Journal: Viruses

Article Title: Population-Specific ACE2 Single-Nucleotide Polymorphisms Have Limited Impact on SARS-CoV-2 Infectivity In Vitro

doi: 10.3390/v13010067

Figure Lengend Snippet: ACE2 single-nucleotide polymorphisms (SNPs) found in different populations.

Article Snippet: To generate the plasmid (pCMV6-hACE2-FLAG) expressing C-terminally FLAG-tagged wild-type human ACE2 (WT ACE2), a DNA fragment encoding the full-length human ACE2 open reading frame (ORF) was amplified by PCR using a human ACE2-expressing plasmid (HG10108-UT; Sino Biological, Beijing, China) as a template and then inserted in-frame into the pCMV6-Entry vector (OriGene, Rockville, MD, USA) between the SgfI and EcoRV sites connecting the ACE2 ORF and FLAG-tag cDNA sequences.

Techniques:

Journal: Viruses

Article Title: Population-Specific ACE2 Single-Nucleotide Polymorphisms Have Limited Impact on SARS-CoV-2 Infectivity In Vitro

doi: 10.3390/v13010067

Figure Lengend Snippet: Mapping of the amino acid residues corresponding to the selected SNPs used in this study. The positions of the amino acid residues corresponding to the selected SNPs are marked in red on the crystal structure of ACE2 (1R42). Three amino acid positions (N637, L656, and N720) are not shown here because the regions containing these amino acids are not present in this crystal structure. The three separate regions reported to be important for binding to SARS-CoV-S are colored in yellow, magenta, and orange.

Article Snippet: To generate the plasmid (pCMV6-hACE2-FLAG) expressing C-terminally FLAG-tagged wild-type human ACE2 (WT ACE2), a DNA fragment encoding the full-length human ACE2 open reading frame (ORF) was amplified by PCR using a human ACE2-expressing plasmid (HG10108-UT; Sino Biological, Beijing, China) as a template and then inserted in-frame into the pCMV6-Entry vector (OriGene, Rockville, MD, USA) between the SgfI and EcoRV sites connecting the ACE2 ORF and FLAG-tag cDNA sequences.

Techniques: Binding Assay

Journal: Viruses

Article Title: Population-Specific ACE2 Single-Nucleotide Polymorphisms Have Limited Impact on SARS-CoV-2 Infectivity In Vitro

doi: 10.3390/v13010067

Figure Lengend Snippet: Effects of amino acid substitutions corresponding to SNPs in ACE2 on binding to SARS-2-S and cell entry mediated by SARS-2-S in vitro. ( A ) Schematic diagram of the construct used to express the ACE2 variants in this study. Amino acid substitutions corresponding to the SNPs were independently introduced into C-terminally FLAG-tagged human ACE2. Red, signal peptide; yellow, ADAM17 cleavage site; gray, transmembrane and cytoplasmic regions. ( B ) HEK293T cells were transfected with plasmids encoding either FLAG-tagged WT or variant ACE2 proteins. The pCMV6-Entry vector only (Empty) was used as the negative control. ACE2 and GAPDH levels in the total cell lysates were analyzed by western blotting. ( C ) HEK293T cells transfected with plasmids expressing either FLAG-tagged WT or variant ACE2 proteins were biotin-labeled and the cell-surface proteins were pulled down with streptavidin beads. The levels of WT ACE2 and ACE2 variants on the cell surface (in the pulled down samples) and in the total cell lysate were then analyzed by western blotting. The levels of transferrin receptor 1 (TFRC) on the cell surface and in the total cell lysate were also analyzed as an internal control. The ratios of the levels of the protein on the cell surface to that in the total cell lysate were calculated for the WT and each variant after first normalizing the data to the corresponding ratios for TFRC. The value for WT ACE2 was set to 1. ( D ) HEK293T cells transfected with plasmids expressing either FLAG-tagged WT or variant ACE2 proteins were incubated with rSARS-2-S1(D614G), and the levels of rSARS-2-S1(D614G) binding to these cells were analyzed by flow cytometry. The value for WT ACE2 was set to 100%. ( E ) HEK293T cells transfected with plasmids expressing either FLAG-tagged WT or variant ACE2 proteins were inoculated with rVSV∆G-GFP/SARS-2-S(D614G). After 16 h, the proportions of GFP-positive cells were determined with a high-content imaging system. (C–E) Data represent the means + S.D. of at least three independent experiments. White bars, controls (vector-only transfected or WT ACE2); black bars, East Asian population-specific SNPs; gray, non-East Asian population-specific SNPs. Empty, transfected with pCMV6-Entry vector only as the negative control; ND, not detected; WT, wild-type ACE2. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: To generate the plasmid (pCMV6-hACE2-FLAG) expressing C-terminally FLAG-tagged wild-type human ACE2 (WT ACE2), a DNA fragment encoding the full-length human ACE2 open reading frame (ORF) was amplified by PCR using a human ACE2-expressing plasmid (HG10108-UT; Sino Biological, Beijing, China) as a template and then inserted in-frame into the pCMV6-Entry vector (OriGene, Rockville, MD, USA) between the SgfI and EcoRV sites connecting the ACE2 ORF and FLAG-tag cDNA sequences.

