Journal: Antiviral Research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Structural elements in the ACE2-Fc constructs. a Schematic depiction of the main parts in an engineered ACE2-Fc molecule and their functional properties. b Design of the ACE2-Fc fusion protein; ACE2 parts in light and dark blue, IgG-Fc part in gray, spike (S) protein trimer in green and the receptor-binding domain (RBD) located at the tip of each spike protein in orange and dark green. The binding region as well as active site residues H374 and H378 important for the enzymatic activity of ACE2 are highlighted. Structures of the following Protein Data Bank (PBD) identifiers were used for modeling: 6M17, 6M0J, 6VSB, 5DK3. c Nomenclature and structural variations in the ACE2-Fc constructs. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL-185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 °C and 5% CO2.

Techniques: Construct, Functional Assay, Binding Assay, Activity Assay

Journal: Antiviral Research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Structural and functional characteristics of the ACE2-Fc proteins. a Far-UV CD spectra (left) and Near-UV CD spectra (right) of ACE2-Fc constructs indicating that all proteins exhibit similar secondary and tertiary structures. b Chromatograms and molecular mass from size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) indicating that the ACE2-Fc molecules form homodimers. c Non-reducing (top) and reducing (bottom) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showing that intermolecular disulfide bonds in the homodimers are formed. d Comparison of fluorescence signals over time obtained in an assay testing the cleavage of a fluorescent peptidyl-4-methylcoumaryl-7-amide (MCA). Relative fluorescent units (RFU) are given. e Amount of MCA cleaved after 30 min of incubation with the ACE2-Fc constructs. Ref1 and Ref2 are two different commercially available ACE2-Fc proteins from Genscript and Acrobiosystems, respectively. Bars are mean values; error bars depict the 95% confidence interval of six independent experiments shown as circles.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL-185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 °C and 5% CO2.

Techniques: Functional Assay, Circular Dichroism, Construct, Size-exclusion Chromatography, Multi-Angle Light Scattering, Polyacrylamide Gel Electrophoresis, SDS Page, Comparison, Fluorescence, Incubation

Journal: Antiviral Research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Interaction of the ACE2-Fc constructs with the SARS-CoV-2 RBD. a Surface plasmon resonance (SPR) was performed to obtain binding curves of ACE2-Fc fusion constructs and an unrelated Fc fusion protein (aflibercept) to an immobilized RBD from SARS-CoV-2. An exemplary binding curve is shown (RU = Response Units). b Binding constants of the ACE2-Fc constructs towards the RBD of SARS-CoV-2 (Mean ± SD of triplicate measurements). c ACE2-Fc fusion proteins were pre-incubated with the SARS-CoV-2 spike S1 protein and tested in a competition ELISA for their ability to neutralize S1 binding to immobilized ACE2 protein. Potent inhibition of SARS-CoV-2 spike S1 protein by ACE2-IgG4-Fc constructs (left) and ACE2-IgG1-Fc constructs (right). Data are represented as means ± SD of at least two independent experiments.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL-185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 °C and 5% CO2.

Techniques: Construct, SPR Assay, Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: Antiviral Research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Binding affinities of ACE2-Fc constructs to Fc-receptors.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL-185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 °C and 5% CO2.

Techniques: Binding Assay, Construct

Journal: Antiviral Research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Effect of ACE2-Fc on SARS-CoV-2-GFP infection and inhibition of SARS-CoV-2 primary isolates. a ACE2-IgG4-Fc reduces SARS-CoV-2-GFP replication. Representative fluorescent images of Vero E6 cells infected with SARS-CoV-2-GFP (multiplicity of infection (MOI) = 0.6 IU/cell) pre-incubated with ACE2-IgG4-Fc fusion construct 1 (632 nM). b ACE2-Fc fusion proteins potently neutralize coronaviruses. Serial dilutions of ACE2-Fc fusion proteins were pre-incubated with different coronaviruses and tested for their ability to neutralize the virus before infection of Vero E6 cells. Neutralization of SARS-CoV (top), SARS-CoV-2-Jan (middle) and SARS-CoV-2-April (bottom) by ACE2-IgG4-Fc constructs (left) and ACE2-IgG1-Fc constructs (right) is shown. Data given are means ± SEM of three independent experiments each. 50% inhibitory concentrations (IC50) determined as well as the 95% confidence interval (CI 95%) are given for each construct. The dashed lines indicate the IC50 values on the corresponding curves.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL-185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 °C and 5% CO2.

