Journal: Frontiers in Microbiology

Article Title: Niclosamide as a chemical probe for analyzing SARS-CoV-2 modulation of host cell lipid metabolism

doi: 10.3389/fmicb.2023.1251065

Figure Lengend Snippet: Workflow for lipidomic and transcriptomic profiling of Vero E6 cells after SARS-CoV-2 infection with and without niclosamide treatment. We used a time-of-addition assay experimental design to (i) capture the lipidomic profile of SARS-CoV-2 infected Vero E6 cells, and (ii) explore the effect of niclosamide on the lipidomic profile of Vero E6 cells when added in the absence of infection, with SARS-CoV-2 virus, or at 24 h post-infection. Each replicate experimental condition ( n = 3) was processed for LC-HRMS/MS or RNASeq analyses. Samples were seeded 48 h prior to the start of the experiment ( t = 0 h). For all samples, media was changed at the start of the experiment ( t = 0 h) and infected with virus (for infected sample groups). Sample collection is denoted by an up arrow and tube above the timeline, media changes are denoted by red bottles, addition of virus and DMSO/drug are denoted as well. Separate samples for each condition were collected for LC-HRMS/MS and RNAseq analysis, respectively. Created with BioRender.com .

Article Snippet: Vero clone E6 cells (ATCC: CRL-1586) were obtained from Dr. Pei-Yong Shi (University of Texas Medical Branch).

Techniques: Infection, Virus

Journal: Frontiers in Microbiology

Article Title: Niclosamide as a chemical probe for analyzing SARS-CoV-2 modulation of host cell lipid metabolism

doi: 10.3389/fmicb.2023.1251065

Figure Lengend Snippet: Niclosamide modulates lipid metabolism in Vero E6 cells in the absence of SARS-CoV-2 infection. (A,B) Bar graphs combining the log P-value (colored in blue) and the Log2 fold change (colored in orange) for total lipid expression at 16h vs 16h NIC (A) and 48h vs 48h NIC (B), data is sorted by low to high fold change. (C) Heatmap clustering of the top 50 lipids across all classes measured in Vero E6 cells showing the differential lipid regulation from 16h to 48h. NIC treatment increased several phosphatidylcholines (PC) and decreased several triglycerides (TG) comparing 16hr to 48hr. TG and PC Plasmalogens were increased at 16hr and 48hr without NIC but were decreased at 16hr and 48hr following NIC treatment.

Article Snippet: Vero clone E6 cells (ATCC: CRL-1586) were obtained from Dr. Pei-Yong Shi (University of Texas Medical Branch).

Techniques: Infection, Expressing

Journal: Frontiers in Microbiology

Article Title: Niclosamide as a chemical probe for analyzing SARS-CoV-2 modulation of host cell lipid metabolism

doi: 10.3389/fmicb.2023.1251065

Figure Lengend Snippet: Clustering analysis of virus free Vero E6 cell cultures comparing early growth (16 h) and late growth (48 h) with and without NIC. Pearson correlation and PCA including all lipids identified (A,B) . Pearson correlation and PCA including only TG lipids detected (C,D) . Clustering was evident in all cases, but correlation with all lipid expressions was primarily based on time points while correlation using only TG expression was from NIC treatment.

Article Snippet: Vero clone E6 cells (ATCC: CRL-1586) were obtained from Dr. Pei-Yong Shi (University of Texas Medical Branch).

Techniques: Virus, Expressing

Journal: Frontiers in Microbiology

Article Title: Niclosamide as a chemical probe for analyzing SARS-CoV-2 modulation of host cell lipid metabolism

