Journal: Journal of Nanobiotechnology

Article Title: A bivalent spike-targeting nanobody with anti-sarbecovirus activity

doi: 10.1186/s12951-025-03243-y

Figure Lengend Snippet: Monovalent and bivalent 7F constructs neutralize authentic SARS-CoV-2 in A549 cells and HAE cell cultures and authentic SARS-CoV in Vero cells. A . Neutralization of SARS-CoV-2 in A549 ACE2+TMPRSS2+ cells. Virus was pre-incubated with serial diluted nanobody, or 10 µM remdesivir, for 30 min before infecting A549 ACE2+TMPRSS2+ cells. Infection was quantified by measuring the virus yield (viral RNA copies/ml, as determined with RT-qPCR) in cell culture supernatants of SARS-CoV-2 infected cells. B . Neutralization of SARS-CoV-2 in HAE cell culture. HAE cultures were incubated with SARS-CoV-2 and 100 nM nanobodies or 10 µM remdesivir on the apical side for 2 h. Nanobody incubation was repeated every 24 h. Graph showing the quantification of viral replication in the cultures, evaluated by RT-qPCR. C . Neutralization of SARS-CoV in Vero cells. Virus was pre-incubated with serial diluted nanobody, or 10 µM remdesivir, for 30 min before infecting Vero cells. Infection was quantified by measuring the virus yield (viral RNA copies/ml, as determined with RT-qPCR) in cell culture supernatants of SARS-CoV infected cells

Article Snippet: A549 cells ( Homo sapiens , lung carcinoma, ATCC CCL-185) expressing human ACE2 receptor protein and TMPRSS2 activating protease (A549 ACE2+TMPRSS2+ ) [ ], Vero cells ( Cercopithecus aethiops , kidney epithelial, ATCC CCL-81) and VeroE6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% FBS for A549 and Vero cells and 10% FBS for VeroE6 cells, 1 mM sodium pyruvate (Gibco), nonessential amino acids (Lonza), penicillin (100 IU/ml), and streptomycin (100 IU/ml).

Techniques: Construct, Neutralization, Virus, Incubation, Infection, Quantitative RT-PCR, Cell Culture

Journal: iScience

Article Title: Impact of mutations defining SARS-CoV-2 Omicron subvariants BA.2.12.1 and BA.4/5 on Spike function and neutralization

doi: 10.1016/j.isci.2023.108299

Figure Lengend Snippet: Impact of BA.2.12.1 and BA.4/5 mutations on Spike-mediated infection (A) Infection kinetics of Caco2, A549-ACE2, and A549-ACE2-TMPRSS2 cells infected by VSVpp carrying the indicated mutant S proteins. Infected GFP+ cells were automatically quantified over a period of 22 h. Exemplary kinetic representing one out of four independent experiments (from B), where each dot indicates the mean of technical duplicates. (B) Automatic quantification of infection events by counting GFP positive cells of Caco2, A549-ACE2, and A549-ACE2-TMPRSS2 cells infected by VSVpp carrying SARS-CoV-2 Spike protein of Hu-1 (gray), BA.1 (light green), BA.2 (yellow), BA.2.12.1 (orange), or BA.4/5 (dark red). Bars represent the mean of four independent experiments (±SEM). Statistical significance was tested by one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (C) Automatic quantification of infection events of Caco2 (upper panel), A549-ACE2 (middle panel), and A549-ACE2-TMPRSS2 (lower panel) cells transduced with VSVΔG-GFP pseudotyped with SARS-CoV-2 S of Hu-1 (gray), BA.1 (light green), BA.2 (yellow), BA.2.12.1 (orange), or BA.4/5 (dark red) or indicated mutant S proteins. Graphs in the center show Hu-based S mutants, infection events are normalized on the Hu-1 S (set at 1) and p values indicate difference to the Hu-1 S. Graphs on the right show BA.2-based S mutants and infection events are normalized on the BA.2 S (set at 1) and p values indicate difference to the BA.2 S. Bars represent the mean of four independent experiments (±SEM). Statistical significance was tested by one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (D) Correlation of the pseudotype infection events of the wild-type S or indicated mutant S proteins in A549-ACE2-TMPRSS2/A549-ACE2 (upper panel), A549-ACE2/Caco2 (middle panel), and A549-ACE2-TMPRSS2/Caco2 (lower panel) cells. Each symbol represents the average value derived from four infection experiments (shown in C). Numbers indicate the combined mutations of BA.4/5 S proteins whose infection events do not correlate between A549 and Caco2 cells. Coefficient of correlation (r), coefficient of determination (R 2 -values) and two tailed p values are provided (see also Figure S2 ).

Article Snippet: Primer/Probe for qRT-PCR of Human TMPRSS2: TaqManTM Gene Expression Assay (FAM-MGB) Hs01122322_m1 , Thermo Fisher , Cat#4331182.

Techniques: Infection, Mutagenesis, Transduction, Derivative Assay, Two Tailed Test

Journal: iScience

Article Title: Impact of mutations defining SARS-CoV-2 Omicron subvariants BA.2.12.1 and BA.4/5 on Spike function and neutralization

doi: 10.1016/j.isci.2023.108299

Figure Lengend Snippet: Impact of BA.2.12.1 and BA.4/5 mutations on TMPRSS2 dependency (A) Automatic quantification of infection events HEK293T-ACE2 cells alone or transfected with TMPRSS2, cathepsin L or B, and infected with VSVpp carrying SARS-CoV-2 S of Hu-1 (gray), BA.1 (light green), BA.2 (yellow), BA.2.12.1 (orange), or BA.4/5 (dark red) or indicated mutant S proteins. The left part of the graphs shows Hu-based S mutants, infection events are normalized on the Hu-1 S (set at 1) and p values indicate difference to the Hu-1 S. Graphs on the right show BA.2-based S mutants and infection events are normalized to BA.2 S (set to 1) and p values indicate difference to the BA.2 S. Bars represent the mean of three independent experiments (±SEM). Statistical significance was tested by one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (B) Correlation of VSVpp infection events of the wild-type S or indicated mutant S proteins in HEK293T overexpressing ACE2 alone or together with TMPRSS2 or cathepsins. Each dot represents the average value derived from three infection experiments (shown in A). Coefficient of correlation (r), coefficient of determination (R 2 -values), and two tailed p values are provided. (C) Infection kinetics (left panel) or infection events (right panel) of A549-ACE2 cells infected by VSVpp containing wild type or mutant Hu-based S proteins. The left panel shows an exemplary kinetic representing one out of three independent experiments (from right panel), where each dot indicates the mean of technical duplicates. In the bar diagram, infection events are normalized on the Hu-1 S (set at 1) and p values indicate difference to the Hu-1 S. Bars represent the mean of three independent experiments (±SEM). Statistical significance was tested by one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (D) Infection kinetics (left panel) or infection events (right panel) of A549-ACE2-TMPRSS2 cells infected by VSVpp containing the wild type or the indicated Hu-based S proteins. The left panel shows an exemplary kinetic representing one out of three independent experiments (from right panel), where each dot indicates the mean of technical duplicates. In the bar diagram, infection events are normalized on the Hu-1 S (set at 1) and p values indicate difference to the Hu-1 S. Bars represent the mean of three independent experiments (±SEM). Statistical significance was tested by one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (E) Correlation of the pseudotype infection events of the wild-type S or indicated Hu-based mutant S proteins harboring mutations characteristic of BA.1 and/or BA.2 S in A549 overexpressing ACE2 alone/with TMPRSS2. Each dot represents the average value derived from three infection experiments (shown in C and 3D). Coefficient of correlation (r), coefficient of determination (R 2 -values) and two tailed p values are provided. (F) Automated quantification of GFP fluorescence of A549-ACE2-TMPRSS2 (left panel) or Caco2 (right panel) cells infected with VSVΔG-GFP pseudotyped with the indicated S variants. Cells were pre-treated (1 h, 37°C) with 20 μM of camostat or E64d in the highest concentration and diluted in a 1:5 titration row. Symbols represent the mean of three independent experiments (±SEM). (G) Quantification of viral N RNA levels in the supernatant of A549-ACE2-TMPRSS2 (left panel) and Caco2 (right panel) cells infected with the indicated SARS-CoV-2 variants (MOI = 0.05), collected at 48 h post-infection. Cells were pre-treated (1 h, 37°C) with 20 μM of camostat or E64d. The right graph of each panel shows the reduction of vRNA levels in the supernatants relative to the untreated control (set at 1). Bars represent the mean of three independent experiments (±SEM). Non-infected controls were below the quantification level (<1000). Statistical significance was tested by two-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S3 .

Article Snippet: Primer/Probe for qRT-PCR of Human TMPRSS2: TaqManTM Gene Expression Assay (FAM-MGB) Hs01122322_m1 , Thermo Fisher , Cat#4331182.

