fusion with the host cell membrane. (A) Schematic illustration of the binding of SARS-CoV-2 spike receptor binding domain (RBD) fused to GFP (RBD-GFP) to ACE2 protein overexpressed in H1299 cells. (B) Examination of the effect of AK extract and AI on the interaction between RBD-GFP and ACE2 on the surface of H1299 cells by flow cytometry after treatment with indicated concentration of AK extract and AI. The grey peaks indicate the control experiments without RBD-GFP addition. (C) ACE2 + H1299 cells were treated with 1 or 5 μM AI for 1 h and washed with fresh media (Wash) or maintained (No wash), followed by the addition of pSARS-CoV-2. The data from pSARS-CoV-2 entry assay were representative of two independent experiments. The error bars indicate the SEM (n > 3). P values were determined by the unpaired, two-tailed Student's t-test (NS, not significant). (D) Schematic illustration of the SARS-CoV-2 S protein-mediated cell fusion. Addition of the cell suspension of H1299 cells stably expressing S protein and GFP (Spike-H1299) to a monolayer of ACE2/TMPRSS2 + H1299 cells with mRuby fluorescence (ACE2/TMPRSS2-H1299) leads to cell-cell fusion. (E) Still images at different time points from time-lapse imaging of S-mediated cell fusion. (F) The fusion between Spike-H1299 and ACE2/TMPRSS2 + H1299 was determined by counting the number of cells double-positive for GFP and mRuby by flow cytometry. H1299 cells expressing only GFP (no spike) were used for control experiment. All compounds were used at the concentration of 10 μM. The data were representative of three independent experiments. (G, H) Filipin cholesterol staining of ACE2 + after treatment with DMSO, AI (5 μM), and other indicated compounds (10 μM) for 1 h (G) and quantification of the intensity of membrane filipin staining using Image J software (n = 20 for each group). Error bars in the graphs indicate the SEM. P values were determined by one-way ANOVA followed by Tukey's post hoc test. ****P < 0.0001; NS not significant (H).

Article Snippet: TMPRSS2 lentiviral expression vector, RRL.sin.cPPT.SFFV/TMPRSS2 (variant 1).IRES-neo.WPRE (MT130) was gifted by Caroline Goujon (Addgene plasmid # 145843).

Techniques: Isolation, Binding Assay, Flow Cytometry, Concentration Assay, Two Tailed Test, Stable Transfection, Expressing, Fluorescence, Imaging, Staining, Software

Journal: Antiviral Research

Article Title: Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibit the infection of SARS-CoV-2 variants and syncytium formation

doi: 10.1016/j.antiviral.2022.105428

Figure Lengend Snippet: Astersaponin I prevents SARS-CoV-2 S-induced syncytia formation. (A) Schematic illustration of Split-GFP assay. Ectopic expression of SARS-CoV-2 spike protein into cultures of the mixture of ACE2/TMPRSS2 + H1299 cells expressing GFP1-10 or GFP11 leads to cell-cell fusion, generating GFP fluorescence. (B) Images of the S-mediated cell-to-cell fusion using Split-GFP. GFP and blue fluorescence indicate the cell-to-cell fusion and DAPI-stained nuclei, respectively. The nuclei are automatically pseudocolored in white and overlapped with GFP fluorescence by CellReporterXpress software. (C) Examination of effect of AI on the S-mediated cell-cell fusion using Split-GFP assay. Representative images of GFP-positive cell-to-cell fusion. The GFP signal and DAPI nuclei staining are automatically pseudocolored in green and red by CellReporterXpress software. (D) Quantitative evaluation for cell-cell fusion using Split-GFP assay. Images of GFP and nuclei stained with DAPI were obtained in five random fields per well. The percentage of fusion cells were calculated by dividing number of nuclei in GFP-positive cells by total number of nuclei. The data were representative of three independent experiments. The error bars indicate the SEM (n > 3). P values were determined by one-way ANOVA followed by Tukey's post hoc test. *P < 0.05; ****P < 0.0001; NS not significant. (E) Protein lysates from co-culture experiments were assessed by Western blot. GAPDH was used as a loading control.

Article Snippet: TMPRSS2 lentiviral expression vector, RRL.sin.cPPT.SFFV/TMPRSS2 (variant 1).IRES-neo.WPRE (MT130) was gifted by Caroline Goujon (Addgene plasmid # 145843).