Techniques: Infection, Inhibition, Incubation, Construct, Virus, Neutralization

Journal: Antiviral Research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Neutralization potency of ACE2-IgG4-Fc fusion proteins increases with evolution of pandemic SARS-CoV-2 variants. Serial dilutions of ACE2-IgG4-Fc fusion constructs 1 and 3 were pre-incubated with the indicated SARS-CoV-2 primary isolates or VoCs and tested for their ability to prevent cytotoxicity following infection of A549-hACE2 cells. Neutralization of SARS-CoV-2-Jan, SARS-CoV-2-April and SARS-CoV-2 VoCs alpha, beta and delta by enzymatically active ACE2-IgG4-Fc construct 1 (left) and enzymatically inactive ACE2-IgG4-Fc construct 3 (right). Data given are means ± SEM of three independent experiments each. 50% inhibitory concentrations (IC50 values) determined as well as the 95% confidence interval (CI 95%) are shown for each construct. The dashed lines indicate the IC50 values on the corresponding curves.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL-185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 °C and 5% CO2.

Techniques: Neutralization, Construct, Incubation, Infection

Journal: bioRxiv

Article Title: SARS-CoV-2 Restructures the Host Chromatin Architecture

doi: 10.1101/2021.07.20.453146

Figure Lengend Snippet: a. A diagram showing the typical contact map patterns in Hi-C (and Hi-C 3.0 or other modified Hi-C approaches) that define A/B compartments, Topologically Associating Domains (TADs), chromatin loops or intra-TAD interactions (which perhaps include most enhancer-promoter contacts). This is an overall summary of these structures, but the exact definition of some structures may be subjected to variable interpretation, and the terminology may not always be used consistently – , . Often, A/B compartmentalization is illustrated by a checkerboard pattern of Hi-C contact matrices over large genomic sizes, indicating preferential interactions between genomic regions belonging to the same type of compartments (A: euchromatin and transcriptionally active; B: heterochromatin, transcriptionally inactive). TADs or chromatin domains are often characterized as a square or triangle-like structure on chromatin contact maps, reflecting a higher contact frequency between any regions inside the same TAD than with regions outside of the TAD. Intra-TAD enhancer-promoter contacts are considered to be facilitated by TADs, while TAD boundaries prevent aberrant interaction with regions outside of TADs. In Hi-C maps, the dot-shaped structures on the tip of domains suggests local enrichment of spatial interaction between a pair of two loci over nearby regions, and is regarded as a chromatin loop in this work. But loops may be subjected to other definitions in other studies. For example, enhancer-promoter contacts often do not appear as dot-shaped structures in Hi-C, but may be defined as loops by other work or other methods. Additional discussion, see , . b. Cartoon diagrams describe A-A and B-B association preference within regions of similar epigenetic features, which compartmentalizes chromosomes into A and B (the left part of the diagram). The diagram in the middle depicts a current model of cohesin loop extrusion inside TADs that generated such structures. The right side shows a zoom-in view of a part of a TAD that harbors enhancer-promoter contact that may play roles in gene transcriptional regulation. c. A workflow showing the experimental design. d. A barplot showing the percentage of RNA-Seq reads mapped to SARS-CoV-2 genome in Mock, 6-hr post infection (6hpi, 0.1 MOI), and 24 hpi (0.1 MOI) conditions. Mean and standard deviation (error bar) were calculated based on two biological replicates of RNA-Seq. e . Confocal images showing immunofluorescence staining of DAPI (DNA, blue) and the Spike protein of SARS-CoV-2 (red) in Mock and 24hpi (0.1 MOI) infected A549-ACE2 cells. Scale bars are shown.