Techniques: Infection, Transfection, Mutagenesis, Derivative Assay, Two Tailed Test, Fluorescence, Concentration Assay, Titration, Control

Journal: iScience

Article Title: Impact of mutations defining SARS-CoV-2 Omicron subvariants BA.2.12.1 and BA.4/5 on Spike function and neutralization

doi: 10.1016/j.isci.2023.108299

Figure Lengend Snippet:

Article Snippet: Primer/Probe for qRT-PCR of Human TMPRSS2: TaqManTM Gene Expression Assay (FAM-MGB) Hs01122322_m1 , Thermo Fisher , Cat#4331182.

Techniques: Virus, Recombinant, Saline, Mutagenesis, Binding Assay, Expressing, Construct, Gene Expression, Plasmid Preparation, Software, Modification, Membrane, Semi Dry Blot, Transfection

Journal: Cell Reports

Article Title: Structural and functional impact by SARS-CoV-2 Omicron spike mutations

doi: 10.1016/j.celrep.2022.110729

Figure Lengend Snippet:

Article Snippet: TMPRSS2 human untagged clone , Origene , Cat#: SC323858.

Techniques: Recombinant, Reporter Gene Assay, Luciferase, Expressing, Construct, Strep-tag, Sequencing, Variant Assay, Software

Journal: Journal of extracellular vesicles

Article Title: Human Transmembrane Serine Protease 2 (TMPRSS2) on Human Seminal Fluid Extracellular Vesicles Is Proteolytically Active.

doi: 10.1002/jev2.70061

Figure Lengend Snippet: FIGURE 1 (A) Graphical overview of the experimental set-up, starting with the isolation of seminal fluid extracellular vesicles (SF-EVs) from leftover semen samples via ultracentrifugation and size-exclusion chromatography. The purified SF-EV stocks showed distinctive TMPRSS2 enzyme activity and were characterised for size distribution via nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Then, we confirmed surface-exposed TMPRSS2 on the SF-EVs via high-sensitivity flow cytometry, triggering us to develop an SF-EV based in vitro screening assay for TMPRSS2-inhibitors. (B) Architecture of TMPRSS2. Native TMPRSS2 constitutes the N-terminal cytoplasmic, the transmembrane, the LDL- receptor class A, the scavenger receptor cysteine-rich and the peptidase S1 domains. The recombinant TMPRSS2 (dasTMPRSS2, 44 kDa) constitutes the LDL-receptor class A, the scavenger receptor cysteine-rich, the peptidase S1 domain (26 kDa) and an AviTag and C-terminal 8xHIS-tag (not shown), with the enterokinase-cleavable sequence DDDDK255 at the red dot replacing the native SRQSR255 amino acid sequence. TM: transmembrane domain (amino acids 85–105); N and C: N- and C-terminus, respectively. Numbers indicate amino acid position according to UniProt O15393. Figure created with BioRender.com.

Article Snippet: Our engineered TMPRSS2 protein expression construct is available on Addgene (plasmid no. 207850).

Techniques: Isolation, Size-exclusion Chromatography, Purification, Activity Assay, Transmission Assay, Electron Microscopy, Flow Cytometry, In Vitro, Screening Assay, Recombinant, Sequencing

Journal: Journal of extracellular vesicles

Article Title: Human Transmembrane Serine Protease 2 (TMPRSS2) on Human Seminal Fluid Extracellular Vesicles Is Proteolytically Active.

doi: 10.1002/jev2.70061

Figure Lengend Snippet: FIGURE 3 SF-EVs and recombinant TMPRSS2 both display specific trypsin-like enzyme activity. (A) Michaelis–Menten plot of initial reaction velocities (V0) for kinetic parameter estimation of the generic Boc-QAR-AMC fluorogenic substrate cleaved by SF-EVs (± 3.3 × 1010 vesicles/mL), after curve fitting in GraphPad (Supporting Information Equation 1). Michaelis constant (Km: 278 ± 52 µM) is indicated by dotted line. (B) Plot of the fractional enzyme activity in SF-EVs (3.3 × 1010 vesicles/mL) versus [Nafamostat mesylate] by measuring the initial reaction velocities at 37◦C in the presence of 100 µM Boc-QAR-AMC fluorogenic substrate and curve fitting for absolute IC50 determination in GraphPad (Supporting Information Equation 2). IC50 (31 ± 5 pM) is indicated by dotted line. (C) Michaelis–Menten plot of initial reaction velocities for kinetic parameter estimation of the generic Boc-QAR- AMC fluorogenic substrate cleaved by recombinant TMPRSS2 (50 pM), after curve fitting in GraphPad (Supporting Information Equation 1). Michaelis constant (Km: 133 ± 16 µM) is indicated by dotted line. (D) Plot of the fractional enzyme activity of recombinant TMPRSS2 (50 pM) versus [Nafamostat mesylate] by measuring the initial reaction velocities at 37◦C in the presence of 100 µM Boc-QAR-AMC fluorogenic substrate and curve fit for absolute IC50 in GraphPad (Supporting Information Equation 2). IC50 (68 ± 3 pM) is indicated by dotted line. All data are shown as mean ± s.d. and were all performed in biological triplicate (n = 3). Graphs were created with GraphPad Prism v9.1.1.

Article Snippet: Our engineered TMPRSS2 protein expression construct is available on Addgene (plasmid no. 207850).

Techniques: Recombinant, Activity Assay

Journal: Journal of extracellular vesicles

Article Title: Human Transmembrane Serine Protease 2 (TMPRSS2) on Human Seminal Fluid Extracellular Vesicles Is Proteolytically Active.

doi: 10.1002/jev2.70061

Figure Lengend Snippet: FIGURE 4 High-sensitivity flow cytometry analysis of SF-EVs. Density plots (R670/30-A vs. SP SSC-H) of purified SF-EVs co-stained with CFDA- SE and (A) anti-human TMPRSS2-APC or (B) anti-human CD26-APC, followed by bottom-up density gradient ultracentrifugation. The plots show a 60 s analysis of fraction 6 (1:50 dilution in PBS) and are representative of technical duplicate or triplicate (n = 2–3). All axes are denoted in arbitrary units. Gates were set as described in the MIFlowCyt checklist (Table S5). (A) The density plot of CFDA-SE positive events labelled with anti-human TMPRSS2 antibody demonstrates a moderate increase in the APC signal (R670/30-A). (B) The density plot of CFDA-SE positive events labelled with anti-human CD26 antibody demonstrates a low increase in the APC signal (R670/30-A).

Article Snippet: Our engineered TMPRSS2 protein expression construct is available on Addgene (plasmid no. 207850).

Techniques: Flow Cytometry, Purification, Staining

Journal: Journal of extracellular vesicles

Article Title: Human Transmembrane Serine Protease 2 (TMPRSS2) on Human Seminal Fluid Extracellular Vesicles Is Proteolytically Active.

doi: 10.1002/jev2.70061

Figure Lengend Snippet: FIGURE 5 Detection of TMPRSS2 in the seminal fluid-derived extracellular vesicle preparations. The detected TMPRSS2 band perfectly matches the theoretical weight of the Peptidase S1 domain (26 kDa) as calculated with ExPASy ProtParam, with the less prominent band probably arising due to off-target auto-activation of the protein (Gasteiger et al. 2005). The band detected in the PC3 cells most closely correlates to the extracellular topological domain of TMPRSS2 and can also slightly be detected in the SF-EVs. A molecular weight ladder (Std) was loaded on each gel. Std, PageRuler Plus Prestained Protein Ladder; EV, seminal-fluid extracellular vesicles; PC3, prostate cancer cell lysate.

Article Snippet: Our engineered TMPRSS2 protein expression construct is available on Addgene (plasmid no. 207850).

Techniques: Derivative Assay, Activation Assay, Molecular Weight

Journal: bioRxiv

Article Title: Contemporary seasonal human coronaviruses display differences in cellular tropism compared to laboratory-adapted reference strains

doi: 10.1101/2025.04.13.648626

Figure Lengend Snippet: Sequence alignments of A) NL63 and B) 229E virus stocks comparing passage 1 (P1) and passage 2 (P2) nucleotide sequences to the same virus isolated in HNEC (for NL63) and BCi (for 229E). Black lines indicate nucleotide changes, red triangles indicate amino acid changes. Infectious virus titres (C) and genome copies (D) in LLC-MK2 and LLC-AT cells infected with lab-adapted (Am-1) and contemporary NL63 isolates (18091206, 18111908, 19071116). Infectious virus titres (E) and genome copies (F) in Huh7 and Huh7-T2 cells infected with lab-adapted (VR740 (Inf1)) and contemporary 229E isolates (21021519, 21042820, 22050721). Data is a representative of at least 2 experimental repeats each with 3 replicates. Mean±SD is shown. Data were analysed using a two-way ANOVA with a Dunnett’s post-test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

Article Snippet: Huh7 overexpressing TMPRSS2 (Huh7-T2) were made by transducing Huh7 cells with a lentiviral construct expressing TMPRSS2 (pscALPSblasti-TMPRSS2 Blasti, Addgene plasmid, Cat. 158088, a gift from Jeremy Luban).