Techniques: Split GFP Assay, Expressing, Fluorescence, Staining, Software, Co-Culture Assay, Western Blot

Journal: Antiviral Research

Article Title: Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibit the infection of SARS-CoV-2 variants and syncytium formation

doi: 10.1016/j.antiviral.2022.105428

Figure Lengend Snippet: Astersaponin I equally inhibits the infection of SARS-CoV-2 WT and D614G mutant. (A) pSARS-CoV-2 entry assay in ACE2 + and ACE2/TMPRSS2 + H1299 cells. The data were representative of two independent experiments. The error bars indicate the SEM (n > 3). P values were determined by the unpaired, two-tailed Student's t-test (****P < 0.0001). (B) The effects of AI on the infection of WT and D614G mutant of pSARS-CoV-2 in ACE2 + and ACE2/TMPRSS2 + H1299 cells. The data were representative of three independent experiments. The error bars indicate the SEM (n > 3).

Article Snippet: TMPRSS2 lentiviral expression vector, RRL.sin.cPPT.SFFV/TMPRSS2 (variant 1).IRES-neo.WPRE (MT130) was gifted by Caroline Goujon (Addgene plasmid # 145843).

Techniques: Infection, Mutagenesis, Two Tailed Test

Journal: Antiviral Research

Article Title: Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibit the infection of SARS-CoV-2 variants and syncytium formation

doi: 10.1016/j.antiviral.2022.105428

Figure Lengend Snippet: Astersaponin I inhibits the infection of SARS-CoV-2 and its variants with a similar efficiency. (A, C) Dose-response curves for AI (A) and chloroquine (C) against ancestral SARS-CoV-2 (A, black circle) and SARS-CoV-2 variants Alpha (B.1.1.7, red square), Beta (B.1.351, orange triangle), Delta (B.1.617.2, pink inverted triangle), and Omicron (B.1.1.529, green diamond) in Vero cells. The blue diamond represents cell viability. The mean ± SEM was calculated from duplicate experiments. (B, D) Confocal images of SARS-CoV-2 N protein (green) and cell nuclei (de Vries et al.) at concentrations near the IC 50 of AI (B) and chloroquine (D) in Vero cells. (E) Dose-response curve for AI against replicable SARS-CoV-2 recombinant viruses expressing Nanoluciferase (Nluc) into A549 cells that overexpress ACE2 and TMPRSS2. The blue diamond represents cell viability.

Article Snippet: TMPRSS2 lentiviral expression vector, RRL.sin.cPPT.SFFV/TMPRSS2 (variant 1).IRES-neo.WPRE (MT130) was gifted by Caroline Goujon (Addgene plasmid # 145843).

Techniques: Infection, Recombinant, Expressing

Journal: Cell

Article Title: High-throughput sequencing of single neuron projections reveals spatial organization in the olfactory cortex.

doi: 10.1016/j.cell.2022.09.038

Figure Lengend Snippet: Figure 1. MAPseq and BARseq projection mapping of individual olfactory bulb neurons (A) Schematics of the MAPseq strategy which uses RNA barcodes to label neurons and map their brain-wide projections. (B) Infection of mitral and tufted cells by Sindbis virus carrying the barcodes and a fluorophore (EGFP). (C) Laser Capture Micro-Dissection of target brain regions from Nissl stained coronal sections registered to the Allen Brain reference atlas. (D) Illustration of laminar positions of mitral, tufted, and deep cells (left) and an example BARseq sequencing image of the barcoded cells (center). The first several bases of the barcode sequences in two example neurons analyzed via BARseq and their projection patterns across 6 bulb target regions (right). Scale bar = 100mm. (E) (Left) projection patterns (415 neurons, 2 mice) identified via BARseq and their soma locations relative to the mitral cell layer (MCL). Columns represent bulb projection target regions and rows indicate individual neurons. (right) Cell identities based on soma positions. Projection strength of each barcoded neuron has been normalized so that the maximum strength is 1 (row). (F) (Left) Soma positions of template neurons shown relative to MCL (y axis) and to glomerular layer (x axis) that were used to train the projection-based classifier. The sectioning planes are not necessarily perpendicular to the mitral cell layer, and thus the distances measured may be inflated. Neuronal identity (colors) is based on laminar positions (tufted, mitral, and deep cells). (Right) Classification confusion matrix of all three cell classes using the BARseq-based classifier versus position-defined classes. See also Figures S1 and S2.

Article Snippet: Barcoded Sindbis viral library Abarcoded Sindbis virus library (JK100L2, Addgene plasmid #79785) was generated and used forMAPseq andBARseq experiments as described previously (Chen et al., 2019; Han et al., 2018; Huang et al., 2020; Kebschull et al., 2016).