Article Snippet: SARS-CoV-2 isolate USA-WA1/2020 (NR-52281; BEI Resources, Manassas, VA) was employed to infect human A549-ACE2 cells (NR53821; BEI Resources).

Techniques: Hi-C, Modification, Generated, RNA Sequencing Assay, Infection, Standard Deviation, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: SARS-CoV-2 Restructures the Host Chromatin Architecture

doi: 10.1101/2021.07.20.453146

Figure Lengend Snippet: a. A diagram illustrating the design of spike-in calibrated ChIP-Seq using mouse ESCs as spike-in for human A549 cells (with or without infection). b,c. Barplots showing the human/mouse reads ratio in both Mock and SARS-CoV-2 conditions which permit calibrated ChIP-Seq analyses of H3K27ac, H3K4me3, H3K27me3, and H3K9me3. A scale factor for each histone mark ChIP-seq was denoted above each plot. d,e,f,g. Scatter plots show virus-caused genome-wide changes of histone mark ChIP-Seq signals at 100kb bins for H3K27ac, H3K4me3, H3K9me3 and H3K27me3. The x,y-axis are natural logarithmically scaled reads densities from calibrated ChIP-Seq data. Dotted lines denote changes by two folds. h. Snapshots of Pearson’s correlation matrices, E1 compartmental scores, sliding correlation scores (SC), as well as ChIP-Seq tracks of H3K27ac and H3K9me3 in Mock or SARS-CoV-2 infected cells. The right side shows the difference of Pearson’s correlation matrices between SARS-CoV-2 and Mock (pink shows decrease). Red arrowheads on top of the H3K27ac peaks show strong reduction of this active mark after infection, which was accompanied by quantitative increase of H3K9me3 signals at the same region (black arrowheads). Accordingly, this entire A compartment shows reduced PCA E1 scores (yellow in the E1 track), showing less compartmental interactions within the same compartment but more interactions with nearby B compartments (see the differential Pearson’s correlation matrices to the right).

Article Snippet: SARS-CoV-2 isolate USA-WA1/2020 (NR-52281; BEI Resources, Manassas, VA) was employed to infect human A549-ACE2 cells (NR53821; BEI Resources).

Techniques: ChIP-sequencing, Infection, Genome Wide

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Time-dependent global cellular sphingolipid (SL) changes upon infection with three different CoVs. ( A ) Experimental design of the sphingolipidome analysis. Huh-7-ACE2 cells were mock infected or infected with the indicated CoV (multiplicity of infection [MOI] = 3) for 1, 6, and 12 hpi. ( B and C ) Corresponding growth kinetics and immunofluorescence images. Scale bars = 100 µm. ( D ) Heat maps showing fold changes of deregulated SL species at the indicated time points in relation to uninfected control (significant differences [ P ≤ 0.05] in bold and marked with asterisks) calculated from the replicates by one-way analysis of variance (ANOVA) with Dunnett´s test for multiple comparisons. ( E ) Corresponding Venn diagrams. Experiments were done in quintuplicates ( n = 5). ( F ) Simplified illustration of SL metabolism. Ceramide (Cer), as the centerpiece of the SL metabolic pathway, can be synthesized de novo via dhCer, via salvage pathway through hydrolysis of glycosphingolipids or by the sphingomyelinase (SMases) pathway through the hydrolysis of SM. Cer, ceramide; dhCer, dihydroceramide; dhSM, dihydrosphingomyelin; dhSph, dihydrosphingosine; HexCer, hexosylceramide; LacCer, lactosylceramide; S1P, sphingosine-1-phosphate; SM, sphingomyelin; Sph, sphingosine.