Techniques: Sequencing, Virus, Isolation, Infection

Journal: bioRxiv

Article Title: Structural and functional impact by SARS-CoV-2 Omicron spike mutations

doi: 10.1101/2022.01.11.475922

Figure Lengend Snippet: ( A ) Time-course of cell-cell fusion mediated by various full-length S proteins, as indicated, with the target HEK293 cells transfected with 10 μg ACE2. ( B ) Time-course of cell-cell fusion mediated by various full-length S proteins, as indicated, using HEK293 cells without exogenous ACE2. ( C ) Cell-cell fusion mediated by various full-length S proteins with HEK293 cells transfected with various levels (0-5 μg) of the ACE2 expression construct. ( D ) Cell-cell fusion mediated by various full-length S proteins expressed in HEK293 cells cotransfected with 5 μg furin expression construct and the ACE2-expressing target cells cotransfected with 5 μg TMPRSS2 expression construct. The experiments were repeated at least twice with independent samples giving similar results.

Article Snippet: The furin and TMPRSS2 expression constructs were purchased from Origene (Rockville, MD, Cat# SC118550 and CAT# SC323858).

Techniques: Transfection, Expressing, Construct

Journal: bioRxiv

Article Title: Conserved role of spike S2 domain N-glycosylation across beta-coronavirus family

doi: 10.1101/2024.09.05.611372

Figure Lengend Snippet: A. Workflow for producing SARS-CoV-2 VLPs using the 2-plasmid (‘2P’) system. VLPs are formed upon co-transfecting 293T cells with spike and LVDP CMV-NME EF-1α-Luc-PS9 plasmid. The latter construct contains two gene cassettes, one encoding for nucleocapsid (N), membrane (M) and envelope (E) proteins, and the second encoding luciferase with a cis-acting packaging signal ‘PS9’. 48 hours post-transfection, supernatant containing VLPs was harvested, clarified and concentrated. In functional assays, VLPs were added to target cells overnight, before measuring luciferase activity in cell lysate. B. Viral entry upon application of VLPs expressing different S2 mutants into three types of target cells, A549-ACE2-TMPRSS2, Calu-3, and 293T-ACE2. All G1 mutants displayed reduced viral entry, with greater reduction being observed upon incorporating more than one glycan mutation. C. Western blots of the VLPs. Densitometry was performed to normalize based on the M protein band. Spike incorporation into VLPs was reduced upon implementing S2 stem N-glycan mutations. D. Western blots of a sub-group of the selected spike mutant VLPs. Spike on VLPs were almost completely cleaved. S1 band/S2 band ratio was used for evaluating S1 shedding on VLP spike, with lower value indicating increased S1 shedding. A decreasing trend was observed as more stem N-glycans were deleted. VLPs Data are Mean ± STD. ** P <0.01, *** P <0.001 with respect to parent.

Article Snippet: Human adenocarcinoma alveolar basal epithelial A549 cells overexpressing ACE2 and TMPRSS2 (‘A549-ACE2-TMPRSS2’) (Cat#: a549-hace2tpsa) was purchased from Invivogen (San Diego, CA).

Techniques: Plasmid Preparation, Construct, Membrane, Luciferase, Transfection, Functional Assay, Activity Assay, Expressing, Glycoproteomics, Mutagenesis, Western Blot

Journal: ACS Infectious Diseases

Article Title: Antiviral Potential of the Antimicrobial Drug Atovaquone against SARS-CoV-2 and Emerging Variants of Concern

doi: 10.1021/acsinfecdis.1c00278

Figure Lengend Snippet: Atovaquone restricts wtVSV and VSV-SARS spike infectivity in VeroE6 and VeroE6 hTMPRSS2-hACE2 cells. VeroE6 cells or VeroE6 cells stably transduced with hACE2 and TMPRSS2 were seeded at 2.5 × 10 4 cells/cm 2 in 96-well plates and treated with various concentrations of atovaquone. Fifteen minutes later, cells were infected at an MOI of 1 with wtVSV-spike (Whelan strain) or wtVSV expressing GFP. Eleven hours postinfection, cells were imaged and GFP counts were obtained using the ArrayScan High Content Platform (Thermo Scientific Cellomics). Forty-eight hours postinfection, the viability was determined using resazurin sodium salt (Sigma-Aldrich). (A, D) Graphs show % GFP counts (left axis) normalized to untreated, infected conditions for VSV-spike (green squares, N = 5) and wtVSV (blue triangles, N = 2). The right axis shows % viability normalized to untreated, uninfected conditions for atovaquone alone (black circles, N = 3), atovaquone-treated, VSV-spike-infected cells (green squares, N = 5), or atovaquone-treated, wtVSV-infected cells (blue triangles, N = 2). Symbols boxed in red demonstrate a significant difference over untreated cells ( p < 0.05 and p < 0.005 for all atovaquone-treated, VSV-spike-infected GFP counts using a t -test). Representative fluorescent images are shown for each condition (C, F), and IC 50 values are indicated for VSV-spike and wt-VSV (B,E). Vehicle = medium containing the atovaquone-diluting agent, DMSO. Media = culture medium only.

Article Snippet: The assay was initiated with 2 μL of recombinant active human TMPRSS2 (Cusabio Biotech) at 10 μM, after which the 384-well plate was shaken at 500 rpm for 1 min for complete mixing.

Techniques: Infection, Stable Transfection, Transduction, Expressing

Journal: ACS Infectious Diseases

Article Title: Antiviral Potential of the Antimicrobial Drug Atovaquone against SARS-CoV-2 and Emerging Variants of Concern

doi: 10.1021/acsinfecdis.1c00278

Figure Lengend Snippet: Atovaquone partially requires TMPRSS2 to drive its antiviral action against SARS-CoV-2 and reduces the interaction between the spike protein and its surface receptor ACE2. (A) Schematic of atovaquone administration. (B) VeroE6 hTMPRSS2 cells were treated with atovaquone (10 μM) for 2 h before infection (full time), at the time of infection (entry), or 1 h after infection (postentry), before challenging with original SARS-CoV-2 at an MOI of 0.1. Infection was carried out for 48 h in the presence of the drug and for all conditions. Infection was assessed by immunoblotting of the spike protein within cell lysates. (C) Structure of constructs for a split NanoLuc-based bioreporter. RBD or S1 from either SARS-CoV1 or SARS-CoV2 was linked to Large BiT (LgBiT) on its N-terminus to form LgBiT-RBD or LgBiT-S1; similarly, the Small BiT (SmBiT) peptide was linked to human ACE2 to form SmBiT-ACE2. ACE2 and RBD or S1 constructs were transfected separately or cotransfected into HEK293 cells for 48 h and lysed with a passive lysis buffer. Mixed lysates or lysates from cotransfected cells were incubated with coelenterazine, and the luminescence measured using a plate reader. (D) Following plasmid transfection into HEK293 cells, the cells were lysed in a NanoLuc-compatible passive lysis buffer and lysates were dispensed into a 96-well plate, to which atovaquone was added at a final concentration of 4 μM. The impact of atovaquone on SARS receptor binding was assessed in two ways: (1) with atovaquone added to LgBiT-RBD or LgBiT-S1 for 50 min followed by the addition of an equal quantity of SmBiT-ACE2 for another 10 min (“mixed lysates)” or (2) with atovaquone added to the preformed SmBiT-ACE2 + LgBiT-RBD/S1 complex for 1 h (“cotransfected).” Following incubation, a nanoluciferase substrate was added and the luminescence was measured. HEK293 cells were also transfected with a nanoluciferase control plasmid and lysates were incubated with 4 μM atovaquone. N = 4 per condition. The graph shows % bioreporter luminescence with the highest value in each of the respective untreated conditions taken as 100% ( N = 4 per condition, the p values were determined using a t -test). (E, F). The different cell lines mentioned were subjected to immunoblotting (E) and flow cytometry analysis (F) of TMPRSS2 and ACE2 expression. For ACE2, unstained samples of the matched cell line were used as a control. For TMPRSS2, secondary antibody-stained samples of the matched cell line were used as a control. Controls for the A549 hACE2 cell line are represented in the figure. The same controls were used for all cell lines studied. (G) Vero hTMPRSS2, Calu-3, and A549 hACE2 cells were pretreated with atovaquone (100 μM) for 2 h before infection with the original SARS-CoV-2 (MOI of 0.1). Viral RNA levels were determined 48 h postinfection by qPCR. The data represent the means ± SEM of one experiment performed in biological triplicates. (H) HEK293T cells expressing hACE2 cells were mock-transfected or transfected with a plasmid encoding hTMPRSS2. Twenty-four hours post-transfection, cells were seeded in 96-well plates and preincubated with the different drugs E64d (10 μM), camostat (25 μM), and atovaquone (100 μM) + 5 μg/mL polybrene for 1 h before infection with purified SARS-CoV-2 pseudotypes. LacZ + cells were quantified using the Beta-Glo assay system and luminescence measurement. The data are the means ± SEM of two experiments performed in biological triplicates. Similar results were obtained by X-gal staining. (I) Effector cells (zipV2+) expressing SARS-CoV-2 spike and target cells (zipV1+) expressing ACE2 with or without TMPRSS2 were cocultured for 3 h in the presence of the indicated concentration of drugs or DMSO. Cell–cell fusion was assessed by measuring the fluorescence of the Venus protein complementation (zipV1 + zipV2). Data were normalized to the fusion obtained with target cells expressing ACE2 but not TMPRSS2 (ACE2 + TMPRSS2) and are the means ± SEM of two experiments performed in triplicates. The p values were calculated using a t -test where * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The assay was initiated with 2 μL of recombinant active human TMPRSS2 (Cusabio Biotech) at 10 μM, after which the 384-well plate was shaken at 500 rpm for 1 min for complete mixing.

