Journal: Science advances

Article Title: Role for N -glycans and calnexin-calreticulin chaperones in SARS-CoV-2 Spike maturation and viral infectivity.

doi: 10.1126/sciadv.abq8678

Figure Lengend Snippet: Fig. 1. N-glycans on Spike are essential for pseudoviral entry. (A) Molecular model of Spike (cyan) bound to ACE2 (yellow). N-glycans are space-filled. Glycans studied in this work are color-coded either green (high-mannose) or magenta (complex structures) on the basis of published abundance data [Watanabe et al. (5)]. Distance of these glycans to S1-S2 (red) and S2′ (blue) cleavage sites are indicated using red and blue dashed lines. (B) Spike mutation rates at N-glycosylation sites (N-X-S/T) based on analysis of ~7.6 × 106 genomic sequences from gisaid.org. Mutation rates are shown both at Asn (N) and Ser/Thr (S/T) sites in the N-glycosylation sequon. Median mutation rate across all amino acids is set to 1 (dashed line). (C) Pseudotyped lentivirus (PVs) were generated using the parent Spike (D614G mutant), along with a panel of N-glycosylation site single, double, and triple N-to-Q mutations on the same background. Western blot quantified Spike incorporation into virions on the basis of de- tection of C-terminal FLAG-tag present in all constructs. p24 capsid protein serves as loading control. (D and E) Viral infectivity was evaluated at 72 hours, using ACE2-overexpressing 293T [293T/ACE2, (D)] and Vero E6 cells [Vero/ACE2, (E)]. Mean fluorescence intensity (MFI) due to DsRed reporter is quantified. Data from multiple viral preparations were normalized to parent virus DsRed signal for the same batch. Data are means ± SD for n ≥ 3. *P < 0.05 with respect to all other treatments, †P < 0.05 with respect to parent, and ‡P < 0.05 with respect to all other treatments except that bars marked by ‡ are not different.

Article Snippet: 8, eabq8678 (2022) 23 September 2022 10 of 14 full-length human ACE2 with blue fluorescence protein reporter (Addgene 164219), was available from prior work (9).

Techniques: Mutagenesis, Glycoproteomics, Genomic Sequencing, Generated, Western Blot, FLAG-tag, Construct, Control, Infection, Fluorescence, Virus

Journal: Science advances

Article Title: Role for N -glycans and calnexin-calreticulin chaperones in SARS-CoV-2 Spike maturation and viral infectivity.

doi: 10.1126/sciadv.abq8678

Figure Lengend Snippet: Fig. 2. SARS-CoV-2 VLPs with N-glycan mutations display reduced viral entry. (A) Plasmids encoding for the four structural proteins containing distinct tags or EGFP reporters were either transfected alone or pooled and transfected together into 293Ts. (B) Confocal micrographs show the intracellular colocalization of M, N, E, and Spike in perinuclear compartments that likely includes the ERGIC (arrowhead). (C) Flow cytometry analysis performed with live and “fixed-permeabilized cells” demonstrate cell surface expression of Spike and M proteins. N and E were intracellular. Here, the structural proteins were either expressed alone or altogether: “+” and “−” indicate presence/ absence of indicated proteins. (D) All proteins were expressed in VLPs (detected using epitope-tag Abs), but Spike carrying N61Q and N801Q mutations displayed somewhat reduced expression. (E) Luc-VLPs containing firefly luciferase reporter were produced by cotransfecting 293T cells with plasmids encoding for N (R203M mutant), M-IRES-E, Luc-PS9, and Spike constructs. Luc-VLP viral entry into 293T/ACE2 cells was reduced upon implementing many of the N-to-Q glycosylation mutations. “No-spike” VLPs were made using all plasmids except Spike. Mock infection did not contain VLP. Data were normalized to parent-VLP luminescence in each run [both for (E) and (F)]. (F) Luc-VLP with parent-Spike, produced using 293T cells, was deglycosylated using either Endo H or peptide N-glycosidase (PNGase) F. PNGase F reduced viral entry into 293T/ACE2 and Calu-3 cells, with a smaller effect being observed for Endo H. Data are means ± SD for n ≥ 3. *P < 0.05 with respect to all other treatments and †P < 0.05 with respect to parent.

Article Snippet: 8, eabq8678 (2022) 23 September 2022 10 of 14 full-length human ACE2 with blue fluorescence protein reporter (Addgene 164219), was available from prior work (9).

Techniques: Glycoproteomics, Transfection, Flow Cytometry, Expressing, Luciferase, Produced, Mutagenesis, Construct, Infection

Journal: Science advances

Article Title: Role for N -glycans and calnexin-calreticulin chaperones in SARS-CoV-2 Spike maturation and viral infectivity.

doi: 10.1126/sciadv.abq8678

Figure Lengend Snippet: Fig. 3. Glycan mutations did not affect Spike cell surface expression and ACE2 binding function. (A) Spike parent and mutants were transiently expressed in 293T cells. Spike cell surface expression was quantified on a portion of the live cells at 48 hours by measuring anti-RBD binding using flow cytometry. ACE2-Fc (0.7 g/ml) binding was also independently quantified in live cells. A portion of the cells were “fixed-permeabilized” using paraformaldehyde-methanol method. Anti-RBD and anti-FLAG Ab binding quantified the presence of the Spike S1 and S2 subunits, respectively. (B) Spike S1 expression on 293T cells quantified using anti-RBD in live and fixed-permeabilized cells. (C) ACE2-Fc binding data in live cells. (D) Spike/S2 expression quantified using anti-FLAG Ab. All Spike variants were equally expressed on cell surface with Spike- (without RRAR site) displaying more RBD/S1 presentation. ACE2-Fc binding correlated with RBD expression. Data are means ± SD (n = 3), after nor- malization with respect to parent Spike MFI in different runs. *P < 0.05 with respect to all other treatments.

