Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: SARS-CoV-2 Spike protein trimer (pink) bound to ACE2 (green). ( A ) Without glycans. ( B ) With N-glycans (red) identified using LC-MS on Spike and ACE2. ( C ) Molecular dynamics simulation analyzed the range of movement of each glycan. The space sampled by glycans is represented by a gray cloud. Glycans cover the Spike-ACE2 interface. They also surround the putative proteolysis site of furin (‘S1-S2’, yellow) and S2’ (blue).

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: Liquid Chromatography with Mass Spectroscopy, Glycoproteomics

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) Full-length proteins expressed on cells include wild-type Spike-protein [v1] and human ACE2 [v2]. N-glycosylation sites are indicated by lollipop. Fc-his soluble proteins encode for S1-subunit [v3], RBD [v4] and soluble ACE2 [v5]. All constructs were co-expressed with fluorescent reporters separated by P2A. Note that the Fc-section also contains one N-glycosylation site. ( B ) Western blot for purified Fc-proteins from HEK293T probed with anti-Fc, anti-RBD or anti-ACE2 Ab. CD44-Fc is positive control. ( C ) Flow cytometry data showing S1-Fc (1.7 µg/mL) and RBD-Fc (0.35 µg/mL) binding to ACE2 expressed on HEK293T (middle panel). Spike expression enhances ACE2-Fc (1.4 µg/mL) binding (bottom). ( D ) Desialylation of Spike-protein expressed on 293 T/S had minimal effect on ACE2-Fc (0.7 µg/mL) binding. ACE2 desialylation on 293T/ACE2 increased binding of RBD-Fc (0.2 µg/mL) and S1-Fc (1.7 µg/mL) by 26–56% (paired experiments, *p<0.05). ( E ) Pseudovirus with DsRed-reporter were developed with three different envelope proteins. VSVG pseudotyped virus infected both HEK293T (black line) and stable 293T/ACE2 (red line) cells. Virus with Spike-WT and Spike-mutant entered 293T/ACE2 only. ( F ) Same titer of virus (0.3 µg/mL p24-equivalent) were treated with or without sialidase, prior to addition to stable 293T/ACE2 cells. Infection using Spike-mutant was higher compared to Spike-WT. Sialidase treatment of virus had no effect. ( G ) 293T/ACE2 cells were sialidase treated prior to addition of VSVG (0.3 µg/mL p24-equiv.), Spike-WT (1.5 µg/mL p24-equiv.) or Spike-mutant (0.2 µg/mL p24-equiv.) pseudovirus. Sialidase treatment did not affect viral entry. Abbreviations: Spike signal peptide (SP), N-terminal domain (NTD), receptor-binding domain (RBD), receptor-binding motif (RBM), subdomain 1 (SD1), subdomain 2 (SD2), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), heptad repeat 2 (HR2) transmembrane section (TM), cytoplasmic tail (CT), ACE2: Angiotensin-converting enzyme-2; VSVG: Vesicular stomatitis virus G-protein; WT: wild-type; mut: mutant.

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: Glycoproteomics, Construct, Western Blot, Purification, Positive Control, Flow Cytometry, Binding Assay, Expressing, Virus, Infection, Mutagenesis

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) Sialidase protocol validation. All lectins were directly conjugated with Alexa dyes. They were incubated with cells at 1–5 µg/mL for 15 min before a quick wash and cytometry measurement. Compared to untreated control (left), sialidase treatment (right) decreased SNA lectin binding to α2,6 sialylated structures by 15-fold and increased ECL binding to desialylated lactosamine chains (Galβ1,4GlcNAcβ) by an order of magnitude. ( B ) Pseudovirus assay. DsRed fluorescence in HEK293T and stable 293T/ACE2 cells upon addition of VSVG, Spike-WT and Spike-mutant pseudotyped virus. ( C ) Sialidase treatment of pseudovirus. % DsRed positive cell data are shown for study in (main manuscript). Viral entry was sialidase independent. ( D ) Sialidase treatment of HEK/ACE2 cells. Pseudovirus expressing VSVG, Spike-WT and Spike-mutant were added to cells under conditions described in (main manuscript). All error bars are standard deviations. Data are representative of 3 independent runs.