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Infection, Immunofluorescence, Control, Synthesized

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Antiviral activities of a/nSMase inhibition in CoVs replication. ( A through D ) Huh-7-ACE2 cells were mock-infected ( A and C ) or infected with the indicated virus (MOI = 0.1; B and D ) in the presence of increasing concentrations of SMase inhibitors ([ A and B ] ARC39 for aSMase and [ C and D ] PDDC for nSMase2) or dimethyl sulfoxide (DMSO) as solvent control. Cell viability ( A and C ) or virus titers ( B and D ) in the presence of increasing inhibitor concentrations were determined using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay or plaque assay. ( E and F ) Genetic manipulation of SMases or CoV-specific entry receptors using siRNA knockdown. ( E ) Huh-7-ACE2 cells were transfected with the indicated siRNAs, and the target mRNA was analyzed using qPCR. ( F ) Impact of siRNA silencing on viral replication. Huh-7-ACE2 cells were reverse transfected with siRNAs (100 nM) for 48 h before being infected with the indicated virus. Infectivity was assessed by image-based quantification of N-positive cells and was normalized to levels in cells targeted by scrambled (scr) siRNA controls. All experiments were performed in Huh-7-ACE2 cells mock-infected or infected with the indicated virus at an MOI of 0.1 in three independent replicates. All bar graphs show mean ± SD; asterisks indicate P values (* P < 0.05; ** P < 0.005; *** P < 0.0005) obtained by a two-tailed unpaired t -test.

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Inhibition, Infection, Virus, Solvent, Control, Plaque Assay, Knockdown, Transfection, Two Tailed Test

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Time-dependent antiviral effects of nSMase2 inhibitor on coronaviral RO formation. ( A ) HCoV-229E-infected Huh-7-ACE2 cells (MOI = 3) were treated with PDDC (10 µM) for different time periods post-infection as indicated below. Production of infectious virus progeny was determined using (pooled) cell culture supernatants collected until 12 hpi. Virus titers were determined and compared to the titer determined for infected but untreated cells. ( B through E ) Huh-7-ACE2 cells were infected with HCoV-229E and then either left untreated ( C ), or treated with PDDC (10 µM, D ) or K22 (40 µM, E ) for 8 hpi. Subcellular replication sites were identified by a double-stranded RNA (dsRNA)-specific antibody in the presence or absence of the indicated inhibitor. Nuclei were stained using DAPI. ( B ) Quantification of RO-positive cells by image-based quantification of dsRNA-positive cells in relation to total cell count. All bar graphs show mean ± SD; asterisks indicate P values (n.s., not significant; * P < 0.05; ** P < 0.005; *** P < 0.0005) obtained by a two-tailed unpaired t -test. ( C through E ) Corresponding representative images from one out of three independent experiments. The scale bar in the second row represents 5 µm. All experiments were performed in three independent replicates.

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Infection, Virus, Cell Culture, Staining, Cell Counting, Two Tailed Test

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Colocalization of ROs and sphingolipids in CoV-infected cells. ( A ) Huh-7-ACE2 cells were transfected with an Eqt-SM-oxGFP expression construct (to visualize SM, green) and after 48 h infected with the indicated CoV (MOI = 3). Eight hours post-infection, cells were fixed and stained for viral ROs (red) using an antibody against dsRNA, a specific marker for viral replication intermediates. ( B ) Huh-7-ACE2 cells were infected with the indicated CoV (MOI = 3) for 8 hpi, fixed, and permeabilized using 0.5% saponin. Cells were stained for viral ROs (dsRNA, red) using an antibody against dsRNA and an antibody against Cer (green). DAPI was used for staining of nuclei. Insets indicate regions of interest displayed at higher magnification in the next row. Colocalization signals, rates, and Manders correlation coefficients (MCCs) were calculated for the total images. Scale bars = 5 µm. Representative images from one out of three biologically independent experiments were shown.

