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: Briefly, HEK293T cells that were ∼80% confluent were co-transfected with pHR-CMV-firefly luciferase plasmid, psPAX2, and SARS-CoV-2 S plasmid (Addgene #145032) using Lipofectamine 3000 transfection reagent (Invitrogen, USA).

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: Steroidal saponins and triterpenoid saponins with a phenyl moiety have a limited inhibitory activity against SARS-CoV-2 infection. (A) Chemical structures and the effects of the steroidal saponins obtained from A. cochinchinensis on the entry of pSARS-CoV-2 into ACE2 + H1299 cells. (B) Chemical structures and the effects of onjisaponins obtained from P. tenuifolia on the entry of pSARS-CoV-2 into ACE2 + H1299 cells. The data from pSARS-CoV-2 entry assay were representative of two or three independent experiments. The error bars indicate the SEM (n > 3).

Article Snippet: Briefly, HEK293T cells that were ∼80% confluent were co-transfected with pHR-CMV-firefly luciferase plasmid, psPAX2, and SARS-CoV-2 S plasmid (Addgene #145032) using Lipofectamine 3000 transfection reagent (Invitrogen, USA).

Techniques: Activity Assay, Infection

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: Briefly, HEK293T cells that were ∼80% confluent were co-transfected with pHR-CMV-firefly luciferase plasmid, psPAX2, and SARS-CoV-2 S plasmid (Addgene #145032) using Lipofectamine 3000 transfection reagent (Invitrogen, USA).

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: Briefly, HEK293T cells that were ∼80% confluent were co-transfected with pHR-CMV-firefly luciferase plasmid, psPAX2, and SARS-CoV-2 S plasmid (Addgene #145032) using Lipofectamine 3000 transfection reagent (Invitrogen, USA).

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: Briefly, HEK293T cells that were ∼80% confluent were co-transfected with pHR-CMV-firefly luciferase plasmid, psPAX2, and SARS-CoV-2 S plasmid (Addgene #145032) using Lipofectamine 3000 transfection reagent (Invitrogen, USA).

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: Briefly, HEK293T cells that were ∼80% confluent were co-transfected with pHR-CMV-firefly luciferase plasmid, psPAX2, and SARS-CoV-2 S plasmid (Addgene #145032) using Lipofectamine 3000 transfection reagent (Invitrogen, USA).

Techniques: Infection, Recombinant, Expressing

Journal: Cell Reports

Article Title: Cryo-EM structure of the SARS-CoV-2 Omicron spike

doi: 10.1016/j.celrep.2022.110428

Figure Lengend Snippet:

Article Snippet: pαH vector , https://www.addgene.org/154754/ , Cat# 154754.

Techniques: Recombinant, Expressing, Plasmid Preparation, Variant Assay, Software

Journal: Cell

Article Title: The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity

doi: 10.1016/j.cell.2020.07.012

Figure Lengend Snippet:

Article Snippet: Plasmid:pcDNA3.1.S2 (codon-optimized S gene of SARS-CoV-2, GenBank: MN_908947 ) , , Addgene ID: 149457.

Techniques: Virus, Reporter Gene Assay, Mutagenesis, Recombinant, Plasmid Preparation, Software

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet: The Frequency of the SARS-CoV-2 S Protein D614G Variant over the Course of the Pandemic Has Increased Nearly to Fixation (A) The global frequency of the S protein D614G variant over time in the GISAID SARS-CoV-2 database as of June 25, 2020. The filled blue plot represents a seven-day rolling average of the fraction of sequences bearing the D614G variant for each collection date. Dates without published sequences are linearly interpolated. The overlaid black line shows the cumulative frequency of D614G in sequences collected up to and including each date. (B) The frequency of the D614G variant over time (blue) in sequences collected from six continental regions, using the same dataset as in (A), plotted as a seven-day rolling average. The frequency of the last date with data is carried forward where recent dates lack data to indicate the most recent calculated frequency (light blue). Red bars show the number of sequences that were used to provide the denominator for calculating the frequency for each date.

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Variant Assay

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet: SARS-CoV-2 D614G S Protein Variant Enhances Infectivity of Pseudotyped Lentiviruses in Cell Culture (A) Lentiviral virions bearing either GFP or Luciferase transgenes, and pseudotyped with either SARS-CoV-2 D614 or D614G S proteins, were produced by transfection of HEK293 cells and used to transduce human Calu3 lung cells, Caco2 colon cells, and either HEK293 or SupT1 cells stably expressing ACE2 and TMPRSS2. Relative infectivity of D614G versus D614, with D614 set at one, was determined based on flow cytometry for percent GFP positivity or on bulk luciferase activity. Each point represents the mean ± SD for transduction with lentiviral stocks derived from independent transfections, each value of which is the mean of three technical replicates. P values are the ratio paired t test (two-tailed). (B) Lentiviral virions bearing a luciferase transgene, pseudotyped with either SARS-CoV-2 D614 or D614G S proteins, were produced by transfection of HEK293 cells and used to transduce human HEK293 cells transiently transfected with plasmids encoding the indicated ACE2 orthologs. Relative infectivity of D614G versus D614, with D614 set at one, was determined based on bulk luciferase activity. Each point represents the mean ± SD after transduction by using lentiviral stock derived from an independent transfection, each of which is the mean of three technical replicates. P values are ratio paired t test (two-tailed).