doi: 10.3389/fmicb.2023.1251065

Figure Lengend Snippet: Global lipidomics analysis in SARS-CoV-2 infected Vero E6 cells at 16 h and 48 h. (A) Hierarchical cluster heatmap analysis depicting the major affected lipid clustering between 16 h and 48 h infection. (B) Volcano plot showing the differential lipid abundance with SARS-CoV-2 infection between 16 h and 48 h. The primary significantly upregulated lipids include plasmalogens, diglycerides and triglycerides. (C) Bar graph showing the total abundance of ether lipids between the time points. Lipid molecule abbreviations (shown in panel B ): PC-76 [1_PC (16,0_20:4) + Na], PC-114 [1_PC (18,1_20:4) + Na | 1_PC (16,0_22:5) + Na], PC-381 [2_PC (16,0_17:1) + Na | 2_PC (15,0_18:1) + Na], TG-584 [2_TG (18,1_22:4_22:4) + NH4], TG-590 [2_TG (20,2_22:3_22:4) + NH4], TG-598 [2_TG (22,3_22:3_22:3) + NH4], SM-524 [2_SM (d17:1/20:3) + H], Cer-NS-317 [2_Cer-NS (d17:1/14:0) + H], DG-15 [1_DG (16,0_18:1) + NH4 | 1_DG (16,1_18:0) + NH4], DG-19 [1_DG (18,1_18,1) + NH4 | 1_DG (16,0_20,2) + NH4 | 1_DG (16,1_20,1) + NH4 | 1_DG (18,0_18,2) + NH4], DG-341 [2_DG (16:1_18:1) + NH4 | 1_DG (16,1_18,1) + NH4 | 1_DG (16,0_18,2) + NH4 | 1_DG (14,0_20,2) + NH4], Ether-LPC-183 [1_Plasmanyl-LPC (O-18:0) + H], Ether-TG-493 [2_plasmanyl-TG (O-16:1_20:0_20:0) + NH4 | 2_plasmenyl-TG (P-16:0_20:0_20:0) + NH4], Plasmanyl-PC-167 [1_Plasmanyl-PC (O-18:0/16:0) + H], Plasmanyl-PC-170 [1_Plasmanyl-PC (O-16:0/22:2) + H], Plasmanyl-PC-175 [1_Plasmanyl-PC (O-16:0/22:1) + H], and Plasmanyl-PC-178 [1_Plasmanyl-PC (O-18:1/22:2) + H | 1_Plasmenyl-PC (P-18:0/22:2) + H].

Article Snippet: Vero clone E6 cells (ATCC: CRL-1586) were obtained from Dr. Pei-Yong Shi (University of Texas Medical Branch).

Techniques: Infection

Journal: Frontiers in Microbiology

Article Title: Niclosamide as a chemical probe for analyzing SARS-CoV-2 modulation of host cell lipid metabolism

doi: 10.3389/fmicb.2023.1251065

Figure Lengend Snippet: The effect of SARS-CoV-2 infection and NIC treatment on host cell lipid metabolism. SARS-CoV-2 infection in Vero E6 cells alters host cell lipid metabolism during early and late stages of infection. Increased transcription of lipid receptors LRP2 and VLDLR, and phosphorylation signaling regulators are observed throughout viral infection. Changes in TG composition from unsaturated to saturated acyl-chains occur as a function of viral replication, with an overall decrease in TG lipids at late infection timepoints. This change corresponds with an increase to DG and BMP lipids that is indicative of energy consumption and incorporation into membranes and vesicles, activation of autophagy pathways, as well as impacting viral replication. Treatment of cells with NIC alters lipid composition and gene regulation corresponding to apoptosis and autophagy related pathways. Decreases to ether lipids (TGs and DGs) and BMP are observed and reflect a decrease to exocytosis pathways for viral egress. Created with BioRender.com .

Article Snippet: Vero clone E6 cells (ATCC: CRL-1586) were obtained from Dr. Pei-Yong Shi (University of Texas Medical Branch).

Techniques: Infection, Phospho-proteomics, Activation Assay

Journal: Pathogens

Article Title: SARS-CoV-2 Exploits Non-Canonical Autophagic Processes to Replicate, Mature, and Egress the Infected Vero E6 Cells

doi: 10.3390/pathogens11121535

Figure Lengend Snippet: Subcellular structures of the cell lines infected with SARS-CoV-2. ( A ) Typical electron microscopy image of control Vero E6 cell showing cytoplasm with organelles near the nucleus. ( B ) Typical EM image of cytoplasm near the nucleus of SARS-CoV-2-infected Vero E6 cell 24 h post-infection. White asterisks—mitochondria, n—nucleus, short arrow—vesicle with attached virus particles, arrowheads—virus particles.