Article Snippet: 8, eabq8678 (2022) 23 September 2022 10 of 14 full-length human ACE2 with blue fluorescence protein reporter (Addgene 164219), was available from prior work (9).

Techniques: Glycoproteomics, Expressing, Binding Assay, Flow Cytometry

Journal: Science advances

Article Title: Role for N -glycans and calnexin-calreticulin chaperones in SARS-CoV-2 Spike maturation and viral infectivity.

doi: 10.1126/sciadv.abq8678

Figure Lengend Snippet: Fig. 4. Small impact of Spike N-glycan mutations on syncytia formation. 293T cells were transiently transfected with the panel of Spike constructs to produce “293T/S.” Twenty-four hours after transfection, these cells were labeled using CMFDA (a green fluorescence dye) and mixed with either 293T/ACE2 or Vero E6 transfected to overexpress ACE2 (“Vero/ACE2”). These ACE2 cells were labeled with red fluorescence dye, CMTMR. Incucyte live cell imaging was performed during a 16-hour cocul- ture experiment to monitor red-green dye overlap/fusion between Spike (green) and ACE2 (red)–expressing cells. A portion of the cells were also trypsinized at 4 hours for cytometry analysis. (A) Representative images for ACE2-expressing cells cocultured with either untransfected cells (top row) or cells expressing parent Spike (middle) or Spike- proteins (bottom). Sixteen-hour images are presented for studies with 293T/ACE2 and Vero/ACE2. Arrow indicates syncytium. (B and C) The left axis presents percent green-red overlap [=100 × (overlap area/total area occupied by cells)], quantified using the Incucyte system at 16 hours. These data are presented for studies conducted with 293T/ACE2 (B) and Vero/ACE2 (C). Syncytia was observed in all cases, except for untransfected cells and cells expressing Spike-. The right axis presents cytometry measured percent green-red events, i.e., % association = (green-red count/total count) × 100. This includes both cells that are bound to each other and those forming syncytium. Data are means ± SD for n = 3. *P < 0.05 with respect to all other treatments, †P < 0.05 with respect to parent, and ‡P < 0.05 with respect to all other treatments except that bars marked by ‡ are not different from each other.

Article Snippet: 8, eabq8678 (2022) 23 September 2022 10 of 14 full-length human ACE2 with blue fluorescence protein reporter (Addgene 164219), was available from prior work (9).

Techniques: Glycoproteomics, Transfection, Construct, Labeling, Fluorescence, Live Cell Imaging, Expressing, Cytometry

Journal: Science advances

Article Title: Role for N -glycans and calnexin-calreticulin chaperones in SARS-CoV-2 Spike maturation and viral infectivity.

doi: 10.1126/sciadv.abq8678

Figure Lengend Snippet: Fig. 5. Chaperone proteins calnexin (CANX) and calreticulin (CALR) regulate SARS-CoV-2 viral entry. (A) Parent Spike was transfected into WT, CANX-KO, and CALR- KO 293Ts. Spike expression was similar in all cells based on anti-FLAG that detects the Spike C terminus (left). A partial (10 to 25%) reduction in anti-RBD Ab/Spike S1 as- sociation (middle) was noted, and this correlated with ACE2-Fc binding (right). (B) 293Ts were transfected with parent Spike. Spike immunoprecipitated from cell lysate 24 hours after transfection using anti-FLAG mAb and coprecipitated both CANX and CALR. CALR (top), CANX (middle), and Spike (bottom) were analyzed both in the immunoprecipitated (protein A/G “bead”) sample and the supernatant remaining after bead removal. Arrows and boxes indicate target proteins. (C) All Spike mutants coprecipitated CANX in the anti-FLAG Spike pulldown assay. Cells without Spike (“untransfected”) served as negative control. (D) Parent pseudotyped lentivirus was produced in WT, CANX-KO, and CALR-KO. Western blot showed reduced Spike incorporation into virions produced in both KOs. (E) Pseudovirus entry into 293T/ACE2, measured 72 hours after infection, was reduced when virus were produced in CANX or CALR-KO 293Ts. (F) VSV-G pseudotyped lentivirus entry into 293T/ACE2 was not reduced by either CANX or CALR. (G) Luc-VLP containing parent Spike were produced in WT, CANX-KO, and CALR-KO 293Ts. Luc-VLPs produced in CANX-KO displayed re- duced entry into A549-ACE2-TMPRSS2, Calu-3, and 293T/ACE2 cells. Luc-VLP viral entry is reported on the basis of luminescence signal, normalized to parent levels in each run. No spike Luc-VLP lacks Spike. Data are means ± SD (n ≥ 3). *P < 0.05 with respect to all other treatments.

Article Snippet: 8, eabq8678 (2022) 23 September 2022 10 of 14 full-length human ACE2 with blue fluorescence protein reporter (Addgene 164219), was available from prior work (9).

Techniques: Transfection, Expressing, Binding Assay, Immunoprecipitation, Negative Control, Produced, Western Blot, Infection, Virus