Journal: Cell Reports

Article Title: Dynamic Ca 2+ sensitivity stimulates the evolved SARS-CoV-2 spike strain-mediated membrane fusion for enhanced entry

doi: 10.1016/j.celrep.2022.110694

Figure Lengend Snippet: Ca 2+ triggers robust SARS-CoV-2 D614G spike-mediated virus-liposome lipid mixing compared to WT spike at acidic pH (A) Pseudovirions formed with SARS-CoV-2 WT spike or D614G spike were labeled with a self-quenching lipophilic fluorophore DiO . Liposomes coated with polyhistidine-tagged hACE2 were incubated with pseudovirion followed by adjustment to the desired pH. Where indicated, 0.5 mM CaCl 2 was included into the reaction mixer. (B) Fluorescence dequenching of DiO was monitored over a 20-min period at the indicated pH and presented as the fold increase over initial fluorescence ( F / F 0 ). Dequenching was observed only in the presence of Ca 2+ with hACE2 at pH 4.6 for both the WT (blue) and D614G spike (green). Maximal dequenching was observed for D614G (green). The data are shown as an average of four independent measurements. (C) The degree of fusion for WT and D614G with hACE2 at pH 4.6 in presence or absence of Ca 2+ has been normalized to fusion efficiency in the presence of 1% Triton X, which was set to 100% ( Table S1 ). (D) The fusion index is the extent of fusion at 20 min after the start of the fusion reaction and is plotted as indicated for conditions (n = 4 independent experiments; error bar represents standard mean). The p values by two-tailed Student’s t test are indicated. p value < 0.0001 is indicated as ( ∗ ), and p value < 0.00001 is indicated as ( ∗∗ ). (E) Pseudovirions of SARS-CoV-2 spike or D614G spike were mixed with liposomes coated with polyhistidine-tagged NRP1-b1, followed by adjustment to the desired pH, in the presence or absence of 0.5 mM CaCl 2 into the reaction mixer. (F) Fluorescence dequenching was observed only in the presence of Ca 2+ with NRP1-b1 at pH 5 and 4.6 for both the WT (blue) and D614G spike (green). The data are shown as an average of four independent measurements. (G) D614G spike shows a high degree of fusion compared to WT in the presence of Ca 2+ at low pH and NRP1-b1 ( Table S1 ). (H) The comparative fusion efficiency after 20 min of fusion reaction for WT and D614G at indicated pH and in presence or absence of Ca 2+ (n = 4 independent experiments; error bar represents standard error of mean). The p values by two-tailed Student’s t test are indicated. (I) Lipid mixing assay between SARS-CoV-2 spike or D614G spike pseudovirions and liposomes coated with polyhistidine-tagged TMPRSS2. (J) Increment of fluorescence over initial fluorescence ( F / F 0 ) was observed only in the presence of Ca 2+ at pH 5 and 4.6 for both the WT (blue) and D614G spike (green). The data are shown as an average of four independent measurements. D614G spike demonstrates higher fusion efficiency (K) and fusion index (L) compared to WT spike ( Table S1 ). (n = 4 independent experiments; error bar represents standard error of mean). The p values by two-tailed Student’s t test are indicated.

Article Snippet: SARS-CoV-2 RBD Rabbit PAb , Sino Biological , Cat# 40592-T62; RRID: AB_2857935.

Techniques: Labeling, Incubation, Fluorescence, Two Tailed Test

Journal: Cell Reports

Article Title: Dynamic Ca 2+ sensitivity stimulates the evolved SARS-CoV-2 spike strain-mediated membrane fusion for enhanced entry