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Infection, Transfection, Expressing, Construct, Staining, Marker

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Colocalization of CoV-induced ROs with nSMase2. ( A ) Huh-7-ACE2 cells were transfected with an nSMase2-eGFP-expressing construct (to visualize sphingomyelinase, green) and infected with HCoV-229E, SARS-CoV-2, or MERS-CoV (MOI = 3) and fixed 8 hpi with 3.7% paraformaldehyde (PFA). Viral ROs (red) were stained using an antibody against dsRNA. ( B ) Huh-7-ACE2 cells were infected with HCoV-229E (MOI = 3) and treated as indicated with the nSMase2 inhibitor PDDC. Viral ROs (red) and ceramide (green) were stained using respective antibodies. Filled arrows indicate colocalization. Outline arrows indicate ceramide spots without a dsRNA signal. DAPI was used for staining of nuclei. Insets indicate regions of interest displayed at higher magnification in the next row. Colocalization signals, rates, and Manders correlation coefficients (MCCs) were calculated for the total images. Scale bars = 5 µm. Representative images from one out of three biologically independent experiments were shown.

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Transfection, Expressing, Construct, Infection, Staining

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Artificially induced ROs by overexpressing a self-cleaving HCoV-229E nsp3-4 construct. ( A ) Schematic illustration of constructs generated to induce artificial HCoV-229E ROs upon transfection. The epitope tags used at the termini of the constructs are indicated as dots. The HA-nsp3-4-V5_K2481A construct contains an alanine substitution in the cleavage site of nsp3-4, therefore avoiding nsp3-mediated polyprotein cleavage. The HA-nsp3-4-V5_C1701A construct contains an alanine substitution that abrogates PL pro activity. ( B ) HEK-293T-ACE2 cells were transfected for 24 h with the indicated expression constructs, lysed, and HA-nsp3 and nsp4-V5-tagged proteins were detected using Western blot analysis. GAPDH served as a loading control. ( C ) Huh-7-ACE2 cells were transfected for 24 h with the indicated expression constructs, fixed, and stained using HA-specific (green) or V5-specific (red) antibodies. Subcellular localization was visualized by confocal microscopy using a Leica SP05. DAPI was used for staining of nuclei. Scale bars = 5 µm. ( D ) Huh-7-ACE2 cells were transfected with the indicated constructs, fixed 24 hours post-transfection, and analyzed via transmission electron microscopy analysis using a Zeiss LEO electron microscope. Scale bars = 500 nm.

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Construct, Generated, Transfection, Activity Assay, Expressing, Western Blot, Control, Staining, Confocal Microscopy, Transmission Assay, Electron Microscopy, Microscopy

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Colocalization of artificially induced ROs and Cer. ( A and B ) Huh-7-ACE2 cells were transfected with the indicated plasmids (0.75 µg DNA) expressing either HA-nsp3-4-V5 or mutants (HA-nsp3-4-V5_K2481A and HA-nsp3-4-V5_C1701A) ( A ) or the single constructs ( B ). After 24 h, the cells were fixed with 3.7% paraformaldehyde (PFA). The cells were then permeabilized with 0.5% saponin. Nsp3 or 4 (red) and Cer (green) were visualized using HA- (nsp3), V5- (nsp4), and Cer- specific antibodies. ( C and D ) Huh-7-ACE2 cells were transfected with the indicated plasmids (0.75 µg DNA) expressing nSMase_eGFP (green) and either HA-nsp3-4-V5 or mutants (HA-nsp3-4-V5_K2481A and HA-nsp3-4-V5_C1701A; C ) or the single constructs ( D ). After 24 h, the cells were fixed with 3.7% PFA. The cells were then permeabilized with 0.5% saponin. Nsp3 or 4 (red) visualized using HA- (nsp3) or V5- (nsp4) specific antibodies. DAPI was used for staining of nuclei. Colocalization signals, rates, and Manders correlation coefficients (MCCs) were calculated for the total images. Representative images from one out of three biologically independent experiments were shown. Scale bars = 5 µm.