Article Snippet: Vero E6 cells (Vero C1008, ATCC CRL 1586) were purchased from ATCC, Manassas, USA, and cultured in a T-25 cell culture flask (Greiner Bio-One, Frickenhausen, Germany) as adherent monolayer until ~80% confluence in DMEM culture medium (Dulbecco’s Modified Eagle’s Medium), supplemented with 5% fetal bovine serum, antibiotics penicillin-streptomycin (Gibco ThermoFisher Scientific, Waltham, MA, USA), and the antimycotic amphotericin B (MilliporeSigma, Darmstadt, Germany) at 37 °C and 5% CO 2 saturation.

Techniques: Infection, Electron Microscopy, Control, Virus

Journal: Pathogens

Article Title: SARS-CoV-2 Exploits Non-Canonical Autophagic Processes to Replicate, Mature, and Egress the Infected Vero E6 Cells

doi: 10.3390/pathogens11121535

Figure Lengend Snippet: Replication of SARS-CoV-2 in Vero E6 cells. The cytoplasmic area shows various membrane vesicles (known as ERGIC) with virus particles (arrowheads), double-membrane vesicles with fibrous material (black asterisks), small mitochondria (white asterisks), mitochondrial fission (arrow), and the dense cytosol with freely distributed ribosomes and RNPs (dotted circle).

Article Snippet: Vero E6 cells (Vero C1008, ATCC CRL 1586) were purchased from ATCC, Manassas, USA, and cultured in a T-25 cell culture flask (Greiner Bio-One, Frickenhausen, Germany) as adherent monolayer until ~80% confluence in DMEM culture medium (Dulbecco’s Modified Eagle’s Medium), supplemented with 5% fetal bovine serum, antibiotics penicillin-streptomycin (Gibco ThermoFisher Scientific, Waltham, MA, USA), and the antimycotic amphotericin B (MilliporeSigma, Darmstadt, Germany) at 37 °C and 5% CO 2 saturation.

Techniques: Membrane, Virus

Journal: Pathogens

Article Title: SARS-CoV-2 Exploits Non-Canonical Autophagic Processes to Replicate, Mature, and Egress the Infected Vero E6 Cells

doi: 10.3390/pathogens11121535

Figure Lengend Snippet: Mitophagy-like structures and degradation of mitochondria in SARS-CoV-2-infected Vero E6 cells. The mitophagosome encapsulates a degrading mitochondrion. Note that the mitophagosome membrane is not fully closed (open arrow); white asterisks—degrading mitochondria; n—nucleus.

Article Snippet: Vero E6 cells (Vero C1008, ATCC CRL 1586) were purchased from ATCC, Manassas, USA, and cultured in a T-25 cell culture flask (Greiner Bio-One, Frickenhausen, Germany) as adherent monolayer until ~80% confluence in DMEM culture medium (Dulbecco’s Modified Eagle’s Medium), supplemented with 5% fetal bovine serum, antibiotics penicillin-streptomycin (Gibco ThermoFisher Scientific, Waltham, MA, USA), and the antimycotic amphotericin B (MilliporeSigma, Darmstadt, Germany) at 37 °C and 5% CO 2 saturation.

Techniques: Infection, Membrane

Journal: Pathogens

Article Title: SARS-CoV-2 Exploits Non-Canonical Autophagic Processes to Replicate, Mature, and Egress the Infected Vero E6 Cells

doi: 10.3390/pathogens11121535

Figure Lengend Snippet: A complex autophagosome in SARS-CoV-2-infected Vero E6 cell. Apposition of mitochondria and a large autophagosome containing an open mitophagosome (open arrow). Open arrowheads—cell debris; solid arrowheads—virus-like particles; white asterisks—mitochondria.