Techniques: Binding Assay, In Vitro, Construct, Transfection, Variant Assay, Plasmid Preparation, Negative Control, Western Blot, Expressing, Labeling, Incubation, Flow Cytometry, Imaging

Journal: Viruses

Article Title: Population-Specific ACE2 Single-Nucleotide Polymorphisms Have Limited Impact on SARS-CoV-2 Infectivity In Vitro

doi: 10.3390/v13010067

Figure Lengend Snippet: Neutralization assay of shedded ACE2 variants. HEK293T/ACE2 cells were inoculated with rVSV∆G-GFP/SARS-2-S(D614G) pre-incubated for 1 h at 37 °C with serially diluted tissue culture supernatants (TCS) from cells transfected with wild-type (WT) ACE2- or ACE2 variant-expressing plasmids. After 16 h, the levels of virally infected (GFP + ) cells were measured with a high-content imaging system. The mean number of GFP + cells inoculated with rVSV∆G-GFP/SARS-2-S(D614G) pre-incubated with the TCS from empty vector-transfected cells was set to 100%. The dilution factors required to attain 50% inhibition of viral infection (IC 50 ) were determined using GraphPad Prism 9 software. Data represent the mean ± S.D. of three independent experiments.

Article Snippet: To generate the plasmid (pCMV6-hACE2-FLAG) expressing C-terminally FLAG-tagged wild-type human ACE2 (WT ACE2), a DNA fragment encoding the full-length human ACE2 open reading frame (ORF) was amplified by PCR using a human ACE2-expressing plasmid (HG10108-UT; Sino Biological, Beijing, China) as a template and then inserted in-frame into the pCMV6-Entry vector (OriGene, Rockville, MD, USA) between the SgfI and EcoRV sites connecting the ACE2 ORF and FLAG-tag cDNA sequences.

Techniques: Neutralization, Incubation, Transfection, Variant Assay, Expressing, Infection, Imaging, Plasmid Preparation, Inhibition, Software

Journal: bioRxiv

Article Title: Characterization of SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.618 on cell entry, host range, and sensitivity to convalescent plasma and ACE2 decoy receptor

doi: 10.1101/2021.09.03.458829

Figure Lengend Snippet: (A) HeLa-ACE2 cells were incubated with recombinant RBD-His proteins bearing mutations of Kappa, Delta and B.1.618 or individual mutation. The binding of RBD-His with cells were analyzed by flow cytometry. Values are expressed as the percent of cells positive for RBD-His among the ACE2-expressing cells (zsGreen1+ cells) and shown as the means ± SD from 3 biological replicates. This experiment was independently performed three times with similar results. (B) The binding kinetics of ACE2 proteins (human or mouse) with recombinant WT or Delta SARS-CoV-2 RBD were obtained using the BIAcore. ACE2 proteins were captured on the chip, and serial dilutions of RBD were then injected over the chip surface. Experiments were performed three times with similar result, and one set of representative data is displayed. (C) Immunoblot analysis of spike protein cleavage of pseudovirus of Kappa and P681R using polyclonal antibodies against spike. Full-length spike (S0), S1, and S2 protein are indicated. The ratio of the S1/S0 or S2/S0 was quantitatively analyzed using ImageJ software. This experiment is repeated twice independently, and data are normalized to the WT (D614G) of individual experiment.