Techniques: Infection, Western Blot, Construct, Transfection, Lysis, Incubation, Plasmid Preparation, Concentration Assay, Binding Assay, Control, Flow Cytometry, Expressing, Staining, Purification, Glo Assay, Fluorescence

Journal: Nature

Article Title: TMPRSS2 is a functional receptor for human coronavirus HKU1

doi: 10.1038/s41586-023-06761-7

Figure Lengend Snippet: a. Alignment of HKU1A and B spikes. TD: N-terminal Domain. CTD: C-terminal domain. FP: Fusion-peptide. HR11/2: Heptad-Repeat 1/2. TMD: Transmembrane Domain. Black: mismatch, white: deletion, boxed text (Red): amino-acids of the putative RBM, S1/S2 and S2/S2’ cleavage site. b. TMPRSS2 mediates HKU1 cell-cell fusion. 293T cells expressing either GFP1–10 or GFP-11 (293T-GFP-split cells) were transfected with HKU1 spike and TMPRSS2 or pQCXIP-empty control (Ctrl) plasmids, fusion was quantified by measuring the GFP area after 20 h. Data are mean ± SD of 5 independent experiments. c. Effect of a panel of proteases on cell-cell fusion. 293T-GFP-split cells were transfected with HKU1 spike and the indicated protease plasmids, fusion was quantified by measuring the GFP area after 48 h. Data are mean ± SD of 3 independent experiments. d. TMPRSS2 has to be on the acceptor cell. TMPRSS2 was transfected either in donor cells and HKU1A spike or in acceptor cells. Fusion was quantified after 20 h. Left: experimental design. Middle: representative images of GFP+ cells. Right: fusion quantification. bar: 400 μm. Data are mean ± SD of 4 independent experiments. e. Role of endogenous TMPRSS2. 293T donor cells expressing HKU1A spike were mixed with Caco2 acceptor cells knocked-out or not for the tmprss2 gene. Left: experimental design. Middle: representative images of GFP+ cells. Right: fusion was quantified by measuring the GFP area after 20 h of coculture. Data are mean ± SD of 3 independent experiments. Statistical analysis: b, d, e: Two-sided unpaired t-test compared to control/parental condition. c: one Way ANOVA on non-normalized data with Tukey’s multiple comparisons.

Article Snippet: The following antibodies were diluted in WB buffer (PBS, 1% BSA, 0.05% Tween, 0.01% Na Azide): rabbit anti-human TMPRSS2 (Atlas antibodies cat# HPA035787, 1:1,000), mouse anti-beta actin (Abcam, 60008–1-Ig, 1:2,000), rabbit anti-HKU1 S1 polyclonal antibody (Thermofisher Scientific Cat #PA5–120768, 1:2,000) and rabbit anti-HKU1 S2 polyclonal antibody (Thermofischer Scientific #PA5–120769, 1:1,000).

Techniques: Expressing, Transfection, Control

Journal: Nature

Article Title: TMPRSS2 is a functional receptor for human coronavirus HKU1

doi: 10.1038/s41586-023-06761-7

Figure Lengend Snippet: a. Binding of the 5 anti-TMPRSS2 VHH on recombinant TMPRSS2 measured by BLI. b, c. Comparison of one commercial TMPRSS2 antibody and of the dimeric anti-TMPRSS2 VHH A01-Fc. 293T cells were transfected with WT TMPRSS2. 24 h later, cells were analysed by flow-cytometry. Staining with the commercial antibody was performed on fixed cells (b) while surface staining with the VHH was performed on live cells (c).

Article Snippet: The following antibodies were diluted in WB buffer (PBS, 1% BSA, 0.05% Tween, 0.01% Na Azide): rabbit anti-human TMPRSS2 (Atlas antibodies cat# HPA035787, 1:1,000), mouse anti-beta actin (Abcam, 60008–1-Ig, 1:2,000), rabbit anti-HKU1 S1 polyclonal antibody (Thermofisher Scientific Cat #PA5–120768, 1:2,000) and rabbit anti-HKU1 S2 polyclonal antibody (Thermofischer Scientific #PA5–120769, 1:1,000).

Techniques: Binding Assay, Recombinant, Comparison, Transfection, Flow Cytometry, Staining

Journal: Nature

Article Title: TMPRSS2 is a functional receptor for human coronavirus HKU1

doi: 10.1038/s41586-023-06761-7

Figure Lengend Snippet: Affinity of the nanobodies for TMPRSS2 S441A and summary of their effect.

Article Snippet: The following antibodies were diluted in WB buffer (PBS, 1% BSA, 0.05% Tween, 0.01% Na Azide): rabbit anti-human TMPRSS2 (Atlas antibodies cat# HPA035787, 1:1,000), mouse anti-beta actin (Abcam, 60008–1-Ig, 1:2,000), rabbit anti-HKU1 S1 polyclonal antibody (Thermofisher Scientific Cat #PA5–120768, 1:2,000) and rabbit anti-HKU1 S2 polyclonal antibody (Thermofischer Scientific #PA5–120769, 1:1,000).

Techniques: Activity Assay, Inhibition, Infection

Journal: Nature

Article Title: TMPRSS2 is a functional receptor for human coronavirus HKU1

doi: 10.1038/s41586-023-06761-7

Figure Lengend Snippet: a. 293T cells were transfected with plasmids encoding for HKU1A or HKU1B spikes and stained 24 h later with mAb10, a pan anti-coronavirus spike antibody. Data are representative of 3 independent experiments. b. Cell-cell fusion mediated by HKU1A or HKU1B spikes. 293T GFP-Split cells were transfected with HKU1 spikes and TMPRSS2, fusion was visualized by the appearance of GFP+ cells. Data are representative of 6 independent experiments. Scale bar: 400 μm c. Cell-cell fusion mediated by the SARS-CoV-2 spike. 293T GFP-Split cells were transfected with SARS-CoV-2 spike, in the presence of ACE2 or TMPRSS2, fusion was visualized by the appearance of GFP+ cells. Data are mean ± SD of 3 independent experiments d. U2OS cell-cell fusion mediated by HKU1A spike. U2OS GFP-Split cells were transfected with HKU1A spike and TMPRSS2, fusion was visualized by the appearance of GFP+ cells 24 h later. Data are mean ± SD of 3 independent experiments. e. Expression levels of myc-tagged proteases. 293T cells were transfected with a control plasmid or the indicated myc-tagged TMPRSS constructs and stained 24 h later with myc antibody 9E10. Left: Representative dot plots. Right: Percentage of positive cells. Data are mean ± SD of 3 independent experiments. f. Surface expression of tagged and untagged TMPRSS2. 293T cells were transfected with WT TMPRSS2 (TMPRSS2-Untagged) or a myc-tagged TMPRSS2 and surface stained for TMPRSS2 using VHH-A01-Fc. Left: Representative dot plots. Right: Percentage of positive cells. Data are mean ± SD of 3 independent experiments. g, h, i . Surface expression of APN, DPP4 and ACE2. 293T cells were transfected with APN, DPP4 or ACE2 plasmids, and surface stained with the respective antibodies 24 h later. Left: Representative dot plots. Right: Percentage of positive cells. Data are mean ± SD of 3 (TMPRSS2, APN, DPP4) or 4 (ACE2) independent experiments. j. Images of time lapse microscopy of HKU1-mediated cell-cell fusion (extracted from Supp. video 1). 293T cells were transfected either with TMPRSS2-scarlet-I or HKU1A S-NeonGreen. After 24 h, cells were mixed and imaged every 2.5 min for 2 h at 37°C.

Article Snippet: The following antibodies were diluted in WB buffer (PBS, 1% BSA, 0.05% Tween, 0.01% Na Azide): rabbit anti-human TMPRSS2 (Atlas antibodies cat# HPA035787, 1:1,000), mouse anti-beta actin (Abcam, 60008–1-Ig, 1:2,000), rabbit anti-HKU1 S1 polyclonal antibody (Thermofisher Scientific Cat #PA5–120768, 1:2,000) and rabbit anti-HKU1 S2 polyclonal antibody (Thermofischer Scientific #PA5–120769, 1:1,000).

