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: ( 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. ]. 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 × 10 6 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 detection 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: A549 lung carcinoma overexpressing human ACE2 and TMPRSS2 (A549-ACE2-TMPRSS2; catalog no. a549-hace2tpsa) were purchased from InvivoGen (San Diego, CA) and cultured according to the manufacturer’s instruction.

Techniques: Mutagenesis, Genomic Sequencing, Generated, Western Blot, FLAG-tag, Construct, 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: ( 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: A549 lung carcinoma overexpressing human ACE2 and TMPRSS2 (A549-ACE2-TMPRSS2; catalog no. a549-hace2tpsa) were purchased from InvivoGen (San Diego, CA) and cultured according to the manufacturer’s instruction.

Techniques: 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: ( 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 μg/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-Δ (without RRAR site) displaying more RBD/S1 presentation. ACE2-Fc binding correlated with RBD expression. Data are means ± SD ( n = 3), after normalization with respect to parent Spike MFI in different runs. * P < 0.05 with respect to all other treatments.

Article Snippet: A549 lung carcinoma overexpressing human ACE2 and TMPRSS2 (A549-ACE2-TMPRSS2; catalog no. a549-hace2tpsa) were purchased from InvivoGen (San Diego, CA) and cultured according to the manufacturer’s instruction.

Techniques: 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: 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 coculture 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-Δ 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-Δ. 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: A549 lung carcinoma overexpressing human ACE2 and TMPRSS2 (A549-ACE2-TMPRSS2; catalog no. a549-hace2tpsa) were purchased from InvivoGen (San Diego, CA) and cultured according to the manufacturer’s instruction.

Techniques: 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: ( 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 association (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 reduced 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: A549 lung carcinoma overexpressing human ACE2 and TMPRSS2 (A549-ACE2-TMPRSS2; catalog no. a549-hace2tpsa) were purchased from InvivoGen (San Diego, CA) and cultured according to the manufacturer’s instruction.

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