Article Snippet: The cDNAs encoding the ACE2 orthologs were synthesized by GenScript and cloned into the pLVX-IRES-zsGreen1 vector (Catalog No. 632187, Clontech Laboratories, Inc) with a C-terminal FLAG tag.

Techniques: Incubation, Recombinant, Mutagenesis, Binding Assay, Flow Cytometry, Expressing, Injection, Western Blot, Software

Journal: JACS Au

Article Title: Engineered Receptor Capture Combined with Mass Spectrometry Enables High-Throughput Detection and Quantitation of SARS-CoV-2 Spike Protein

doi: 10.1021/jacsau.4c00980

Figure Lengend Snippet: Directed evolution of ACE2 ectodomain. (A) Schematic representation of cell surface expressed ACE2 ectodomain. Full-length ACE2 was expressed with an amino-terminal FLAG tag and short linker region. (B) Selection of ACE2 for increased affinity. Flow cytometry plots are shown for cells incubated with RBD and stained for bound RBD using anti-His-phycoerythrin, and ACE2 expression using anti-FLAG-allophycocyanin. Three rounds of selection at the indicated RBD concentrations and diversification were performed. The sort windows are indicated for each round of selection. (C) Amino acid substitutions of evolved ACE2 are shown in red. (D) Position of substitutions in ACE2 structure (PDB entry ID: 6M0J ). Left panel shows part of ACE2 structure (blue) in complex with RBD (yellow) with substitutions in evolved ACE2 shown in red. Position of A368L is shown in orange. Right panel shows structure of RBD binding interface of ACE2 and the position of substitutions. Position of RBD-interacting residues in Wt-ACE2 are indicated in green. (E) Kinetic binding constants for RBD binding by Wt-ACE2, evolved ACE2 and evolved ACE2 with A386L, measured by biolayer interferometry with soluble ACE2 (19–615) and immobilized monomeric RBD. Data are shown as means and SEM for at least two experiments.

Article Snippet: DNA encoding full-length human ACE2 was a gift from Hyeryun Choe (Addgene plasmid # 1786; http://n2t.net/addgene:1786 ; RRID:Addgene_1786).

Techniques: FLAG-tag, Selection, Flow Cytometry, Incubation, Staining, Expressing, Binding Assay

Journal: JACS Au

Article Title: Engineered Receptor Capture Combined with Mass Spectrometry Enables High-Throughput Detection and Quantitation of SARS-CoV-2 Spike Protein

doi: 10.1021/jacsau.4c00980

Figure Lengend Snippet: ACE2 multimerization increases binding to RBD. (A) Schematic representation of monomeric (ACE2–615), dimeric (ACE2–740), and pentameric (ACE2-COMP) ACE2. (B) Negative stain electron microscopy of monomeric, dimeric, and pentameric ACE2 (left to right). Scale bar, 50 nm. (C) Kinetic binding constants for RBD binding by Wt-ACE2 dimer and pentamer measured by biolayer interferometry with soluble ACE2 and immobilized RBD. Data are shown as means and SEM for two experiments. (D) Kinetic association and dissociation curves for monomeric, dimeric, and pentameric ACE2 demonstrating decreased dissociation kinetics for dimer and pentamer, and retention of bound RBD for more than 8 h for pentamer. (E) Negative stain electron microscopy image of ACE2-COMP bound to SARS-CoV-2 virus. Scale bar, 100 nm.

Article Snippet: DNA encoding full-length human ACE2 was a gift from Hyeryun Choe (Addgene plasmid # 1786; http://n2t.net/addgene:1786 ; RRID:Addgene_1786).