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Transfection, Expressing, Construct, Staining

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Overview of sphingolipid (SL) changes upon artificial RO formation upon transfection with constructs expressing nsp3 and nsp4 in Huh-7-ACE2 cells. (A) Experimental design of the sphingolipidome analysis. Huh-7-ACE2 cells were mock transfected with empty vector control (pcDNA3.1) or transfected with either HA-nsp3-4-V5, HA-nsp3-4-V5_K2481A or HA-nsp3-4-V5_C1701A for 24 h. (B) Corresponding immunofluorescence images of transfected cells. The value indicates transfection efficacy of HA-nsp3-positive cells (green) in relation to total cell count. Scale bars = 100 µm. (C) Heatmap showing fold changes of deregulated SL species in relation to mock-transfected control (significant differences in bold and marked with asterisks, P ≤ 0.05). Cer, ceramide; dhCer, dihydroceramide; dhSM, dihydrosphingomyelin; dhSph, dihydrosphingosine; HexCer, hexosylceramide; LacCer, lactosylceramide; S1P, sphingosine-1-phosphate; SL, sphingolipid; SM, sphingomyelin; Sph, sphingosine

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Transfection, Construct, Expressing, Plasmid Preparation, Control, Immunofluorescence, Cell Counting

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Colocalization of CoV-induced ROs and Cer in lung-derived cells. (A) Adenocarcinoma cell line A549-ACE2 (for SARS-CoV-2) or A549-CD13 (for HCoV-229E) or (B) primary lung fibroblasts MRC-5 cells (for HCoV-229E and MERS-CoV) were infected with an MOI of 3 for 8 hpi. The fixed cells were then permeabilized with 0.5% saponin and stained against dsRNA (red) and Cer (green). DAPI was used for staining of nuclei. Colocalization signals, rates and MCCs were calculated for the total images. Scale bars = 5 µm. Representative images from one out of three biologically independent experiments were shown.

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Derivative Assay, Infection, Staining

Journal: mBio

Article Title: Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation

doi: 10.1128/mbio.00084-25

Figure Lengend Snippet: Overview of sphingolipid changes upon infection with HCoV-229E and SARS-CoV-2 in lung-derived cells. (A) Experimental design of the sphingolipidome analysis. A549-ACE2 or A549-CD13 cells were mock-infected or infected with HCoV-229E (A549-CD13) or SARS-CoV-2 (A549-ACE2) with an MOI of 3 for 12 hpi. (B and C) Corresponding viral titers and immunofluorescence images of A549-ACE2 (for SARS-CoV-2) and A549-CD13 (for HCoV-229E) cells (MOI = 3) 12 hpi. Scale bars = 100 µm. (D) Heatmap showing fold changes of deregulated sphingolipid species at the indicated time points in relation to uninfected control based on significant differences (significant differences in bold and marked with asterisks, P ≤ 0.05) calculated from the replicates by t -test (SARS-CoV-2) or one-way ANOVA with Dunnett´s test for multiple comparisons (HCoV-229E). (E) Corresponding Venn diagrams. Experiments were done in biological independent replicates ( n = 5). Cer, ceramide; dhCer, dihydroceramide; dhSM, dihydrosphingomyelin; dhSph, dihydrosphingosine; HexCer, hexosylceramide; LacCer, lactosylceramide; S1P, sphingosine-1-phosphate; SL, sphingolipid; SM, sphingomyelin; Sph, sphingosine.

Article Snippet: Human hepatoma cells (Huh-7; Japanese Collection of Research Bioresources cell bank), human embryonal kidney cells (HEK-293T; ATCC CRL-1573), and human lung adenocarcinoma cells (A549; ATCC CCL-185) overexpressing the ACE2 receptor (Huh-7-ACE2, HEK-293T-ACE2, A549-ACE2; kindly provided by Friedemann Weber, Institute of Virology, Justus Liebig University Giessen, Germany), A549 overexpressing CD13 and TMPRSS2 (A549-CD13; kindly provided by Krzysztof Pyrć, Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland), and primary human lung fibroblasts (MRC-5 cells; ATCC CCL-171) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) and supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL of penicillin, 100 μg/mL of streptomycin and 0.5 μg/mL puromycin).