Article Snippet: Vero E6 cells (Vero C1008, ATCC CRL 1586) were purchased from ATCC, Manassas, USA, and cultured in a T-25 cell culture flask (Greiner Bio-One, Frickenhausen, Germany) as adherent monolayer until ~80% confluence in DMEM culture medium (Dulbecco’s Modified Eagle’s Medium), supplemented with 5% fetal bovine serum, antibiotics penicillin-streptomycin (Gibco ThermoFisher Scientific, Waltham, MA, USA), and the antimycotic amphotericin B (MilliporeSigma, Darmstadt, Germany) at 37 °C and 5% CO 2 saturation.

Techniques: Infection, Virus

Journal: Pathogens

Article Title: SARS-CoV-2 Exploits Non-Canonical Autophagic Processes to Replicate, Mature, and Egress the Infected Vero E6 Cells

doi: 10.3390/pathogens11121535

Figure Lengend Snippet: Phagophores in SARS-CoV-2-infected Vero E6 cells. ( A ) Cytoplasm with various phagophores; multivesicular body (mb), double-membrane vesicles (long arrows), and flattened zipped ER membranes (white arrow). ( B ) Phagosome-like vesicles (short arrows) with virus particles. Note that the membranes of these vesicles do not fully close, and the virus particles are mostly bound to the concave surface.

Article Snippet: Vero E6 cells (Vero C1008, ATCC CRL 1586) were purchased from ATCC, Manassas, USA, and cultured in a T-25 cell culture flask (Greiner Bio-One, Frickenhausen, Germany) as adherent monolayer until ~80% confluence in DMEM culture medium (Dulbecco’s Modified Eagle’s Medium), supplemented with 5% fetal bovine serum, antibiotics penicillin-streptomycin (Gibco ThermoFisher Scientific, Waltham, MA, USA), and the antimycotic amphotericin B (MilliporeSigma, Darmstadt, Germany) at 37 °C and 5% CO 2 saturation.

Techniques: Infection, Membrane, Virus

Journal: Pathogens

Article Title: SARS-CoV-2 Exploits Non-Canonical Autophagic Processes to Replicate, Mature, and Egress the Infected Vero E6 Cells

doi: 10.3390/pathogens11121535

Figure Lengend Snippet: Interaction of phagosomes with lipid droplets in SARS-CoV-2-infected Vero E6 cells. ( A ) Phagosome with virus particles (arrowheads) in close contact with lipid droplets (ld); open arrow—open membrane. ( B ) The lipid droplets (ld) in contact with a phagosome (white arrow), which contains a small phagosome (short arrow) encapsulating virus particles (arrowheads). ( C ) Apposition of mitochondrion and lipid droplet (ld). The long arrow points to the site of mitochondrial membrane degradation.

Article Snippet: Vero E6 cells (Vero C1008, ATCC CRL 1586) were purchased from ATCC, Manassas, USA, and cultured in a T-25 cell culture flask (Greiner Bio-One, Frickenhausen, Germany) as adherent monolayer until ~80% confluence in DMEM culture medium (Dulbecco’s Modified Eagle’s Medium), supplemented with 5% fetal bovine serum, antibiotics penicillin-streptomycin (Gibco ThermoFisher Scientific, Waltham, MA, USA), and the antimycotic amphotericin B (MilliporeSigma, Darmstadt, Germany) at 37 °C and 5% CO 2 saturation.

Techniques: Infection, Virus, Membrane

Journal: Pathogens

Article Title: SARS-CoV-2 Exploits Non-Canonical Autophagic Processes to Replicate, Mature, and Egress the Infected Vero E6 Cells

doi: 10.3390/pathogens11121535

Figure Lengend Snippet: Virus particles in phagosome vesicles in SARS-CoV-2-infected Vero E6 cells. ( A ) Virus particles of different morphology in the phagosome and nearby cytosol (arrowheads). ( B ) A group of budding virus particles in the lumen of a phagosome. Inset—a detail of the stalk between budding virus particles. ( C ) Virus particles adhering to remnants of phagosome membrane. Inset—a detail of the stalks between the virus particle and the phagosome membrane. ( D ) Virus particles in the lumen of the phagosome in direct contact with the lipid droplets. Arrowheads—virus particles; short arrow—phagosome membrane; white arrowheads—spikes; white arrow—nucleocapsids; ps—lumen of phagosomes; mf—a membrane fragment; white asterisk—mitochondrion; ld—lipid droplet.