Techniques: Infection, Virus, Dissection, Staining, Sequencing

Journal: Research Square

Article Title: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs

doi: 10.21203/rs.3.rs-555275/v1

Figure Lengend Snippet: a. Schematic of pooled screen to identify SARS-CoV-2 regulators in Vero E6 cells. b. Scatter plot showing the gene-level mean z-scores of genes when knocked out in Vero E6 cells. The top genes conferring resistance to SARS-CoV-2 are annotated and shown in blue. c. Comparison between this Vero E6 screen to the Vero E6 screen conducted by the Wilen lab 27. Genes that scored among the top 20 resistance hits and sensitization hits in both screens are labeled. d. Venn diagram comparing hits across screens conducted in Vero E6, A549, and Huh7 (or derivatives) cells (ectopically expressing ACE2 and TMPRSS2 or not) 23–28. The top 20 genes from each cell line are included, with genes considered a hit in another cell line if the average z-score was > 3.

Article Snippet: The lentiviral vector expressing ACE2 (pRRL.sin.cPPT.SFFV/ACE2, Addgene 145842) has been described .

Techniques: Comparison, Labeling, Expressing

Journal: Research Square

Article Title: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs

doi: 10.21203/rs.3.rs-555275/v1

Figure Lengend Snippet: Properties of SARS-CoV-2 host factor screens assayed by cell viability. For each library, the number of unique guides per gene is indicated in parentheses. The essential gene QC serves as a metric for screen quality (see Methods ) in the untreated arm, when applicable; the number of days post-library introduction until the end of the experiment is written after the semicolon.

Article Snippet: The lentiviral vector expressing ACE2 (pRRL.sin.cPPT.SFFV/ACE2, Addgene 145842) has been described .

Techniques:

Journal: Research Square

Article Title: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs

doi: 10.21203/rs.3.rs-555275/v1

Figure Lengend Snippet: Calu-3-Cas9 cells were stably transduced to express 2 different sgRNAs (g1, g2) per indicated gene and selected for 10–15 days. a. Cells were infected with SARS-CoV-2 bearing the mNG reporter and the infection efficiency was scored 48h later by flow cytometry. b. The expression levels of ACE2 were analyzed by immunoblot, Actin served as a loading control. c. Relative surface ACE2 expression was measured using a Spike-RBD-Fc fusion and a fluorescent secondary antibody followed by flow cytometry analysis. d. Cells were incubated with SARS-CoV-2 at MOI 5 for 2h at 37°C and then treated with Subtilisin A followed by RNA extraction and RdRp RT-qPCR analysis as a measure of viral internalization. e. Cells were infected with Spike del19 and VSV-G pseudotyped, GFP expressing VSV and infection efficiency was analyzed 24h later by flow cytometry. f. Cells were infected with SARS-CoV-2 at MOI 0.05 and, 24h later, lysed for RNA extraction and RdRp RT-qPCR analysis. g. Aliquots of the supernatants from F were harvested and plaque assays were performed to evaluate the production of infectious viruses in the different conditions. h. Cells were infected with MERS-CoV and 16h later, infectious particle production in the supernatant was measured by TCID 50 . The mean and SEM of at least 5 ( a ), 3 ( c , d , e , f , h ) independent experiments or representative experiments ( b and g ) are shown. The red dashed line represents 50% inhibition ( a , c-f ).

Article Snippet: The lentiviral vector expressing ACE2 (pRRL.sin.cPPT.SFFV/ACE2, Addgene 145842) has been described .

Techniques: Stable Transfection, Infection, Flow Cytometry, Expressing, Western Blot, Control, Incubation, RNA Extraction, Quantitative RT-PCR, Inhibition

Journal: Research Square

Article Title: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs

doi: 10.21203/rs.3.rs-555275/v1

Figure Lengend Snippet: sgRNA sequences used for CRISPR KO perturbations

Article Snippet: The lentiviral vector expressing ACE2 (pRRL.sin.cPPT.SFFV/ACE2, Addgene 145842) has been described .

Techniques: CRISPR, Sequencing

Journal: Research Square

Article Title: Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs

doi: 10.21203/rs.3.rs-555275/v1

Figure Lengend Snippet: sgRNA sequences used for CRISPRa perturbations

Article Snippet: The lentiviral vector expressing ACE2 (pRRL.sin.cPPT.SFFV/ACE2, Addgene 145842) has been described .

Techniques: Sequencing