Techniques: Expressing, Activity Assay, Transfection, Staining, Control, Plasmid Preparation, Construct, Time-lapse Microscopy

Journal: Nature

Article Title: TMPRSS2 is a functional receptor for human coronavirus HKU1

doi: 10.1038/s41586-023-06761-7

Figure Lengend Snippet: The CRISPR-Cas9 targeting site is underlined and the proto-spacer recognition motif (PAM) is in bold. Rectangles represent exons, black for 5’ and 3’ untranslated regions, gray for coding regions. b. Sequence analysis of the knock-out Caco2 clone. Both alleles compared to the original wild-type sequence are shown. The knock-out clone harbors an out-of-frame deletion and an insertion, resulting in a frameshift on both alleles. c. Validation of TMPRSS2 knock-out by TMPRSS2 surface staining. Representative dot plots of VHH anti-TMPRSS2 (VHH-A01-Fc) surface staining of a WT and KO Caco2 obtained following CRISPR gene targeting. d. Role of endogenous TMPRSS2 on cell-cell fusion assessed by siRNA. 293T donor cells expressing HKU1A spike were mixed with Caco2 acceptor cells, silenced or not for TMPRSS2. Left: experimental design. Middle: relative expression of TMPRSS2 in Caco2 cells, assessed by RT-qPCR. Data were normalized to β-Tubulin levels. Relative mRNA expression normalized to siCtrl condition (2−ΔΔCT) was plotted. Right: fusion was quantified by measuring the GFP area after 20 h of coculture. Data are mean ± SD of 3 independent experiments. Statistical analysis: two-sided unpaired t-test compared to siCtrl cells. ***p<0.0001.

Article Snippet: The following antibodies were diluted in WB buffer (PBS, 1% BSA, 0.05% Tween, 0.01% Na Azide): rabbit anti-human TMPRSS2 (Atlas antibodies cat# HPA035787, 1:1,000), mouse anti-beta actin (Abcam, 60008–1-Ig, 1:2,000), rabbit anti-HKU1 S1 polyclonal antibody (Thermofisher Scientific Cat #PA5–120768, 1:2,000) and rabbit anti-HKU1 S2 polyclonal antibody (Thermofischer Scientific #PA5–120769, 1:1,000).

Techniques: Knock-Out, CRISPR, Sequencing, Biomarker Discovery, Staining, Expressing, Quantitative RT-PCR

Journal: Nature

Article Title: TMPRSS2 is a functional receptor for human coronavirus HKU1

doi: 10.1038/s41586-023-06761-7

Figure Lengend Snippet: a. TMPRSS2 protein. TMPRSS2 is composed of a Transmembrane Domain (TMD), a class A LDL receptor domain (LDLR), a Scavenger Receptor Cysteine-2 rich domain (SRCRD) and a serine peptidase. TMPRSS2 precursor autocleaves at R255-L256, resulting in an active protease. b. TMPRSS2 and spike cleavage. WB of 293T transfected with HKU1A spike and indicated TMPRSS2 mutants. One membrane was probed for S1, TMPRSS2 and actin, another for S2 and actin. For gel source data, see SI 1a, b. Representative blots of 3 independent experiments. Molecular weights: kDa. Arrows and # denote the uncleaved and cleaved proteins, respectively. c. Catalytically inactive TMPRSS2 mutants do not trigger HKU1 cell-cell fusion. Fusion of 293T-GFP split transfected with HKU1 spike and mutant TMPRSS2 was quantified after 20 h and normalized to WT TMPRSS2. Ctrl: pQCXIP-Empty. Left: HKU1A. Right: HKU1B. Data are mean ± SD of 3 independent experiments. d. Catalytically inactive TMPRSS2 mutants mediate HKU1 pseudovirus infection. 293T transfected with WT or mutant TMPRSS2 were infected by Luc-encoding HKU1 pseudovirus. Luminescence was read 48 h post infection. Dotted line indicates background in non-infected cells. Ctrl: pQCXIP-Empty. Data are mean ± SD of 6 (HKU1A) or 3 (HKU1B) independent experiments. e. HKU1 pseudovirus infection in cell lines stably expressing TMPRSS2. Left: U2OS cells. Middle: A549 cells. Right: Caco2 cells. The TMPRSS2 KO Caco2 cells were stably transduced with indicated TMPRSS2. Data are mean ± SD of 3 (U2OS), 8 (A549), 4 (Caco2) independent experiments. Statistical analysis: c: one-way ANOVA on non-normalized data with Dunnett’s multi-comparison test compared to WT TMPRSS2 expressing cells. d, e: one-way ANOVA on log-transformed data with Dunnett’s multi-comparison test compared to the untransfected (d), parental (e: U2OS and A549) or KO cells (e: Caco2), ***p<0.0001.

Article Snippet: The following antibodies were diluted in WB buffer (PBS, 1% BSA, 0.05% Tween, 0.01% Na Azide): rabbit anti-human TMPRSS2 (Atlas antibodies cat# HPA035787, 1:1,000), mouse anti-beta actin (Abcam, 60008–1-Ig, 1:2,000), rabbit anti-HKU1 S1 polyclonal antibody (Thermofisher Scientific Cat #PA5–120768, 1:2,000) and rabbit anti-HKU1 S2 polyclonal antibody (Thermofischer Scientific #PA5–120769, 1:1,000).

Techniques: Transfection, Membrane, Mutagenesis, Infection, Stable Transfection, Expressing, Transduction, Comparison, Transformation Assay

Journal: Nature

Article Title: TMPRSS2 is a functional receptor for human coronavirus HKU1

doi: 10.1038/s41586-023-06761-7

Figure Lengend Snippet: 293T cells were transfected with WT and indicated TMPRSS2 mutants. 24 h post transfection, the catalytic activity was assessed using BoC-QAR-AMC fluorogenic substrate. Data are mean ± SD of 4 independent experiments. Statistical analysis: a: one-way ANOVA with Dunnett’s multiple comparisons compared to Ctrl cells (transfected with pQCXIP-Empty). **p<0.01. b. Effect of TMPRSS2 on HKU1 spike expressed on adjacent cells. 293T cells were transfected either with HKU1A spike or with the different TMPRSS2. 20 h post-transfection, cells were mixed at a 1:1 ratio, and let to settle. 3 or 6 h post-mixing, cells were harvested and lysed for WB. One membrane was probed for S1, TMPRSS2 and actin, another membrane was probed for S2 and actin. For gel source data, see SI 1c, d. Representative blots of 2. Molecular weights: kDa. The arrows and # denote the uncleaved and cleaved protein products, respectively. c. Surface levels of WT and mutant TMPRSS2. 293T cells were transfected with the indicated doses of TMPRSS2 plasmid. 18 h post-transfection they were stained intracellularly for TMPRSS2 using the commercial antibody. Left: % of TMP positive cells. Right: Median fluorescent intensity (MFI). #: indicates the chosen plasmid ratios to achieve similar TMPRSS2 levels with WT and indicated mutants. Data are mean ± SD of 3 independent experiments. d. Comparison of the anti-TMPRSS2 commercial antibody and VHH staining in 293T cells. 293T cells were transfected with WT, R255Q and S441A TMPRSS2 plasmids. Cells were stained 24 h later with the indicated antibodies and analysed by flow cytometry. One experiment representative of 3 is shown. Light grey curves correspond to staining with control antibodies or VHH. Parental: untransfected cells. e. Effect of WT and mutant TMPRSS2 on SARS-CoV-2 pseudovirus infection. 293T cells expressing ACE2 were transfected with WT and indicated TMPRSS2 mutants. 24 h later, cells were infected by Luc-encoding SARS-CoV-2 pseudovirus. Luminescence was measured after 48 h. Data are mean ± SD of 4 independent experiments. Statistical analysis: RM one-way ANOVA with Geisser-Greenouse correction on log-transformed data, with Dunnett’s multiple comparisons compared to Ctrl cells (transfected with pQCXIP-Empty). **p<0.01. f, g. Effect of SB412515, E64d or hydroxychloroquine (HCQ) on f. HKU1A or g. HKU1B pseudovirus infection. 293T cells expressing WT or S441A TMPRSS2 were incubated for 2 h with the indicated drugs, before infection with HKU1A or HKU1B pseudoviruses. Luminescence was read 48 h post infection. Left: SB412515. Middle: E64d. Right: HCQ. Data are mean ± SD of 3 (E64d, HKU1B), 4 (E64d HKU1A, SB142515 HKU1B), 5 (HCQ HKU1A) or 6 (HCQ HKU1B, SB412515 HKU1A) independent experiments. Statistical analysis: RM two-way ANOVA with Geisser-Greenouse correction on non-normalized log transformed data, with Dunnett’s multiple comparisons compared to the non-treated conditions. h, i. Surface levels of TMPRSS2 in different cell lines stably expressing WT or S441 TMPRSS2. Cells were stained for TMPRSS2 using VHH-A01-Fc and analyzed by flow cytometry. Representative histograms are shown. h. Left: U2OS. Right: A549. Light grey: cells stained with a non-target control VHH-Fc. Dark grey: Unmodified parental cell lines. Dark and light blue: Cells transduced with either TMPRSS2 or TMPRSS2 S441A mutant. i. Caco2. Light grey: cells stained with a non-target control VHH-Fc. Blue: Unmodified parental cell line. Dark Grey: TMPRSS2 KO Caco2. Dark and light blue: TMPRSS2 KO caco2 stably transduced with TMPRSS2 WT or S441A mutant expression vectors. j. Effect of Camostat on endogenous TMPRSS2 in Caco2 cells. Caco2 cells were incubated in the presence of 100 μM Camostat for 2 h, before infection with HKU1A, HKU1B, or SARS-CoV-2 (D614G or Delta) pseudovirus. Data are normalized to the infection in the absence of the drug. Data are mean ± SD of 3 (SARS-CoV-2) or 4 (HKU1) independent experiments. Statistical analysis: RM two-way ANOVA on non-normalized log transformed data, with Sidak’s multiple comparisons compared to the non-treated conditions. k. Susceptibility of Vero E6 and Vero E6-TMPRSS2 cells to HKU1 pseudovirus infection. Left: pseudovirus infection. Right: TMPRSS2 surface levels. Dark grey: Parental cells. Dark blue: Cells transduced with TMPRSS2. Data are mean ± SD of 4 independent experiments.