Techniques: Infection, Derivative Assay, Immunofluorescence, Control

Journal: bioRxiv

Article Title: A guanidine-based coronavirus replication inhibitor which targets the nsp15 endoribonuclease and combines a direct antiviral effect with subtle boosting of interferon

doi: 10.1101/2024.09.09.611961

Figure Lengend Snippet: (A) Chemical structures of EPB-113 and its structural analogues EPB-102 and JMLv-061. (B) Inhibition of HCoV-229E replication in HEL299 cells was determined by MTS-based CPE reduction assay; infectious virus titration on the supernatant; and RT-qPCR-based viral load assay. Inhibition of SARS-CoV-2 replication in A549 ACE2/TMPRSS2 cells was quantified by viral load assay. Cytotoxicity was determined by MTS cell viability assay in mock-infected cells and is shown in dashed lines (open circles). Data points are the mean ± SEM (N=3-12). (C) Anti-HCoV-229E activity and cytotoxicity in HEL299 cells for EPB-102 and JMLv-061, two structural analogues of EPB-113. Data points are the mean ± SEM (N=3). (D) Table summarizing the values for antiviral activity and cytotoxicity of EPB-113, JMLv-061, EPB-102 and reference compound GS-441524.

Article Snippet: A549 cells expressing ACE2 and TMPRSS2 (A549 ACE2/TMPRSS2 ) were purchased from InvivoGen (No. a549d-cov2r) and cultured in DMEM supplemented with 6 mM L-glutamine; 25 mM glucose; 10% FCS; 100 µg/ml hygromycin and 0.5 µg/ml puromycin.

Techniques: Analogues, Inhibition, Virus, Titration, Quantitative RT-PCR, Viral-load Assay, Viability Assay, Infection, Activity Assay

Journal: bioRxiv

Article Title: A guanidine-based coronavirus replication inhibitor which targets the nsp15 endoribonuclease and combines a direct antiviral effect with subtle boosting of interferon

doi: 10.1101/2024.09.09.611961

Figure Lengend Snippet: (A) Setup of the resistance selection experiment. (B) Alignment of partial nsp15 sequences from 11 coronaviruses, with EPB-113 and 5h resistance sites marked with triangles. Both EPB-113 resistance sites, G248 and H250, are located at the catalytic core of nsp15 EndoU and strictly conserved in all CoVs, with H250 being part of the catalytic triad. Sequence similarities from Clustal Omega-aligned sequences were rendered using ESPript 3.0. (C) Structure of the catalytic core of SARS-CoV-2 EndoU bound to dsRNA (PDB 7TJ2). The residues associated with HCoV-229E resistance to EPB-113 or 5h are marked in blue and green, respectively. (D) Evaluation of EPB-113 and reference compound GS-441524 against nsp15-mutant CoVs, based on RT-qPCR-based viral load assay. Left panels: HEL299 cells infected with HCoV-229E WT; the G248V nsp15 or H250Y nsp15 mutant (obtained in this study); or the K60R nsp15 or T66 nsp15 mutant (previously selected under compound 5h) . Middle panels: 16HBE cells infected with H250A nsp15 mutant HCoV-229E, created by reverse genetics . Right panels: A549 ACE2/TMPRSS2 cells infected with K61R nsp15 or V67I nsp15 mutant SARS-CoV-2, created by reverse genetics. Data points are the mean ± SEM (N=2-3). The panel legends show the EC 90 values and statistical significance of the difference between mutant and WT, based on (left panels) ordinary one-way ANOVA with Dunnett’s corrections for multiple comparisons and (middle panels) two-tailed unpaired t-test.

Article Snippet: A549 cells expressing ACE2 and TMPRSS2 (A549 ACE2/TMPRSS2 ) were purchased from InvivoGen (No. a549d-cov2r) and cultured in DMEM supplemented with 6 mM L-glutamine; 25 mM glucose; 10% FCS; 100 µg/ml hygromycin and 0.5 µg/ml puromycin.

Techniques: Selection, Sequencing, Mutagenesis, Quantitative RT-PCR, Viral-load Assay, Infection, Two Tailed Test