Techniques: Binding Assay, Staining, Electron Microscopy, Virus

Journal: JACS Au

Article Title: Engineered Receptor Capture Combined with Mass Spectrometry Enables High-Throughput Detection and Quantitation of SARS-CoV-2 Spike Protein

doi: 10.1021/jacsau.4c00980

Figure Lengend Snippet: Binding of ACE2-COMP to RBD variants. (A) Kinetic binding constants for RBD binding by ACE2-COMP measured by biolayer interferometry with soluble ACE2 and immobilized monomeric RBD. Data are shown as means and SEM for two experiments. (B) Kinetic association and dissociation curves for binding of ACE2-COMP to B.1.617.1/3 and B.1.351 variant RBD, demonstrating retention of bound RBD for more than 8 h.

Article Snippet: DNA encoding full-length human ACE2 was a gift from Hyeryun Choe (Addgene plasmid # 1786; http://n2t.net/addgene:1786 ; RRID:Addgene_1786).

Techniques: Binding Assay, Variant Assay

Journal: JACS Au

Article Title: Engineered Receptor Capture Combined with Mass Spectrometry Enables High-Throughput Detection and Quantitation of SARS-CoV-2 Spike Protein

doi: 10.1021/jacsau.4c00980

Figure Lengend Snippet: ACE2-COMP trap capture-based mass spectrometric assay for the resolution and detection of SARS-CoV-2 spike peptides. (A) Overlayed chromatogram of saliva with spike protein (157 nM) extracted with the trap assay but with plates that lack ACE2-COMP capture protein. (B) Overlayed chromatogram of saliva with spike protein (157 nM) extracted using plates coated with the ACE2-COMP capture protein. (C) Concentration dependence of spike protein detection in PBS and saliva (as indicated).

Article Snippet: DNA encoding full-length human ACE2 was a gift from Hyeryun Choe (Addgene plasmid # 1786; http://n2t.net/addgene:1786 ; RRID:Addgene_1786).

Techniques: TRAP Assay, Concentration Assay

Journal: JACS Au

Article Title: Engineered Receptor Capture Combined with Mass Spectrometry Enables High-Throughput Detection and Quantitation of SARS-CoV-2 Spike Protein

doi: 10.1021/jacsau.4c00980

Figure Lengend Snippet: Binding of Wt and Evolved ACE2 to RBD Variants

Article Snippet: DNA encoding full-length human ACE2 was a gift from Hyeryun Choe (Addgene plasmid # 1786; http://n2t.net/addgene:1786 ; RRID:Addgene_1786).

Techniques: Binding Assay

Journal: Viruses

Article Title: Population-Specific ACE2 Single-Nucleotide Polymorphisms Have Limited Impact on SARS-CoV-2 Infectivity In Vitro