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: Biomarker Discovery, Incubation, Cytometry, Control, Binding Assay, Fluorescence, Mutagenesis, Virus, Expressing

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) Knocking out C1GALT1 and MGAT1 using CRISPR-Cas9 inhibits O- and N-glycan biosynthesis in HEK293Ts. ( B ) Sanger sequencing results of isogenic 293T clones shows indels on all 3 alleles of C1GALT1 (‘[O] - 293T’) and single allele of MGAT1 (‘[N] - 293T’) knockout cells. Wild-type (WT) sequence is on the first line. Lower line shows base deletions (hyphen) and insertions (black fonts) for individual KOs. sgRNA target sequence is in red and protospacer adjacent motif is underlined. ( C ) Increased VVA and reduced PHA-L binding confirm loss of O-linked glycans in [O] - 293Ts and N-glycans in [N] - 293Ts, respectively. ( D ) Knocking out N-glycans on Spike protein reduced ACE2-Fc binding in cytometry based binding studies. Knocking out Spike O-glycans increased ACE-2 binding. ( E–F ) Truncation of ACE2 N- and O-glycans did not affect either S1-Fc (panel E ) or RBD-Fc (panel F ) binding. ( G ) ACE2 was transiently expressed on 293T, [O] - 293T and [N] - 293 T cells. All pseudotyped virus efficiently entered ACE2 expressing cells. Virus was not titered for these runs, and thus comparison between viruses is not possible. *p<0.05 with respect to all other treatments. # p<0.05 with respect to 293T and [N] - 293T/ACE2 in panel G .

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: CRISPR, Glycoproteomics, Sequencing, Clone Assay, Knock-Out, Binding Assay, Cytometry, Virus, Expressing, Comparison

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) Surface expression of Spike-protein and ACE2. Full-length Spike (top) and human ACE2 (bottom) were expressed in HEK 293T, [N] - 293T and [O] - 293T. Protein expression was measured in EGFP+ cells in the case of Spike (using anti-RBD), and on BFP+ cells in the case of ACE2 (using anti-ACE2), as these fluorescent reporters are co-expressed with surface proteins. Protein expression was comparable in all cells. Untransfected 293Ts serve as negative control. ( B ) Viral entry assay. Pseudovirus expressing VSVG envelope protein, Spike-WT or Spike-mutant were added to HEK 293 T cells transiently transfected to overexpress ACE2 (both wild-type 293T and glycosylation mutants). An additional control included 293 T cells not expressing ACE2, which only allowed entry of VSVG pseudotyped viral particles, but not Spike bearing virus. % cells that were DsRed (reporter) positive is shown at 72 hr. All treatments were statistically different except as indicated by n.s. (‘not significant’). All data are from of N > 3 repeats.

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: Expressing, Negative Control, Mutagenesis, Transfection, Glycoproteomics, Control, Virus

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] - and [N] - 293 T cells. All nine viruses were applied at equal titer to stable 293T/ACE2. ( B–C ) O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. ( D ) Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. ( E ) Western blot of Spike protein using anti-S2 Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] - 293T products due to truncation of glycan biosynthesis. ( F ) Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] - 293Ts is almost fully proteolyzed during viral production (red arrowhead). *p<0.05 with respect to all other treatments.

