Journal: Virus Evolution

Article Title: Deep mutational scanning of SARS-CoV-2 Omicron BA.2.86 and epistatic emergence of the KP.3 variant

doi: 10.1093/ve/veae067

Figure Lengend Snippet: Deep mutational scanning of the SARS-CoV-2 Omicron BA.2.86 RBD. (a) Diagram of the RBD substitutions that distinguish Omicron BA.2 from Wuhan-Hu-1 (top), and BA.2.86 from BA.2 (bottom). Italicized mutations in BA.2.86 indicate secondarily mutated (D339H, A484K) or reverted (R493Q) substitutions that originally changed from Wuhan-Hu-1, and dashed lines show propagation of BA.2. changes to BA.2.86. Wuhan-Hu-1 reference spike numbering is used throughout the manuscript. (b–d) Quality control of the BA.2.86 RBD site-saturation mutagenesis library as assessed by PacBio sequencing, illustrating the distribution of number of amino acid mutations per barcoded variant (b), the average number of mutations of each type across library variants (c), and the distribution of mutations across sites in the RBD over all variants (d). (e, f) FACS gates used to sort RBD + singlet cells for ACE2 titration (e) and RBD expression (f) deep mutational scanning experiments from one representative replicate. (g, h) Correlation in per-mutant deep mutational scanning measurements between independently barcoded replicate libraries for ACE2-binding affinity (g) and RBD expression (h) experiments.

Article Snippet: Induced cells were washed with phosphate buffered saline supplemented with bovine serum albumin (PBS-BSA , BSA 0.2 mg/l), split into 16-OD*ml aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 −6 to 10 −13 m at 1−log intervals, plus a 0 m sample.

Techniques: Control, Mutagenesis, PacBio Sequencing, Variant Assay, Titration, Expressing, Binding Assay

Journal: Virus Evolution

Article Title: Deep mutational scanning of SARS-CoV-2 Omicron BA.2.86 and epistatic emergence of the KP.3 variant

doi: 10.1093/ve/veae067

Figure Lengend Snippet: Effects of mutations in the BA.2.86 receptor-binding domain on ACE2-binding and RBD expression. (a) Heatmap illustrating the impacts of all mutations in the BA.2.86 RBD on ACE2-binding affinity as determined from FACS-seq experiments with yeast-displayed RBD mutant libraries. ACE2 contact residues (top row of yellow squares, bottom heatmap) defined as RBD residues with non-hydrogen atoms <5 Å from ACE2 in the BA.2.86 RBD structure (PDB 8QSQ; Liu et al. 2024 ). Antibody escape residues (bottom row of orange squares, bottom heatmap) defined as those with average >0.125 relative antibody escape from aggregated deep mutational scanning data ( Greaney et al. 2022a ). (b) Deep mutational scanning data from (a) mapped to the ACE2-bound BA.2.86 RBD structure (PDB 8QSQ; Liu et al. 2024 ), illustrating the average effect of mutations at a site (left), the maximal effect of any mutation at a site (center), or the effect of the single-codon deletion (right). Sites of interest are labeled, and ACE2 (key motifs only) is shown as transparent gray cartoon. (c) Heatmap illustrating the impacts of all mutations in the BA.2.86 RBD on yeast-surface expression levels, a proxy for folding and expression efficiency. (d) Scatterplot illustrating the average effect of mutations at each site on ACE2-binding affinity ( y -axis) versus RBD expression ( x -axis). Yellow points indicate direct structural contacts as in (a). Individual measurements from (a) and (c) are reported in , and an interactive version of these heatmaps is available at https://tstarrlab.github.io/SARS-CoV-2-RBD_DMS_Omicron-EG5-FLip-BA286/RBD-heatmaps/ .

Article Snippet: Induced cells were washed with phosphate buffered saline supplemented with bovine serum albumin (PBS-BSA , BSA 0.2 mg/l), split into 16-OD*ml aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 −6 to 10 −13 m at 1−log intervals, plus a 0 m sample.