doi: 10.3390/v13010067

Figure Lengend Snippet: Effects of amino acid substitutions corresponding to SNPs in ACE2 on binding to SARS-2-S and cell entry mediated by SARS-2-S in vitro. ( A ) Schematic diagram of the construct used to express the ACE2 variants in this study. Amino acid substitutions corresponding to the SNPs were independently introduced into C-terminally FLAG-tagged human ACE2. Red, signal peptide; yellow, ADAM17 cleavage site; gray, transmembrane and cytoplasmic regions. ( B ) HEK293T cells were transfected with plasmids encoding either FLAG-tagged WT or variant ACE2 proteins. The pCMV6-Entry vector only (Empty) was used as the negative control. ACE2 and GAPDH levels in the total cell lysates were analyzed by western blotting. ( C ) HEK293T cells transfected with plasmids expressing either FLAG-tagged WT or variant ACE2 proteins were biotin-labeled and the cell-surface proteins were pulled down with streptavidin beads. The levels of WT ACE2 and ACE2 variants on the cell surface (in the pulled down samples) and in the total cell lysate were then analyzed by western blotting. The levels of transferrin receptor 1 (TFRC) on the cell surface and in the total cell lysate were also analyzed as an internal control. The ratios of the levels of the protein on the cell surface to that in the total cell lysate were calculated for the WT and each variant after first normalizing the data to the corresponding ratios for TFRC. The value for WT ACE2 was set to 1. ( D ) HEK293T cells transfected with plasmids expressing either FLAG-tagged WT or variant ACE2 proteins were incubated with rSARS-2-S1(D614G), and the levels of rSARS-2-S1(D614G) binding to these cells were analyzed by flow cytometry. The value for WT ACE2 was set to 100%. ( E ) HEK293T cells transfected with plasmids expressing either FLAG-tagged WT or variant ACE2 proteins were inoculated with rVSV∆G-GFP/SARS-2-S(D614G). After 16 h, the proportions of GFP-positive cells were determined with a high-content imaging system. (C–E) Data represent the means + S.D. of at least three independent experiments. White bars, controls (vector-only transfected or WT ACE2); black bars, East Asian population-specific SNPs; gray, non-East Asian population-specific SNPs. Empty, transfected with pCMV6-Entry vector only as the negative control; ND, not detected; WT, wild-type ACE2. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: To generate the plasmid (pCMV6-hACE2-FLAG) expressing C-terminally FLAG-tagged wild-type human ACE2 (WT ACE2), a DNA fragment encoding the full-length human ACE2 open reading frame (ORF) was amplified by PCR using a human ACE2-expressing plasmid (HG10108-UT; Sino Biological, Beijing, China) as a template and then inserted in-frame into the pCMV6-Entry vector (OriGene, Rockville, MD, USA) between the SgfI and EcoRV sites connecting the ACE2 ORF and FLAG-tag cDNA sequences.

Techniques: Binding Assay, In Vitro, Construct, Transfection, Variant Assay, Plasmid Preparation, Negative Control, Western Blot, Expressing, Labeling, Incubation, Flow Cytometry, Imaging

Journal: medRxiv

Article Title: Targeting ATP2B1 impairs PI3K/Akt/Fox-O3 signaling and reduces SARS-COV-2 replication in vivo

doi: 10.1101/2022.09.02.22279542

Figure Lengend Snippet: (A, B) Linkage disequilibrium (LD) analyses on the top 5 SNPs (rs11105352; rs11105353; rs73437358; rs111337717; rs2681492) in order to select those independent. The SNP rs10777221 was excluded from this analyses because located at most 5’ region in extragenic ATP2B1 locus region ( A ). The graph in ( B ) shows the only SNP not in LD is rs111337717 (black boxes). (C) Sanger sequencing of the genomic region of ATP2B1 locus (chr12:89,643,709-89,643,749) in HEK293T-ACE2 cells to exclude the presence of intronc variance potentially responsible for alterated transcriptional levels of ATP2B1 gene. The red box indicate the nucleotide wild type allele for the SNP here studied. (D) Sanger sequencing of the genomic region of ATP2B1 locus (chr12:89,643,709-89,643,749) in human primary epithelial nasal cells to exclude the presence of intronic variants potentially responsible for alterated transcriptional levels of ATP2B1 gene. (E) Quantification of mRNA abundance relative to that in control (CTR) cells (2−ΔΔCt) for FOXK2, FOXP1 and FOXO3 genes. RT- PCR analysis of RNA extracted from HEK293T-ACE2 cells treated with 20 mM valproc acid (VPA) for 16 hours or 0.01% DMSO as vehicle control. Data are means ± SD. ** p<0.01, ***P<0.001 (unpaired two-tailed student’s t test; N = 3 independent experiments per group). (F) Expression levels of FOXO3 in single-nuclei RNA-seq (snRNA-seq) analyses performed on >116,000 nuclei from n.19 COVID-19 autopsy lungs and n.7 pre-pandemic controls ( Melms et al ., 2021 ). (G) Genome browser screenshots showing accumulation of normalized FOXO3 signal, together with CromHMM state segmentation and H3K4me3 signal (ENCODE), along the ATP2A1 gene in human cells. ForCromHMM state segmentation colors indicate: Bright Red – Promoter; Orange and yellow - enhancer; Green - Transcriptional transition. The expanded view of the highlighted region, on the left, shows FOXO3 peaks over ATP2A1 enhancer regions, as marked by yellow region of CromHMM.