Article Snippet: Vero E6 cells (Vero C1008, ATCC CRL 1586) were purchased from ATCC, Manassas, USA, and cultured in a T-25 cell culture flask (Greiner Bio-One, Frickenhausen, Germany) as adherent monolayer until ~80% confluence in DMEM culture medium (Dulbecco’s Modified Eagle’s Medium), supplemented with 5% fetal bovine serum, antibiotics penicillin-streptomycin (Gibco ThermoFisher Scientific, Waltham, MA, USA), and the antimycotic amphotericin B (MilliporeSigma, Darmstadt, Germany) at 37 °C and 5% CO 2 saturation.

Techniques: Virus, Infection, Membrane

Journal: Pathogens

Article Title: SARS-CoV-2 Exploits Non-Canonical Autophagic Processes to Replicate, Mature, and Egress the Infected Vero E6 Cells

doi: 10.3390/pathogens11121535

Figure Lengend Snippet: Egress of virus particles from SARS-CoV-2-infected Vero E6 cells. ( A ) Accumulation of the egress vacuoles under the cell surface. Note the virus particles anchored at the concave side of the vacuoles. ( B ) Outer surface of a cell with evaginated egress vacuoles. The virus particles are still anchored to the membrane, but now they are at the convex side of the evaginations. The cell segment at the bottom of the image shows a part of an uninfected cell. ( C ) Virus particles adhering to the evaginated cell membrane and clustered like grapes while exposed to the extracellular milieu. ( D ) The cell after the virus egress. Ec—extracellular space; long arrow—the surface membrane; open arrow—the membrane–free surface area; s—the surface lamellae; v—the egress vacuoles; n-nucleus; Vv—vacuoles; white asterisks—mitochondria.

Article Snippet: Vero E6 cells (Vero C1008, ATCC CRL 1586) were purchased from ATCC, Manassas, USA, and cultured in a T-25 cell culture flask (Greiner Bio-One, Frickenhausen, Germany) as adherent monolayer until ~80% confluence in DMEM culture medium (Dulbecco’s Modified Eagle’s Medium), supplemented with 5% fetal bovine serum, antibiotics penicillin-streptomycin (Gibco ThermoFisher Scientific, Waltham, MA, USA), and the antimycotic amphotericin B (MilliporeSigma, Darmstadt, Germany) at 37 °C and 5% CO 2 saturation.

Techniques: Virus, Infection, Membrane

Journal: Pathogens

Article Title: SARS-CoV-2 Exploits Non-Canonical Autophagic Processes to Replicate, Mature, and Egress the Infected Vero E6 Cells

doi: 10.3390/pathogens11121535

Figure Lengend Snippet: SARS-CoV-2 replication dynamics and infectivity in Vero E6 cells. ( A ) The columns show the mean of the viral E gene expression relative to the β-actin gene, n = 3. Error bars represent the standard error of the mean. hpi—hours post-infection. ( B ) TCID 50 assay of cytopathic effect (CPE) of SARS-CoV-2 at 24 hpi. The plate shows cytopathic effects of the virus at dilutions -1 to -10 times and the control (NC) without the virus. The lower the violet signal intensity, the lower number of viable cells attached to the dish.

Article Snippet: Vero E6 cells (Vero C1008, ATCC CRL 1586) were purchased from ATCC, Manassas, USA, and cultured in a T-25 cell culture flask (Greiner Bio-One, Frickenhausen, Germany) as adherent monolayer until ~80% confluence in DMEM culture medium (Dulbecco’s Modified Eagle’s Medium), supplemented with 5% fetal bovine serum, antibiotics penicillin-streptomycin (Gibco ThermoFisher Scientific, Waltham, MA, USA), and the antimycotic amphotericin B (MilliporeSigma, Darmstadt, Germany) at 37 °C and 5% CO 2 saturation.