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: Expressing, Mutagenesis, Produced, Glycoproteomics, Virus, Generated, Titration, Infection, Western Blot

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) VSVG, Spike-WT and Spike-mutant pseudovirus were produced in the presence of 15 µM kifunensine or vehicle control. The six viruses were added to 293T/ACE2 at equal titer. ( B–D ) Microscopy (panel B) and cytometry (panel C, D ) show ~90% loss of viral infection in the case of Spike-WT and Spike-mutant virus upon kifunensine treatment (*p<0.05). ( E ) Spike molecular mass is reduced in the western blots due to high-mannose glycan synthesis in runs with kifunensine. Intact Spike is reduced in the presence of kifunensine, in anti-FLAG blot. ( F ) The polybasic furin ‘RRAR’ site was substituted by a single ‘A’ amino acid in Spike-delta. Virus with Spike-delta were expressed both in the presence of vehicle and kifunensine. Western blot shows lack of S1-S2 cleavage in this construct. In viral entry assay, kifunensine reduced DsRed expression in 293T/ACE2 cells, even in the case of Spike-delta pseudovirus (*p<0.05). Similar observation was made at two different viral titers (0.3 and 0.6 μg/mL p24 equivalent).

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: Mutagenesis, Produced, Control, Microscopy, Cytometry, Infection, Virus, Western Blot, Glycoproteomics, Construct, Expressing

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet: ( A ) ACE2-Fc binding was measured to wild-type or glycoEnzyme-KO 293 T cells expressing Spike. Sialidase treatment of cells was performed in some cases. Similar studies also measured S1-Fc and RBD-Fc binding to cell-surface expressed ACE2. ( B ) SARS-CoV-2 pseudovirus (bearing Spike-WT, Spike-mut, Spike-delta variants) were generated in wild-type or glycoEnzyme-KO 293Ts, in the presence and absence of kifunensine. Main results of binding ( A ) and viral entry ( B ) assay are listed. ( C ) Conceptual model shows that kifunensine can induce S1-S2 site proteolysis on Spike-WT and Spike-mut virus, but not Spike-delta virus. This proteolysis reduces RBD presentation and attenuates viral entry into 293T/ACE2. Without affecting S1-S2 cleavage, kifunensine also partially reduced Spike-delta pseudovirus entry function. The data suggest additional roles for Spike N-glycans during viral entry.

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: Binding Assay, Expressing, Generated, Virus

Journal: eLife

Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

doi: 10.7554/eLife.61552

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , ACE2 [v2] , This paper , Derived from RRID: Addgene_1786 , Plasmid to express full length human ACE2.

Techniques: Binding Assay, Plasmid Preparation, Recombinant, Knock-Out, Derivative Assay

Journal: Nature genetics

Article Title: A novel ACE2 isoform is expressed in human respiratory epithelia and is upregulated in response to interferons and RNA respiratory virus infection.

doi: 10.1038/s41588-020-00759-x

Figure Lengend Snippet: Fig. 1 | A novel short transcript of ACE2 is expressed in airway epithelia. a, Sashimi plot showing splice junction between exons (Ex) 9 and 10, and between exons 9a and 10, counted from RNA-seq reads from one nasal brushing sample. GENCODE v.33 gene build exons and new SCALLOP transcriptome build exons, showing new exon 9a in a new transcript encompassing exons 9a–19, assembled by SCALLOP tool. Letters ‘A’, ‘B’ and ‘C’ indicate locations of primers for RT–PCR (Fig. 1c). Nucleotide sequence of novel exon 9a, plus 5′ UTR, start codon and splice junction, are shown. b, Box and whisker plot showing expression levels of short and long ACE2 transcripts in primary NECs (reads mapped to exon 9a/10 or exon 9/10 per million mapped reads) (P = 0.0201, paired, two-way Student’s t-test; n = 6 donors). c, Agarose gel electrophoresis image of long-range, transcript-specific PCR products amplifying full short ACE2 transcript and exons 9–19 of long ACE2 transcript from nasal epithelial brushings and BCi-NS1.1 cells, using different pairs of primers (A and C or B and C), specific to regions of ACE2 shown in a. d, Sanger sequencing electropherogram traces showing sequence at exon/ exon boundaries of long ACE2 transcript exons 9–10 and short ACE2 transcript exons 9a–10. Amino acid (aa) translation is shown below.

Article Snippet: Full-length sequence-verified human ACE2 cDNA expression clone with C-terminal GFP tag was purchased from Origene (catalog no. rg208442).

Techniques: RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Sequencing, Whisker Assay, Expressing, Agarose Gel Electrophoresis