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Variant Assay, Infection, Cell Culture, Luciferase, Produced, Transfection, Transduction, Stable Transfection, Expressing, Flow Cytometry, Activity Assay, Derivative Assay, Two Tailed Test

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet: SARS-CoV-2 D614G S Protein Variant Binds ACE2 Weaker than the Ancestral Protein (A–D) SPR measurement of D614-ACE2 binding ([A] and [C]) and D614G-ACE2 binding ([B] and [D]) at 25°C ([A] and [B]) or 37°C ([C] and [D]). (E) Summary of kinetic parameters measured in (A)–(D). D614G binds ACE2 5-fold weaker than D614 at both temperatures tested.

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Variant Assay, Binding Assay

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet: Neutralization Potency of Monoclonal Antibodies Targeting the SARS-CoV-2 S Protein Receptor-Binding Domain Is Not Attenuated by D614G (A and B) Vero cells were challenged with pVSV-SARS-CoV-2-S-mNeon pseudoparticles encoding either D614 (A) or D614G (B) S protein variants in the presence of serial dilutions of the indicated human monoclonal antibodies targeting the SARS-CoV-2 S protein receptor-binding domain or IgG1 isotype control. mNeon protein fluorescence was measured 24 h post-infection as a readout for virus infectivity. Data are graphed as percent neutralization relative to virus only infection control. Data represent the mean ± SD of three technical replicates. (C) Neutralization potency (IC 50 ) of individual monoclonals and of combinations of monoclonals, against the SARS-CoV-2 D614G and D614G S protein variants, as indicated.

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Neutralization, Bioprocessing, Binding Assay, Control, Fluorescence, Infection, Virus

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet: Summary of Cryo-EM Data Collection, Three-Dimensional Reconstruction, and Model Refinement

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Imaging

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet: Structural Determination of Spike D614G, Related to Figure 6 (A) Size exclusion column elution profiles of D614G (blue line) and D614 (red line). (B) Coomassie-stained SDS-PAGE of the peaks collected in (A) shows equivalent, full-length monomeric S proteins at around 180 kD. (C) A still frame from a raw micrograph movie. Individual particles can be clearly visualized. (D) 2D-clustering of trimeric D614G particles. Dagger denotes a top-view. Asterisk denotes a side-view. (E) Fourier shell correlation diagram for D614G (unmasked). (F) Half map Fourier shell correlation diagram (unmasked). (G) Model to map Fourier shell correlation diagram. (H) Local resolution of the ensemble map for Spike D614G.

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Staining, SDS Page

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet: Structural Determination of Spike D614G (A) D614G envelope from the three-dimensional reconstruction. EMDB: EMD-22301. (B) Published D614 envelope (EMD-21452). Arrows point to the density corresponding to the receptor-binding domains, which is missing in the corresponding positions in (A). (C) Domain arrangement of the SARS-CoV-2 S protein. (D) Atomic model for D614G without the receptor-binding domain. PDB: 6XS6 . (E and F) Comparison of the D614G S1 subunit with the closed conformation (E) and open conformation (F) of the D614 S1 subunit. Arrows indicate the relative movement of the S1 subunit of D614G. (G) Position of amino acid 614 on the S protein. (H and I) Substitution of Asp614 with glycine changes hydrogen bonding around residue 614. In the case of D614 (H), an inter-protomer hydrogen bond is detected. For D614G (I), the Asp614-Thr859 hydrogen bond is eliminated, and interaction with intradomain Ala647 is strengthened. See also and .

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Binding Assay, Comparison, Residue

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet: Structural Determination of Spike D614G, Related to Figure 6 (A) Representative density map detail of an alpha helix region. (B) Representative density map detail of a loop region. (C) Representative density map detail of a beta strand region. Note: the relative positioning between (B) and (C) is the same as in the real structural model. No cryo-EM density is observed in between the loop where Gly614 resides and the beta strand where Thr859 resides.

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Cryo-EM Sample Prep

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet: D614G Populates More Open Conformations Than Does the Ancestral S Protein (A) Cryo-EM density maps of the two conformations of D614G protomer. The first is a closed conformation with a buried RBD. The second is an open conformation with the RBD standing up. (B) Atomic models for the closed (left) and open (right) conformations for the two D614G protomers shown in (A). (C) Comparison of the two D614G protomer S1 subunit conformations with the corresponding conformations of the D614 protomer S1 subunit. (D) The D614G S protein trimer adopts four conformations. In addition to the all-closed and one open conformation detected with the D614 S protein trimer, the D614G S protein trimer adopts two-open and three-open conformations.

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Cryo-EM Sample Prep, Comparison

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet:

Article Snippet: pcDNA3.1 SARS-CoV-2 S D614G , This publication , Addgene #158075.

Techniques: Binding Assay, Virus, Recombinant, Transfection, Luciferase, Bradford Protein Assay, Plasmid Preparation, Software, Single Particle, Microscopy