Journal: bioRxiv

Article Title: LRRC15 is an inhibitory receptor blocking SARS-CoV-2 spike-mediated entry in trans

doi: 10.1101/2021.11.23.469714

Figure Lengend Snippet: (A) Schematic of a focused CRISPR activation screen for surface proteins interacting with SARS-CoV-2 spike S1-Fc fusion protein. (B) Volcano plots showing sgRNAs enriched or depleted in cells binding with SARS-CoV-2 spike S1-Fc or human IgG isotype control. Results from two biologically independent replicates are shown.

Article Snippet: For SARS-CoV-2 spike S1-Fc binding screen, 5 × 10 7 cells per sample were washed with FACS buffer (1x PBS supplemented with 2% FBS and 1 mM EDTA) and incubated with 50 μg/mL SARS-CoV-2 spike S1 subunit-Fc fusion protein (R&D systems, #10623-CV-100) or human IgG1 isotype control (BioXCell, #BE0297) for 30 min at 4°C.

Techniques: CRISPR, Activation Assay, Binding Assay

Journal: bioRxiv

Article Title: LRRC15 is an inhibitory receptor blocking SARS-CoV-2 spike-mediated entry in trans

doi: 10.1101/2021.11.23.469714

Figure Lengend Snippet: (A) A375 cells were transduced with indicated activating sgRNAs and incubated with SARS-CoV-2 spike S1-Fc fusion protein. Protein binding was measured by flow cytometry. (B) HeLa cells were transduced with indicated activating sgRNAs and incubated with SARS-CoV-2 spike S1-Fc fusion protein. Protein binding was measured by flow cytometry. (C) Dose-dependent binding of SARS-CoV-2 spike protein (Wuhan-Hu-1) to both ACE2 and LRRC15 with a Fc tag was determined by ELISA. Human IgG1 was included as a negative control. Dots indicate means of duplicates. (D) HeLa cells were transduced with indicated activating sgRNAs and incubated with SARS-CoV-2 spike NTD-Fc or RBD-Fc fusion protein. Protein binding was measured by flow cytometry. (E) The binding of the SARS-CoV-2 RBD and NTD to LRRC15 was measured by ELISA.

Article Snippet: For SARS-CoV-2 spike S1-Fc binding screen, 5 × 10 7 cells per sample were washed with FACS buffer (1x PBS supplemented with 2% FBS and 1 mM EDTA) and incubated with 50 μg/mL SARS-CoV-2 spike S1 subunit-Fc fusion protein (R&D systems, #10623-CV-100) or human IgG1 isotype control (BioXCell, #BE0297) for 30 min at 4°C.

Techniques: Transduction, Incubation, Protein Binding, Flow Cytometry, Binding Assay, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: bioRxiv

Article Title: LRRC15 is an inhibitory receptor blocking SARS-CoV-2 spike-mediated entry in trans

doi: 10.1101/2021.11.23.469714

Figure Lengend Snippet: (A) HeLa-ACE2 cells were transduced with indicated activating sgRNAs and infected with VSV pseudoviruses, VSVΔG-S-SARS2 or VSVΔG-G. GFP signal was measured at 20 hpi by flow cytometry and normalized to mock control (n=4). (B) HeLa-ACE2 cells expressing LRRC15 or empty vector were infected with VSV psuedoviruses and GFP signal was measured at 20 hpi by flow cytometry normalized to empty control (n=6 for VSVΔG-S-SARS2 and n=5 for VSVΔG-G). (C) LRRC15-induced or mock HeLa-ACE2 cells were infected with VSV pseudoviruses harboring spike proteins of different SARS-CoV-2 variants. GFP signal was measured at 20 hpi by flow cytometry (n=3). (D) LRRC15-induced or mock HeLa-ACE2 cells were infected with VSV pseudoviruses harboring SARS-CoV-1 spike. GFP signal was measured at 20 hpi by flow cytometry (n=3). Data represent means ± SEM (A-D). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test (A) or unpaired two-tailed t test (B-D). ns, not significant; *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: For SARS-CoV-2 spike S1-Fc binding screen, 5 × 10 7 cells per sample were washed with FACS buffer (1x PBS supplemented with 2% FBS and 1 mM EDTA) and incubated with 50 μg/mL SARS-CoV-2 spike S1 subunit-Fc fusion protein (R&D systems, #10623-CV-100) or human IgG1 isotype control (BioXCell, #BE0297) for 30 min at 4°C.