Article Snippet: Then, in order to validate the role of FOXO3 as a positive transcriptional regulator of ATP2B1 and ATP2A1 , we transiently overexpressed a FOXO3-encoding plasmid (containing the coding region of FOXO3; #14937, Addgene) in HEK293T-ACE2 cells.

Techniques: Sequencing, Control, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, Expressing, RNA Sequencing

Journal: medRxiv

Article Title: Targeting ATP2B1 impairs PI3K/Akt/Fox-O3 signaling and reduces SARS-COV-2 replication in vivo

doi: 10.1101/2022.09.02.22279542

Figure Lengend Snippet: (A) In-silico analysis for the presence of noncoding variants in the genomic region of the ATP2B1 locus acting as expression quantitative traits (eQTLs) located in putative elements responsible for transcriptional regulation. Here, 77 SNPs were selected that were eQTLs for the ATP2B1 gene (P<1 ×10 -6 ) using the GTex database. These SNPs were then annotated with prediction functional scores calculated by the GWAVA tool. The top five SNPs were selected to verify their linkage disequilibrium (LD). The rs111337717 SNP was not in linkage disequilibrium. (B) Alignment of the sequence genomic region flanking rs111337717 SNP (NC_000012.12:g.89643729T>C) SNP [(CACATG(T/C)ACATTAT)] shows the conservation through evolution across different species, with a degree of sequence identity and homology. (C) Literature search of publically available datasets obtained from single-nuclei RNA-seq on >116,000 nuclei from 19 COVID19 autopsy lungs and 7 pre-pandemic controls ( https://singlecell.broadinstitute.org/single_cell/study/SCP1052/covid-19-lung-autopsy-samples [ Melms et al ., 2021 ]) to determine whether FOXOs transcriptional factors showed distinct fractional and dysfunctional changes across the lungs from the COVID19 decedents. (D) Genome browser screenshots showing accumulation of normalized FOXO3 signal, together with CromHMM state segmentation and the H3K4me3 signal (ENCODE), along the ATP2B1 gene in human cells. For the CromHMM state segmentation: bright red, promoter; orange and yellow, enhancer; green, transcriptional transition; red arrow, polymorphism-containing region. The expanded view of the highlighted region (left) shows FOXO3 peaks over the ATP2B1 promoter and enhancer regions, as marked by H3K4me3 and CromHMM (red and orange regions) respectively. (E) Representative immunoblotting analysis using antibodies against the indicated proteins on total protein lysates obtained from HEK293T-ACE2 cells infected with SARS-CoV-2 VOC Delta at 0.026 MOI for 72 h. Noninfected cells were used as negative control. All experiments were performed in triplicate. Densitometry analysis of the indicated band intensities in blots from three independent experiments. Data are means ±SD. * p <0.05, *** p <0.001 (unpaired two-tailed student’s t -tests; N = 3 independent experiments per group). (F) Representative immunoblotting analysis using antibodies against the indicated proteins on total protein lysates obtained from HEK293T-ACE2 cells transiently transfected with the human FOXO3 encoding plasmid for 48 h. Empty vector transfected cells were used as negative control. All experiments were performed in triplicate. Densitometry analysis of the indicated band intensities in blots from three independent experiments. Data are means ±SD. * p <0.05, *** p <0.001 (unpaired two-tailed student’s t -tests; N = 3 independent experiments per group). (G) Cartoon representation to illustrate our hypothesis for downregulation of ATP2B1 during SARS-CoV-2 infection via the FOXO3 transcriptional factor. During SARS-CoV-2 infection, the PI3K/Akt pathway is activated and enhances the phosphorylation of FOXO3, thus excluding it from the nucleus. As a consequence, the expression of the FOXO3 targets, including ATP2B1, are also reduced, thus increasing the intracellular Ca 2+ levels and further promoting SARS-CoV-2 replication.