Techniques: Infection, Gene Expression, Virus, Control

Journal: Pathogens

Article Title: SARS-CoV-2 Exploits Non-Canonical Autophagic Processes to Replicate, Mature, and Egress the Infected Vero E6 Cells

doi: 10.3390/pathogens11121535

Figure Lengend Snippet: Autophagic gene array in Vero E6 cells 24 hpi with SARS-CoV-2. Dots in the volcano plot represent the mean of the fold-change (virus-infected vs. non-infected cells, n = 3) in the gene expression levels normalized using a panel of the internal housekeeping genes (red dots—upregulated genes, green dots—downregulated genes). The grey area indicates the cut-off level of p ≤ 0.05.

Article Snippet: Vero E6 cells (Vero C1008, ATCC CRL 1586) were purchased from ATCC, Manassas, USA, and cultured in a T-25 cell culture flask (Greiner Bio-One, Frickenhausen, Germany) as adherent monolayer until ~80% confluence in DMEM culture medium (Dulbecco’s Modified Eagle’s Medium), supplemented with 5% fetal bovine serum, antibiotics penicillin-streptomycin (Gibco ThermoFisher Scientific, Waltham, MA, USA), and the antimycotic amphotericin B (MilliporeSigma, Darmstadt, Germany) at 37 °C and 5% CO 2 saturation.

Techniques: Virus, Infection, Gene Expression

Journal: medRxiv

Article Title: Different HMGCR-inhibiting statins vary in their association with increased survival in patients with COVID-19

doi: 10.1101/2022.04.12.22273802

Figure Lengend Snippet: (a) The NeMoCAD gene network analysis tool is a drug repurposing algorithm that uses Bayesian statistical network analysis combined with data from publicly available datasets (e.g., LINCS, KEGG, CTD, TRRUST) for reference transcriptional signatures and to define regulatory network architecture. The algorithm identifies transcriptome-wide differential expression profiles between two biological states (e.g., healthy vs. diseased) in experimental or published transcriptomic datasets and defines the target normalization signature, i.e., the subset of genes that would need to reverse their expression to revert one state to the other. The output of NeMoCAD includes correlation and causation predictions for numerous chemical compounds and approved drugs in the LINCS database based on their ability to reverse the differential expression profile of interest. (b) Statins are predicted to shift the COVID-19 state to a healthy state, with simvastatin predicted for all datasets analyzed (14). Rosuvastatin was the only statin not predicted (n.p.) for any COVID-19 transcriptomics signatures. (c) 8 of 9 statins in the LINCS database were in the top 25% of drugs predicted for at least one dataset investigated. (d) Frequency of prediction for each statin when input datasets are stratified by sample source and tissue origin. Prediction frequency is normalized by the number of input datasets from each sample source and tissue origin. (e) Inhibitory and cytotoxicity parameters from SARS-CoV-2 infection of Vero6 wild-type or GFP-expressing cells for a subset of statins contained in the LINCS database. Mean parameters for each statin are derived from two independent experiments.

Article Snippet: All drug screens to assess SARS-CoV-2 inhibition and cytotoxicity were performed with Vero E6 (Vero6) cells (ATCC# CRL 1586) using published methods ( Supplemental Methods ).

Techniques: Quantitative Proteomics, Expressing, Infection, Derivative Assay

Journal: medRxiv

Article Title: Different HMGCR-inhibiting statins vary in their association with increased survival in patients with COVID-19

doi: 10.1101/2022.04.12.22273802

Figure Lengend Snippet: (a) Dose-response curves demonstrating the ability for simvastatin to inhibit GFP-SARS-CoV-2 infection (MOI=0.1) in a dose-dependent manner in Vero6 cells. (b) Simvastatin also inhibits the human coronavirus, OC43, in HUVEC cells when added at 1 μ M or 5 μ M concentrations (***p<0.001, **p<0.01). Error bars represent average +/- s.d.; repeated in n=2 independent biological experiments displayed in light (experiment 1) and dark (experiment 2) shaded data points.

Article Snippet: All drug screens to assess SARS-CoV-2 inhibition and cytotoxicity were performed with Vero E6 (Vero6) cells (ATCC# CRL 1586) using published methods ( Supplemental Methods ).

Techniques: Infection