Techniques: Binding Assay, Expressing, Mutagenesis, Labeling

Journal: Virus Evolution

Article Title: Deep mutational scanning of SARS-CoV-2 Omicron BA.2.86 and epistatic emergence of the KP.3 variant

doi: 10.1093/ve/veae067

Figure Lengend Snippet: Epistatic shifts in mutational effects on ACE2 binding. (a) Epistatic shift in the effects of mutations on ACE2 binding at each RBD position as measured in the Wuhan-Hu-1 [previously reported in ( Starr et al. 2022a )] or BA.2.86 background compared to those previously measured in Omicron BA.2 ( Starr et al. 2022b ). Shaded gray bars indicate sites of strong antibody escape, as defined in Fig. 2A . (b) Mutation-level plots of epistatic shifts between BA.2 and BA.2.86 at sites of interest. Each scatterplot shows the measured ACE2-binding affinity of each amino acid (plotting character) in the BA.2.86 versus BA.2. backgrounds. Horizontal and vertical red dashed lines mark the wildtype RBD affinities on each axis, and the diagonal gray dashed line indicates the additive (non-epistatic) expectation. Interactive plots enabling the comparison of all SARS-CoV-2 variants and scatterplots for all RBD sites are available at https://tstarrlab.github.io/SARS-CoV-2-RBD_DMS_Omicron-EG5-FLip-BA286/epistatic-shifts/ .

Article Snippet: Induced cells were washed with phosphate buffered saline supplemented with bovine serum albumin (PBS-BSA , BSA 0.2 mg/l), split into 16-OD*ml aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 −6 to 10 −13 m at 1−log intervals, plus a 0 m sample.

Techniques: Binding Assay, Mutagenesis, Comparison

Journal: Virus Evolution

Article Title: Deep mutational scanning of SARS-CoV-2 Omicron BA.2.86 and epistatic emergence of the KP.3 variant

doi: 10.1093/ve/veae067

Figure Lengend Snippet: Epistatic emergence of the KP.3 variant . (a) Cladogram showing relationships among select SARS-CoV-2 Omicron variants, with amino acid substitutions at positions 455, 456, and 493 indicated (other mutations not shown). (b) Triple mutant cycle diagram illustrating epistatic interactions between L455S, F456L, and Q493E underlying KP.3 variant evolution. Transparent points indicate duplicate measurements of each variant’s binding strength for human ACE2 (determined as the EC50 from titrations of monomeric human ACE2 over yeast-displayed RBD variants), and solid points and lines connect the averaged binding values for each genotype. Red-orange lines highlight the impact of introducing the Q493E mutation in different sequence backgrounds. Asterisk indicates expected triple-mutant binding affinity assuming additivity of the single-mutant effects as measured in the BA.2.86 wildtype background. (c) Subset of the sarbecovirus RBD sequence alignment showing unique combinations of residues at positions 455, 456, and 493 that have evolved across different sarbecoviruses. Sequence names are colored according to RBD phylogenetic clade as in Starr et al. (2022c ).

Article Snippet: Induced cells were washed with phosphate buffered saline supplemented with bovine serum albumin (PBS-BSA , BSA 0.2 mg/l), split into 16-OD*ml aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 −6 to 10 −13 m at 1−log intervals, plus a 0 m sample.

Techniques: Variant Assay, Mutagenesis, Binding Assay, Sequencing

Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Serum ACE2 activity is significantly correlated with SBP in stroke-alert patients and healthy young adults, but not AIS patients. Correlation graphs of ACE2 activity and SBP among stroke-alert patients (a) and healthy young adults (b) as compared to stroke patients (c). Young adult blood plasma samples in panel (b) were from a biorepository established by Wegman et al., which were obtained from research participants undergoing baseline measurements. (d) Correlation graph of ACE activity and mRS at discharge from hospital among AIS patients. ACE2: angiotensin converting enzyme 2; AIS: acute ischemic stroke; mRS: modified Rankin score; RFU: relative fluorescence unit; SBP: systolic blood pressure.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay, Clinical Proteomics, Modification, Fluorescence

Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Activity of ACE2 and ACE in serum is altered following stroke. For human serum, bar graphs are means ± SEM and represent enzyme activity levels of ACE2 (a) and ACE (c) from control, stroke-alert, or AIS patients at an average of 3.6 hours and again at 3 days after stroke. Individual differences and means ± SEM in ACE2 (b) and ACE (d) are shown. * P <0.05 versus control and † P <0.05 versus stroke-alert. ‡ P <0.05 versus AIS <6 hours. ACE: angiotensin converting enzyme; ACE2: angiotensin converting enzyme 2; AIS: acute ischemic stroke; RFU: relative fluorescence unit.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay, Control, Fluorescence

Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Predictors of acute ischemic stroke by multiple linear regression analysis.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay

Journal: Pharmacological research

Article Title: An innovative strategy to investigate microbial protein modifications in a reliable fast and sensitive way: A therapy oriented proof of concept based on UV-C irradiation of SARS-CoV-2 spike protein.

doi: 10.1016/j.phrs.2023.106862

Figure Lengend Snippet: Fig. 7. SPR analysis of the UV- or UV+ S protein binding to sensorchip- immobilized ACE2. (A) Overlay of blank-subtracted sensorgrams derived from a single cycle analysis of UV- (gray) or UV+ (blue) S proteins at 9.4, 18.8, 40, 37.5, 75, 150 mM injected on the ACE2 biosensor. White and black arrows point to the start and end of the injections, respectively. (B) Overlay of the steady-state analysis of UV- (gray) or UV+ S (blue) injected onto the ACE2 biosensor. The results shown are representative of three n = 3 experiments that gave similar results. (C) Kd of the UV- or UV+ S protein interaction with sensorchip-immobilized ACE2. The difference between the calculated Kd mean value is statistically significant (** p < 0.006).

Article Snippet: Human ACE2 protein was purchased from AcroBiosystems (Newark, DE).

Techniques: Protein Binding, Derivative Assay, Injection

Journal: Frontiers in Pharmacology

Article Title: Honeysuckle ( Lonicera japonica ) and Huangqi ( Astragalus membranaceus ) Suppress SARS-CoV-2 Entry and COVID-19 Related Cytokine Storm in Vitro

doi: 10.3389/fphar.2021.765553

Figure Lengend Snippet: The effect of honeysuckle, Huangqi, and their combination on the binding of SARS-CoV2-spike with ACE2 and syncytia formation. (A,C) Imaging of EGFP/spike-positive BHK-21 effector cell binding (upper panel) and fusion (lower panel) with Calu-3 target cells in the absence or presence of honeysuckle (A) , Huangqi (C) , or combination of honeysuckle-EtOH and either APS or APS-L. The binding efficiency of SARS-CoV2-spike to ACE2 (gray bars) and the formation of syncytium indicating fusion efficiency (white bars) was quantified in the cells treated with honeysuckle (B) , Huangqi (D) , and honeysuckle-EtOH combined with either APS or APS-L (E) . *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s: no significance. Scale bar equals 1.0 mm in all figures.

Article Snippet: Next, 100 μL of biotinylated human ACE2 protein (10 ng/ml; cat. AC2-H82E6-25ug; ACRO Biosystems, OX, UK) was added to each well and incubated for another 1 h at 37°C.

Techniques: Binding Assay, Imaging

Journal: Viruses

Article Title: Design of Three Residues Peptides against SARS-CoV-2 Infection

doi: 10.3390/v14102103

Figure Lengend Snippet: BLI interaction analysis. RBD (0.2 µM) ( A , B ) either alone in solution or mixed with VFI ( A ) and TLH ( B ) peptides, tested at different concentrations (40 µM, blue lines; 80 µM, green lines and 160 μM, orange lines). BLI interaction was performed at 25 °C in PBST (10 mM phosphate, 150 mM NaCl, 0.05% Tween 20, pH 7.4). The corresponding plots ( C , D ) of steady-state binding from the end of the association phases (nm), after the subtraction of RBD signal, against analyte concentration were used to calculate the steady-state affinity by nonlinear regression analysis using GraphPad 5 software.

Article Snippet: An Octet ® Red 96 system ® (ForteBio, Fremont, CA, USA) was used to detect interactions between the His-tagged Spike RBD protein (Elabscience Biotechnology, Biomedical Park, Wuhan, China; Cat. No.: PKSH032068) and peptides using Octet His2 biosensors according to the standard instructions with minor modifications.

Techniques: Binding Assay, Concentration Assay, Software