Article Snippet: The following antibodies were diluted in WB buffer (PBS, 1% BSA, 0.05% Tween, 0.01% Na Azide): rabbit anti-human TMPRSS2 (Atlas antibodies cat# HPA035787, 1:1,000), mouse anti-beta actin (Abcam, 60008–1-Ig, 1:2,000), rabbit anti-HKU1 S1 polyclonal antibody (Thermofisher Scientific Cat #PA5–120768, 1:2,000) and rabbit anti-HKU1 S2 polyclonal antibody (Thermofischer Scientific #PA5–120769, 1:1,000).

Techniques: Mutagenesis, Infection, Activity Assay, Transfection, Membrane, Plasmid Preparation, Staining, Comparison, Flow Cytometry, Control, Expressing, Transformation Assay, Incubation, Stable Transfection, Transduction

Journal: Nature

Article Title: TMPRSS2 is a functional receptor for human coronavirus HKU1

doi: 10.1038/s41586-023-06761-7

Figure Lengend Snippet: U2OS-TMPRSS2 cells were treated with indicated concentration of neuraminidase from Arthrobacter ureafaciens for 24 h. a. HKU1A and HKU1B pseudovirus infection in neuraminidase treated cells. Cells were infected with HKU1 A or B pseudoviruses. Luminescence was read 48 h post infection. Data were normalized to the non-treated condition. Data are mean ± SD of 3 (4 mU/mL) or 4 (0, 20, 100 mU/mL) independent experiments. Statistical analysis: Mixed-effect analysis with Geisser-Greenhouse corrections (handles missing values) on non-normalized log transformed data, with Dunnett’s multi-comparison test compared to non-treated cells. b. Surface levels of TMPRSS2 in neuraminidase treated cells. Left: % of TMP positive cells. Right: Median fluorescent intensity (MFI). Data are mean of 2 independent experiments. c. Sambucus Nigra Lectin (SNA) binding on neuraminidase treated cells. Treated cells were stained with fluorescent Lectin SNA which preferentially binds α2,6- over α-2,3 linked sialic acids. Left: Representative histograms. Right: MFI. Data are mean of 2 independent experiments. d. Siglec-E binding on neuraminidase treated cells. Treated cells were stained with recombinant Siglec-E-Fc protein which preferentially binds α2,8- over α2,3- and α2,6-linked sialic acids. Left: Representative histograms. Right: MFI. Data are mean ± SD of 3 independent experiments.

Article Snippet: The following antibodies were diluted in WB buffer (PBS, 1% BSA, 0.05% Tween, 0.01% Na Azide): rabbit anti-human TMPRSS2 (Atlas antibodies cat# HPA035787, 1:1,000), mouse anti-beta actin (Abcam, 60008–1-Ig, 1:2,000), rabbit anti-HKU1 S1 polyclonal antibody (Thermofisher Scientific Cat #PA5–120768, 1:2,000) and rabbit anti-HKU1 S2 polyclonal antibody (Thermofischer Scientific #PA5–120769, 1:1,000).

Techniques: Infection, Concentration Assay, Transformation Assay, Comparison, Binding Assay, Staining, Recombinant

Journal: Nature

Article Title: TMPRSS2 is a functional receptor for human coronavirus HKU1

doi: 10.1038/s41586-023-06761-7

Figure Lengend Snippet: a-b. Binding of soluble HKU1 spikes to 293T cells expressing TMPRSS2. Cells were transfected with TMPRSS2 mutants and incubated or not with 10 μM of Camostat. a. TMPRSS2 levels (assessed with a commercial Ab) b. Binding of soluble biotinylated trimeric spikes measured by flow cytometry. One representative experiment of 3 is shown. Ctrl: pQCXIP-Empty control plasmid. c. Binding of HKU and SARS-CoV-2 spikes on immobilized WT TMPRSS2 measured by ELISA. Mean of 2 independent experiments. d. Binding of S441A TMPRSS2 to RBD coated receptors quantified by Bio-layer interferometry (BLI). One representative experiment of 4 is shown. e, f, g. Properties of W515A and R517A mutant HKU1 spikes. Binding of TMPRSS2 to wild type or mutant RBD coated receptors quantified by BLI. One representative experiment of 4 is shown. f. 293T GFP-split cells were transfected with TMPRSS2 and the indicated HKU1 mutant spikes, fusion was quantified by measuring the GFP area after 24 h. g. 293T cells expressing TMPRSS2 were infected by Luc-encoding mutant HKU1 pseudovirus. Luminescence was read 48 h post infection. Dotted line indicates background. Data are mean ± SD of 3 independent experiments. Statistical analysis: f: one-way ANOVA on non-normalized data with Dunnett’s multi-comparison test compared to WT TMPRSS2 expressing cells. g: one-way ANOVA on log-transformed data with Dunnett’s multi-comparison test compared to WT Spike pseudotypes *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The following antibodies were diluted in WB buffer (PBS, 1% BSA, 0.05% Tween, 0.01% Na Azide): rabbit anti-human TMPRSS2 (Atlas antibodies cat# HPA035787, 1:1,000), mouse anti-beta actin (Abcam, 60008–1-Ig, 1:2,000), rabbit anti-HKU1 S1 polyclonal antibody (Thermofisher Scientific Cat #PA5–120768, 1:2,000) and rabbit anti-HKU1 S2 polyclonal antibody (Thermofischer Scientific #PA5–120769, 1:1,000).

Techniques: Binding Assay, Expressing, Transfection, Incubation, Flow Cytometry, Control, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Mutagenesis, Infection, Comparison, Transformation Assay

Journal: Nature

Article Title: TMPRSS2 is a functional receptor for human coronavirus HKU1

doi: 10.1038/s41586-023-06761-7

Figure Lengend Snippet: Cells were transfected with WT or mutant TMPRSS2 and incubated or not overnight with 10 μM of Camostat. The spikes were then incubated for 0.5 h and their binding was revealed with streptavidin-647 and measured by flow cytometry. The % of cells binding to TMPRSS2 was quantified. Data are mean ± SD of 3 (HKU1A) or 4 (HKU1B, SARS-CoV-2) independent experiments. Two Way ANOVA with Dunnett’s multiple comparisons compared to control cells with or without Camostat. Exact p-values: HKU1A: TMPRSS2 WT-: *0.029, TMPRSS2 WT+:***<0.0001, TMPRSS2 R255Q-: **0.0010, TMPRSS2 R255Q+: **0,0013, TMPRSS2 S441A-: ***0,0001, TMPRSS2 S441A+: ***<0.0001. HKU1B: ***<0.0001. b. Binding of the indicated recombinant spikes to 293T cells expressing ACE2. Cells were transfected with ACE2. The spikes were then incubated for 0.5 h and their binding was revealed with streptavidin-647 and measured by flow cytometry. The % of cells binding to ACE2 was quantified. Data are mean of 2 independent experiments. c. Binding of the indicated soluble spikes on immobilized ACE2 measured by ELISA. d. Binding of S441A TMPRSS2 to HKU1A, HKU1B or SARS-CoV-2 RBD measured by BLI. The response was measured at the indicated concentrations of spikes. Left: HKU1A. Middle: HKU1B. Right: SARS-CoV-2. One representative experiment of 4 is shown. e. Determination of the affinity of HKU1A and B RBD for TMPRSS2 using the steady state method. Circles: Experimental values. Black: Fitting of the experimental data f. Binding of ACE2 to SARS-CoV-2 or HKU1B RBD quantified by BLI, at different concentrations of spikes. g. Binding of S441A TMPRSS2 to HKU1B mutants. Response was measured by BLI at different concentrations of spikes. Left: HKU1B RBD mutant W515A. B: HKU1B RBD mutant R517A. h. Determination of the affinity of HKU1B-R517A RBD for TMPRSS2 using the steady state method. Circles: Experimental values. Black: Fitting of the experimental data. i. Cell surface levels of WT and mutant HKU1 spikes. 293T were transfected with the indicated WT or mutant HKU1A or B spikes, expression was measured by flow cytometry after 24 h, using the anti-spike mAb10.