doi: 10.1016/j.celrep.2022.110694

Figure Lengend Snippet: Ca 2+ drives the spike-mediated full fusion between virus and liposome, and D614G is highly fusogenic (A) DiO-labeled pseudovirions of SARS-CoV-2 spike (WT) or D614G spike were incubated with proteoliposomes and coated with polyhistidine-tagged hACE2, NRP1-b1, and TMPRSS2 at desired pH and 0.5 mM CaCl 2 , as indicated. (B) Fluorescence dequenching was observed only in the presence of Ca 2+ at pH 5 and 4.6 for both the WT (blue) and D614G spike (green) in presence of all receptors ( Table S1 ). The data are shown as an average of four independent measurements. (C) Normalized fusion efficiency shows D614G spike virion is 2-fold fusogenic compared to the WT spike. (D) Comparative fusion efficacy after 20 min of the fusion reaction for D614G and WT spike in presence of all receptors, at indicated pH and calcium. (n = 4 independent experiments; error bar represents standard error of mean). The p values by two-tailed Student’s t test are indicated. p value < 0.0001 is indicated as ( ∗ ), and p value < 0.00001 is indicated as ( ∗∗ ). (E) Individual receptor’s role in fusion efficacy between D614G and WT in presence of low pH and 0.5 mM CaCl 2 . (n = 4 independent experiments; error bar represents standard error of mean). (F) FRET-based assay for probing the full fusion and inner leaflet fusion between the viral and liposomal membrane. NBD-PE (donor) and Rho-PE (acceptor) lipids were included along with other lipids in the liposome . Virus and proteoliposomes were incubated at desired pH and 0.5 mM CaCl 2 , as indicated. Inner leaflet fusion is being reported only from the dequenching of the donor dye present in the inner leaflet . The full fusion and inner leaflet fusion kinetics were followed for 20 min for D614G virion (G) and WT spike virion (H) ( Table S2 ). The data are shown as an average of three independent measurements. The comparative inner leaflet fusion efficiency in presence individual receptor ACE2, NRP1-b1, and TMPRSS2 with D614G spike virion (I) and WT spike virion (J) at pH 4.6 and 0.5 mM CaCl 2 ( Table S2 ). The data are shown as an average of three independent measurements. (K) D614G spike variant has higher inner layer fusion efficacy in any circumstance. (n = 3 independent experiments; error bar represents standard error of mean). The p values by two-tailed Student’s t test are indicated.

Article Snippet: SARS-CoV-2 RBD Rabbit PAb , Sino Biological , Cat# 40592-T62; RRID: AB_2857935.

Techniques: Labeling, Incubation, Fluorescence, Two Tailed Test, Variant Assay

Journal: Cell Reports

Article Title: Dynamic Ca 2+ sensitivity stimulates the evolved SARS-CoV-2 spike strain-mediated membrane fusion for enhanced entry

doi: 10.1016/j.celrep.2022.110694

Figure Lengend Snippet: Ca 2+ promotes the spike’s fusion peptide for membrane penetration with high efficacy compared to WT (A) Fluorescence anisotropy assay design for probing fusion peptide binding to target membrane by incubating pseudovirions having SARS-CoV2-S ∗ or SARS-CoV2-D614G-S ∗ with the proteoliposome with ACE2/NRP1/TMPRSS2 during triggering with Ca 2+ and low pH 4.6 . (B) The change in anisotropy value in fusion peptide with time during membrane fusion in presence of Ca 2+ and in absence of calcium at low pH 4.6 for WT and D614G spikes. The data are shown as an average of three independent measurements. (C and D) Fluorescence anisotropy measurements were performed at different Ca 2+ concentrations, as indicated, for both WT and D614G spikes. The point at which calcium was added (150 s) is indicated. The drastic anisotropy change indicates the binding of the fusion peptide with the membrane due to calcium stimulation, occurring only at 500 μM Ca 2+ . The data are shown as an average of three independent measurements. (E) The effect of Ca 2+ concentration in anisotropy reflects the fusion peptide binding efficiency with the membrane. At 500 μM Ca 2+ , the fusion peptide binding to membrane is maximum for both the WT and D614G, but D614G spike fusion peptide shows 150% more binding efficiency compared to WT. (n = 3 independent experiments; error bar represents standard error of mean). (F) Amino acid sequences of the fusion loop for SARS-2S are indicated; the fusion loop is comprised of FP1 and FP2, and the acidic amino acids are highlighted (red) as the plausible Ca 2+ binding site. Structural features of pre-fusion trimeric spike SARS-CoV-2 WT (PDB: 6XR8 ) and SARS-CoV-2 D614G spike (PDB: 7KRQ ) and the close-up view of fusion peptide domain (S2 816−855 ) for SARS-CoV-2 WT spike and SARS-CoV-2 D614G spike showing the conformational difference in the loop and different orientation of side chain of D810 and D839 amino acids. (G) Mechanistic model of fusion peptide translocation into membrane, where D614G spike variant can penetrate more into the target membrane due to efficient Ca 2+ binding, compared to WT spike.

Article Snippet: SARS-CoV-2 RBD Rabbit PAb , Sino Biological , Cat# 40592-T62; RRID: AB_2857935.