Article Snippet: Then, in order to validate the role of FOXO3 as a positive transcriptional regulator of ATP2B1 and ATP2A1 , we transiently overexpressed a FOXO3-encoding plasmid (containing the coding region of FOXO3; #14937, Addgene) in HEK293T-ACE2 cells.

Techniques: In Silico, Expressing, Functional Assay, Sequencing, RNA Sequencing, Western Blot, Infection, Negative Control, Two Tailed Test, Transfection, Plasmid Preparation, Phospho-proteomics

Journal: medRxiv

Article Title: Targeting ATP2B1 impairs PI3K/Akt/Fox-O3 signaling and reduces SARS-COV-2 replication in vivo

doi: 10.1101/2022.09.02.22279542

Figure Lengend Snippet: (A) Experimental plan for human primary epithelial cells treated with 1 μM PI-7 or 0.001% DMSO as vehicle control. After 1 h, the cells were infected with SARS-CoV-2 viral particles of VOC Omicron 2 at 0.04 MOI for 70 h. The cells were then used for qPCR analysis. (B) Quantification of mRNA abundance relative to that in control (Vehicle) cells (2 −ΔΔCt ) for the indicated cytokines in HEK293T-ACE2 cells treated as in ( A ). Data are means ±SD. * P <0.05, ** P <0.01 (as determined by unpaired two-tailed Student’s t -tests; n = three independent experiments per group). Uninfected cells are used as negative control. (C) Cartoon representation to illustrate our hypothesis for the role of ATP2B1 during SARS-CoV-2 infection upon treatment with compound PI-7. On the left: during SARS-CoV-2 infection, the PI3K/Akt pathway is activated via Akt phosphorylation, thus enhancing the phosphorylation of FOXO3 to exclude it from the nucleus ( , ). As a consequence, the expression of the FOXO3 targets, including ATP2B1 and ATP2A1, are reduced, thus increasing the intracellular Ca 2+ levels and further promoting SARS-CoV-2 replication. Furthermore, SARS-CoV-2 infection also leads to NF-κB activation (also due to reduction of active FOXO3; ) and, as a consequence, the expression of the inflammatory cytokines (e.g., IL-1β, TNF-α and IL-12) belonging to the cytokine storm resulted increased. On the right: Treatment with the caloxin-derivative (compound PI-7) inhibits the viral propagation of SARS-CoV-2 (VOC: Delta and Omicron 2) by blocking ATP2B1 pump activity, thus reducing both the extra- and intracellular Ca 2+ levels that are necessary for SARS-CoV-2 replication. Compound PI-7 also increases ATP2B1 and ATP2A1 levels, thus further reducing the intracellular Ca 2+ levels because of its extracellular export via ATP2B1 (on the plasma membrane), and potentially the endoplasmic reticulum import through ATP2A1 . These increased levels of ATP2B1 and ATP2A1 occurred because of the enhancement of transcriptionally active FOXO3. The levels of active unphosphorylated-FOXO3 are increased due to the inactivation of PI3K/AKT pathway (due to decreased viral infection upon compound PI-7). As a consequence, the levels of nuclear active FOXO3 results increased, thus enhancing the transcriptional activation of ATP2B1 and ATP2A1. Treatment with PI-7 also reduces NF-κB activation (due to enhancement of FOXO3) and as a consequence, the expression of the inflammatory cytokines belonging to the cytokine storm resulted decreased IL-1β, TNF-α and IL-12. All the gene names whose expression resulted upregulated are depicted in bold.

Article Snippet: Then, in order to validate the role of FOXO3 as a positive transcriptional regulator of ATP2B1 and ATP2A1 , we transiently overexpressed a FOXO3-encoding plasmid (containing the coding region of FOXO3; #14937, Addgene) in HEK293T-ACE2 cells.

Techniques: Control, Infection, Two Tailed Test, Negative Control, Phospho-proteomics, Expressing, Activation Assay, Blocking Assay, Activity Assay, Clinical Proteomics, Membrane