Article Snippet: The following antibodies were diluted in WB buffer (PBS, 1% BSA, 0.05% Tween, 0.01% Na Azide): rabbit anti-human TMPRSS2 (Atlas antibodies cat# HPA035787, 1:1,000), mouse anti-beta actin (Abcam, 60008–1-Ig, 1:2,000), rabbit anti-HKU1 S1 polyclonal antibody (Thermofisher Scientific Cat #PA5–120768, 1:2,000) and rabbit anti-HKU1 S2 polyclonal antibody (Thermofischer Scientific #PA5–120769, 1:1,000).

Techniques: Binding Assay, Recombinant, Expressing, Transfection, Mutagenesis, Incubation, Flow Cytometry, Control, Enzyme-linked Immunosorbent Assay

Journal: Nature

Article Title: TMPRSS2 is a functional receptor for human coronavirus HKU1

doi: 10.1038/s41586-023-06761-7

Figure Lengend Snippet: Affinity (K d ) of the indicated RBD for TMPRSS2 or ACE2. ND (Non-Detectable) denotes proteins for which interaction with the loaded sensor was similar or below the interaction with an empty sensor.

Article Snippet: The following antibodies were diluted in WB buffer (PBS, 1% BSA, 0.05% Tween, 0.01% Na Azide): rabbit anti-human TMPRSS2 (Atlas antibodies cat# HPA035787, 1:1,000), mouse anti-beta actin (Abcam, 60008–1-Ig, 1:2,000), rabbit anti-HKU1 S1 polyclonal antibody (Thermofisher Scientific Cat #PA5–120768, 1:2,000) and rabbit anti-HKU1 S2 polyclonal antibody (Thermofischer Scientific #PA5–120769, 1:1,000).

Techniques:

Journal: Nature

Article Title: TMPRSS2 is a functional receptor for human coronavirus HKU1

doi: 10.1038/s41586-023-06761-7

Figure Lengend Snippet: 293T cells were transfected with TMPRSS2 S441A. Binding of the indicated VHHs (0.5 μM) was assessed by flow cytometry. Left: representative dot plots. Right: Quantification of the percentage of positive cells and MFI (Median Fluorescent Intensity of positive cells). Data are mean ± SD of three independent experiments b. Effect of VHHs or Camostat on TMPRSS2 enzymatic activity. Recombinant soluble TMPRSS2 was incubated with the indicated VHHs at different concentrations or with Camostat (1 μM). The initial rate of enzymatic activity, measured with a fluorescent substrate is plotted. One representative experiment of 3 is shown. c. Effect of VHHs on HKU1A or HKU1B cell-cell fusion. 293T GFP-Split cells were cotransfected with TMPRSS2 and HKU1 spike in the presence of indicated amounts of VHH. Fusion was quantified by measuring the GFP area after 20 h. Data were normalized to the non-treated condition for each spike. d. Effect of VHHs on HKU1A or HKU1B pseudovirus infection. 293T cells transfected with S441A TMPRSS2 were incubated with the indicated amounts of VHH for 2 h and infected by Luc-encoding pseudovirus. Luminescence was read 48 h post infection. Data were normalized to the non-treated condition for each virus. Data are from one experiment representative of 3. Statistical analysis: c: One Way ANOVA with Dunnett’s multiple comparisons compared to cells stained with a non-target antibody (VHH93). d: One Way ANOVA on log-transformed data with Dunnett’s multiple comparisons compared to cells stained with a non-target antibody (VHH93).

Article Snippet: The following antibodies were diluted in WB buffer (PBS, 1% BSA, 0.05% Tween, 0.01% Na Azide): rabbit anti-human TMPRSS2 (Atlas antibodies cat# HPA035787, 1:1,000), mouse anti-beta actin (Abcam, 60008–1-Ig, 1:2,000), rabbit anti-HKU1 S1 polyclonal antibody (Thermofisher Scientific Cat #PA5–120768, 1:2,000) and rabbit anti-HKU1 S2 polyclonal antibody (Thermofischer Scientific #PA5–120769, 1:1,000).

Techniques: Binding Assay, Expressing, Transfection, Flow Cytometry, Activity Assay, Recombinant, Incubation, Infection, Virus, Staining, Transformation Assay

Journal: Nature

Article Title: TMPRSS2 is a functional receptor for human coronavirus HKU1

doi: 10.1038/s41586-023-06761-7

Figure Lengend Snippet: a. Effect of VHHs on binding of HKU1B RBD to TMPRSS2 measured by BLI. One representative experiment of 2 is shown b. Effect of VHHs on HKU1-mediated cell-cell fusion. 293T GFP-Split cells were transfected with TMPRSS2 and HKU1 spike in the presence of 1 μM of VHH, fusion was quantified 20 h later. Data were normalized to the non-VHH treated condition (dotted line). Data are mean ± SD of 4 independent experiments. c. Effect of VHHs on HKU pseudovirus infection. 293T cells transfected with S441A TMPRSS2 were treated with 1 μM VHH 2 h before infection. Luminescence was read 48 h post infection. Data were normalized to the non-treated condition for each virus (dotted line). Data are mean ± SD of 3 independent experiments. Statistical analysis: b: one Way ANOVA data with Dunnett’s multiple comparisons compared to non-target VHH c: one Way ANOVA on log-transformed data with Dunnett’s multiple comparisons compared to non-target VHH *p<0.05, **p<0.01, ***p<0.001. p-values. Fusion, HKU1A: A07 0.0005, C11: 0.025, D01: 0.004, HKU1B: A07: 0.029, Infection: HKU1A: A01: 0.016, A07: <0.0001, C11:0.0009, D01:<0.0001 HKU1B: A07/C11/D01: <0.0001

Article Snippet: The following antibodies were diluted in WB buffer (PBS, 1% BSA, 0.05% Tween, 0.01% Na Azide): rabbit anti-human TMPRSS2 (Atlas antibodies cat# HPA035787, 1:1,000), mouse anti-beta actin (Abcam, 60008–1-Ig, 1:2,000), rabbit anti-HKU1 S1 polyclonal antibody (Thermofisher Scientific Cat #PA5–120768, 1:2,000) and rabbit anti-HKU1 S2 polyclonal antibody (Thermofischer Scientific #PA5–120769, 1:1,000).

Techniques: Binding Assay, Transfection, Infection, Virus, Transformation Assay

Journal: Nature

Article Title: TMPRSS2 is a functional receptor for human coronavirus HKU1

doi: 10.1038/s41586-023-06761-7

Figure Lengend Snippet: a. TMPRSS2 staining of HBE cells. Red: Phalloidin, Blue: DAPI, Green: TMPRSS2 stained with VHH A01-Fc. Scale bars: top, 10 μm; bottom, 5 μm. Images are representative of 3 independent experiments. b. Effect of the anti-TMPRSS2 VHH (A07) on HKU1 infection. The experimental design is represented. Infected cells were visualized with an anti-spike antibody and scored. Representative images of spike staining 48 h post-infection are shown. Spike pixel intensity in 5 random fields per experiment was measured and normalized to the intensity in the infected but non-treated condition. Data are mean ± SD of 3 independent experiments for infected conditions, and mean of 2 for uninfected condition. Scale bar: 20 μm. Statistical analysis: Two-sided unpaired t-test compared to non-target VHH.

Article Snippet: The following antibodies were diluted in WB buffer (PBS, 1% BSA, 0.05% Tween, 0.01% Na Azide): rabbit anti-human TMPRSS2 (Atlas antibodies cat# HPA035787, 1:1,000), mouse anti-beta actin (Abcam, 60008–1-Ig, 1:2,000), rabbit anti-HKU1 S1 polyclonal antibody (Thermofisher Scientific Cat #PA5–120768, 1:2,000) and rabbit anti-HKU1 S2 polyclonal antibody (Thermofischer Scientific #PA5–120769, 1:1,000).