Techniques: Transduction, Infection, Flow Cytometry, Expressing, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: LRRC15 is an inhibitory receptor blocking SARS-CoV-2 spike-mediated entry in trans

doi: 10.1101/2021.11.23.469714

Figure Lengend Snippet: (A-B) Cell type-specific expression of ACE2 and LRRC15 , assessed by scRNA-seq of lungs from deceased COVID-19 patients (A) Delorey et al, 2021 or (B) Melms et al, 2021 . (C) Boxplots of the relative frequencies of fibroblast subtypes among total fibroblasts, comparing COVID-19 patients (blue) to non-COVID-19 controls (red). Statistical significance was assessed by two-tailed unpaired Mann-Whitney test. (D) Volcano plot of differentially expressed genes in the lungs of deceased COVID-19 patients, comparing samples with high vs low SARS-CoV-2 RNA levels at the time of death . Genes with positive log2 fold changes are associated with high viral burden, while genes with negative log2 fold changes are associated with low viral burden. (E) LRRC15 expression in lung cell lines (A549, A549-ACE2, Calu-3), comparing mock controls vs SARS-CoV-2 infected samples. Statistical significance was assessed by two-tailed unpaired Welch’s t-test.

Article Snippet: For SARS-CoV-2 spike S1-Fc binding screen, 5 × 10 7 cells per sample were washed with FACS buffer (1x PBS supplemented with 2% FBS and 1 mM EDTA) and incubated with 50 μg/mL SARS-CoV-2 spike S1 subunit-Fc fusion protein (R&D systems, #10623-CV-100) or human IgG1 isotype control (BioXCell, #BE0297) for 30 min at 4°C.

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY, Infection

Journal: bioRxiv

Article Title: LRRC15 is an inhibitory receptor blocking SARS-CoV-2 spike-mediated entry in trans

doi: 10.1101/2021.11.23.469714

Figure Lengend Snippet: (A) Schematic of trans-inhibition assay with ACE2+ and LRRC15+ cells. (B) HeLa-ACE2 cells were co-cultured with HeLa or HeLa-sgLRRC15 cells at 1:4 ratio and infected with VSV pseudovirus harboring spike of SARS-CoV-2 Wuhan-hu-1 strain at high or low titer. GFP signal was measured at 20 hpi by flow cytometry (n=4). Representative of three independent experiments are shown. (C) Trans-inhibition assay was performed as in (B) with VSV pseudovirus harboring spike of SARS-CoV-2 Delta variant (B.1.617.2 strain) (n=4). Representative of three independent experiments are shown. (D) Representative images of immunofluorescence staining of SARS-CoV-2 spike (green), LRRC15 (red), and DAPI (blue). HeLa-ACE2 cells were co-cultured with HeLa or HeLa-sgLRRC15 cells, inoculated with VSVΔG-S-SARS2 for 1 h on ice and incubated at 37°C to allow internalization. Cells were harvested at indicated timepoints and subjected to staining. The white arrowheads indicate spikes. The scale bar indicates 20 μm. (E) Quantification was performed by calculating the number of spikes on LRRC15- or LRRC15+ cells per mm 2 area from multiple images per sample. (F) Proposed working model of the LRRC15-mediated inhibition of SARS-CoV-2 entry. Data represent means ± SEM and were analyzed by unpaired two-tailed t test (B, C). *p < 0.05; ***p < 0.001; ****p < 0.0001.

Article Snippet: For SARS-CoV-2 spike S1-Fc binding screen, 5 × 10 7 cells per sample were washed with FACS buffer (1x PBS supplemented with 2% FBS and 1 mM EDTA) and incubated with 50 μg/mL SARS-CoV-2 spike S1 subunit-Fc fusion protein (R&D systems, #10623-CV-100) or human IgG1 isotype control (BioXCell, #BE0297) for 30 min at 4°C.

Techniques: Inhibition, Cell Culture, Infection, Flow Cytometry, Variant Assay, Immunofluorescence, Staining, Incubation, Two Tailed Test