Techniques: Fluorescence, Binding Assay, Concentration Assay, Translocation Assay, Variant Assay

Journal: Cell Reports

Article Title: Dynamic Ca 2+ sensitivity stimulates the evolved SARS-CoV-2 spike strain-mediated membrane fusion for enhanced entry

doi: 10.1016/j.celrep.2022.110694

Figure Lengend Snippet:

Article Snippet: SARS-CoV-2 RBD Rabbit PAb , Sino Biological , Cat# 40592-T62; RRID: AB_2857935.

Techniques: Recombinant, Mutagenesis, Plasmid Preparation, Software

Journal: PLoS Pathogens

Article Title: Intranasal gene therapy to prevent infection by SARS-CoV-2 variants

doi: 10.1371/journal.ppat.1009544

Figure Lengend Snippet: A. Yeast display (YD) system B . Decoy affinity maturation and candidate selection process. C . Flow cytometry analysis of the naïve (purple) and sorted populations (gray) from the secondary YD library. D . NGS analysis of plasmid populations recovered from rounds of YD. E . Mutations accumulated in the top five decoy-Fc4 candidates. F and G . SPR binding analysis for CoV-2 RBD injected over surface-immobilized ACE2-wt (F) or CDY14HL (G). H . Wuhan CoV-2-Pseudotyped lentiviral reporter neutralization assay of ACE2-wt-Fc4, and CDY14HL-Fc4. Data for at least three independent measurements are presented as average ± standard error of the mean.

Article Snippet: SARS-CoV-2 Spike Protein RBD (Sinobio #40592-V08H) was immobilized on a 96-well plate (0.25ug/mL in PBS, 100ul/well) at 4°C overnight.

Techniques: Selection, Flow Cytometry, Plasmid Preparation, Binding Assay, Injection, Neutralization

Journal: PLoS Pathogens

Article Title: Intranasal gene therapy to prevent infection by SARS-CoV-2 variants

doi: 10.1371/journal.ppat.1009544

Figure Lengend Snippet: A. Structural model (7DF4.pdb ) of Wuhan CoV-2 RBD (yellow) bound to human ACE2 (blue) RBD. The six RBD residues frequently mutated in emerging CoV-2 variants are shown in red spheres. B . Wuhan CoV-2 model as in (A) but with RBD residues highlighted at the ACE2 interface (red spheres) which differ from CoV-1. C . SPR measurements: ACE2-wt-Fc4 or CDY14HL-Fc4 binding to various purified recombinant RBD proteins. D . CDY14HL-Fc4 titrations using pseudotyped lentivirus reporters encoding CoV-2 spike proteins from four US-CDC Variants of Concern. RBD mutations are indicated in brackets, however in many cases other spike mutations exist that are not listed. E . Pseudotype titrations for six additional CoV-2 spike variants. G . Pseudotype titration for CoV-1 spike reporter virus. Reporter virus activity data are presented as mean ± standard error of the mean for at least three replicate titrations.

Article Snippet: SARS-CoV-2 Spike Protein RBD (Sinobio #40592-V08H) was immobilized on a 96-well plate (0.25ug/mL in PBS, 100ul/well) at 4°C overnight.

Techniques: Binding Assay, Purification, Recombinant, Titration, Activity Assay

Journal: PLoS Pathogens

Article Title: Intranasal gene therapy to prevent infection by SARS-CoV-2 variants

doi: 10.1371/journal.ppat.1009544

Figure Lengend Snippet: BAL from vector-treated animals analyzed for: A. decoy protein by MS; B. SARS-CoV-2 spike ELISA; C. neutralization of SARS-CoV-2 pseudotyped lentivirus. D. Challenge study design. E. Weight loss in the animals that were sustained for 7 days; one animal in the vehicle and vector treated groups required euthanasia. F . MS assay of expression in ASF (corrected for BAL dilution). G. Pulmonary inflammation histopathology scores of tissues harvested at days 4 and 7. H. Viral RNA in BAL. I. Viral RNA in lung. J. Sub-genomic RNA in lung. Outliers are indicated with X.

Article Snippet: SARS-CoV-2 Spike Protein RBD (Sinobio #40592-V08H) was immobilized on a 96-well plate (0.25ug/mL in PBS, 100ul/well) at 4°C overnight.

Techniques: Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Neutralization, Expressing, Histopathology