Techniques: Staining, Infection

Journal: Nature Microbiology

Article Title: SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

doi: 10.1038/s41564-022-01143-7

Figure Lengend Snippet: a , Virus-induced fusion of co-cultured cells expressing split GFP (GFP-10 and GFP-11, respectively) reconstitutes fluorescence (image generated in BioRender, agreement SX23HJ6GY8SX23HJ6GY8). b , Co-cultured GFP-10 and GFP-11 A549-ACE2-TMPRSS2 cells were infected with B.1, Delta and Omicron/BA.1, photomicrographs taken at 22 h post infection: GFP, green; nucleocapsid (N), red; DAPI nuclei, blue; scale bars, 100 µm. c , Fluorescence over 20 h ( n = 4 technical repeats, 3 independent experiments, 2 virus stocks). d , Calu-3 cell infection with B.1, Delta and Omicron/BA.1. Supernatants were assessed by RT-qPCR ( n = 3 technical repeats, 2 independent experiments). e , SARS-CoV-2 entry routes: route 1 – fusion at the cell surface following processing by TMPRSS2; route 2 – fusion occurs post endocytosis after processing by cathepsins. Routes 1 and 2 are inhibited by Camostat and E64d, respectively. f , SARS-CoV-2 pseudotype infection of cell lines, mean luciferase values (1 experiment, n = 8 technical repeats, representative of 3 independent experiments). Route 1 predominates in Calu-3, route 2 in HEK, and both routes are supported by A549-ACE2-TMPRSS2. Control is Pangolin CoV (route 2 only), negative control is no glycoprotein pseudotypes (No). g , Relative pseudotype infection (compared to untreated) of 10 μM protease inhibitor-treated cells; mean of 4 biological repeats, **** P < 0.0001, one-way ANOVA, Dunnett’s multiple comparisons test. h , Camostat (left) and E64d (right) titration against Delta, Omicron/BA.1 and Pangolin CoV in A549-ACE2-TMPRSS2; mean relative infection compared to untreated control ( n = 2 biological repeats). i , Immunoblot of spike-expressing HEK lysates with anti-spike (S2) and anti-actin. j , HEK infection by Omicron/BA.2 pseudotype; mean luciferase values (1 experiment, n = 8 technical repeats, representative of 3 independent experiments). k , hNEC infection with Delta and Omicron/BA.1 clinical isolates, supernatants assessed by RT-qPCR; mean of 2 independent biological repeats. l , Immunoblots of uninfected (left) and infected (right) hNEC and Calu-3 lysates. Immunoblots probed for ACE2, TMPRSS2, Cathepsin-L, GAPDH, Spike S1 and N. m , Cell–cell fusion (as in a ) in Omicron/BA.1-transfected cells treated with trypsin. Plot displays mean fluorescence (1 experiment, 3 technical repeats, representative of 2 independent experiments). n , Cell–cell fusion in Delta or Omicron/BA.1-transfected cells, ±0.3 µg ml −1 trypsin. Data are relative to fusion by Omicron/BA.1 spike (−trypsin, 8 h), mean of 2 biological repeats. All error bars represent s.e.m.

Article Snippet: Samples were then subjected to SDS–PAGE and immunoblotted for ACE2 (Abcam, ab108252), human Cathepsin-L (AF952, R&D Systems), TMPRSS2 (14437-1-AP, Proteintech), SARS-CoV-2 spike protein S1-NTD (E7M5X, Cell Signalling Technology), mouse anti-SARS-CoV-2 spike protein S2 (clone 1A9, GeneTex), sheep anti-SARS-CoV-2 nucleocapsid protein 3rd bleed , GAPDH (2118, Cell Signalling Technology), mouse anti-beta actin (AC-15, Abcam) or mouse anti-p55 (EVA365, NIBSC).

Techniques: Virus, Cell Culture, Expressing, Fluorescence, Generated, Infection, Quantitative RT-PCR, Luciferase, Control, Negative Control, Protease Inhibitor, Titration, Western Blot, Transfection

Journal: Nature Microbiology

Article Title: SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

doi: 10.1038/s41564-022-01143-7

Figure Lengend Snippet: GFP-10 and GFP-11 A549 ACE2 TMPRSS2 cells were co-cultured and infected with Delta and Omicron BA.1 clinical isolates or with live reverse-genetics virus in which the Delta or Omicron BA.1 spike is presented in the context of the ancestral wildtype B lineage genome. Fusion was quantified by measuring GFP signal, as in Fig. .

Article Snippet: Samples were then subjected to SDS–PAGE and immunoblotted for ACE2 (Abcam, ab108252), human Cathepsin-L (AF952, R&D Systems), TMPRSS2 (14437-1-AP, Proteintech), SARS-CoV-2 spike protein S1-NTD (E7M5X, Cell Signalling Technology), mouse anti-SARS-CoV-2 spike protein S2 (clone 1A9, GeneTex), sheep anti-SARS-CoV-2 nucleocapsid protein 3rd bleed , GAPDH (2118, Cell Signalling Technology), mouse anti-beta actin (AC-15, Abcam) or mouse anti-p55 (EVA365, NIBSC).

Techniques: Cell Culture, Infection, Virus

Journal: Nature Microbiology

Article Title: SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

doi: 10.1038/s41564-022-01143-7

Figure Lengend Snippet: SARS-CoV-2 infection of A549 ACE2 TMPRSS2 cells in the presence of 10 µM Camostat or E64d, data is expressed relative to untreated control, values represent mean across two independent experiments, asterisks indicate statistical significance (Two tailed T-test) between E64d treated Delta and Omicron infections. Error bars indicate standard error of the mean.

Article Snippet: Samples were then subjected to SDS–PAGE and immunoblotted for ACE2 (Abcam, ab108252), human Cathepsin-L (AF952, R&D Systems), TMPRSS2 (14437-1-AP, Proteintech), SARS-CoV-2 spike protein S1-NTD (E7M5X, Cell Signalling Technology), mouse anti-SARS-CoV-2 spike protein S2 (clone 1A9, GeneTex), sheep anti-SARS-CoV-2 nucleocapsid protein 3rd bleed , GAPDH (2118, Cell Signalling Technology), mouse anti-beta actin (AC-15, Abcam) or mouse anti-p55 (EVA365, NIBSC).

Techniques: Infection, Control, Two Tailed Test

Journal: Nature Microbiology

Article Title: SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

doi: 10.1038/s41564-022-01143-7

Figure Lengend Snippet: a , Top: schematic representation of domain swap constructs between WT (lineage B) and Omicron spike. Bottom: linear representation illustrates the junction points between the chimeric proteins. b , Domain swap pseudotype infection of HEK cells. Data represent mean luciferase values from a representative experiment ( n = 8 technical repeats, data are representative of 3 independent experiments). c , Sensitivity of domain swap pseudotypes to camostat and E64d in A549-ACE2-TMPRSS2 cells. Data are expressed relative to the untreated control for each virus. Values represent the mean of 3 biological repeats. d , Immunoblot analyses of lysates from HEK cells producing domain swap pseudotypes. Blots were probed for spike (S1 and processed S2), actin loading control and HIV Gag p55 transfection control. The actin control relates to the S1 blot, while the p55 control relates to the processed S2 blot. e , Quantification of spike proteolytic processing in WT (lineage B), Omicron BA.1 and the reciprocal RBD swaps. Data are expressed relative to the total for a given spike and represent the mean of 5 independent blots. f , Cell fusion assay using cells transfected with WT (lineage B) (left), Omicron BA.1 (right) and the reciprocal RBD swap spikes. Plot displays mean GFP fluorescence signal from 1 representative experiment. g , Cell–cell fusion (as in f ). Data are expressed relative to fusion by WT (lineage B) spike (at 16 h) and are the mean of 4 independent experiments. One-way ANOVA, * P < 0.01, **** P < 0.0001. In all plots, error bars represent s.e.m.

Article Snippet: Samples were then subjected to SDS–PAGE and immunoblotted for ACE2 (Abcam, ab108252), human Cathepsin-L (AF952, R&D Systems), TMPRSS2 (14437-1-AP, Proteintech), SARS-CoV-2 spike protein S1-NTD (E7M5X, Cell Signalling Technology), mouse anti-SARS-CoV-2 spike protein S2 (clone 1A9, GeneTex), sheep anti-SARS-CoV-2 nucleocapsid protein 3rd bleed , GAPDH (2118, Cell Signalling Technology), mouse anti-beta actin (AC-15, Abcam) or mouse anti-p55 (EVA365, NIBSC).

Techniques: Construct, Infection, Luciferase, Control, Virus, Western Blot, Transfection, Cell Fusion Assay, Fluorescence

Journal: Nature Microbiology

Article Title: SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

doi: 10.1038/s41564-022-01143-7

Figure Lengend Snippet: a , Vero ACE2 TMPRSS2 (VAT) and BHK-hACE2 cells were inoculated with diluted clinical samples. Viral progeny was quantified in the medium 5 dpi by RT-qPCR. b , Aliquots of the medium from samples named 204 and 205 were used to generate a P1 in BHK-hACE2 and Calu-3 cells and, limited to sample 205, a P2 in Calu-3 and Caco2 cells. Viral stocks were quantified by RT-qPCR.

Article Snippet: Samples were then subjected to SDS–PAGE and immunoblotted for ACE2 (Abcam, ab108252), human Cathepsin-L (AF952, R&D Systems), TMPRSS2 (14437-1-AP, Proteintech), SARS-CoV-2 spike protein S1-NTD (E7M5X, Cell Signalling Technology), mouse anti-SARS-CoV-2 spike protein S2 (clone 1A9, GeneTex), sheep anti-SARS-CoV-2 nucleocapsid protein 3rd bleed , GAPDH (2118, Cell Signalling Technology), mouse anti-beta actin (AC-15, Abcam) or mouse anti-p55 (EVA365, NIBSC).

Techniques: Quantitative RT-PCR