Journal: Nature
Article Title: Heart-nosed bat alphacoronaviruses use human CEACAM6 to enter cells
doi: 10.1038/s41586-026-10394-x
Figure Lengend Snippet: a , Different human cell lines were assessed for their permissivity to alphacoronavirus pseudotyped spike proteins. CcCoV-KY43 could enter Calu3 (lung) and Caco2 (intestinal) cells. The mean entry (fold change (FC) compared with non-enveloped (NE) controls) from technical triplicates is shown. b , Recombinantly produced CcCoV-KY43 RBD shows potentially high levels of glycosylation. c , Screening an array of human receptor ectodomains identified CEACAM6, CEACAM3 and CEACAM5 as interactors of the CcCoV-KY43 RBD. d , CEACAM proteins were overexpressed in refractory HEK293T cells, and the assays showed that pseudotyped CcCoV-KY43 spike protein could only enter in the presence of CEACAM6. Two biological replicates (technical triplicates) along with their s.d. values, are shown. *** P = 0.0003 using two-way analysis of variance (ANOVA). NS, not significant. e , Interaction of the CcCoV-KY43 RBD and CEACAM6 was confirmed by ELISA, whereas no binding was observed with CEACAM3 or CEACAM5. The mean of two experiments (technical triplicate) is shown with s.d. values. f , ITC (showing differential power (DP) values) of CEACAM6 and CEACAM5 binding the CcCoV-KY3 RBD. Data are representative of two (CEACAM5) or three (CEACAM6) independent experiments. For CEACAM6, mean K d , number of binding sites ( n ) and enthalpy change (Δ H ) are shown. g , Pseudotyped CcCoV-KY43 spike protein was incubated for 1 h with recombinant CEACAM6, followed by titration on HEK293T cells expressing human CEACAM6 (hCEACAM6). Raw data of the mean of two independent experiments (technical triplicate) are shown with s.d. values. h , Monoclonal antibodies against CEACAM6 (B6.2 and clone 439424) were used to neutralize CcCoV-KY43 entry into HEK293T cells expressing hCEACAM6. The mean of two independent experiments (technical triplicate) was normalized to the untreated condition and plotted with s.d. values. i , Left, CEACAM6-specific siRNAs, or negative control, were electroporated into permissive cells and then infected with CcCoV-KY43. To confirm CEACAM6 reduction, cell lysates were analysed by immunoblotting (bottom). Mean entry reduction relative to the scrambled siRNA control from three independent experiments (technical triplicates) along with s.d. values, is shown. Significance of log 10 fold change was determined using a one-sample t -test and P values were adjusted for multiple comparisons (Caco2: P = 0.004 for siRNA-1, P = 0.001 for siRNA-2 and P = 0.009 for siRNA-3; Calu3: P = 0.022 for siRNA-1, P = 0.04 for siRNA-2 and siRNA-3). Right, using lentivirus expressing shRNA, stable knockdown of CEACAM6 expression in Caco2 and Calu3 cell lines was induced and validated by immunoblotting. One biological replicate (technical triplicates) is shown, along with s.d. values. WT, wild type. j , Left, lung-specific single-cell transcriptomic data show the expression of CEACAM6. Right, comparison of the expression of coronavirus receptors in lung cells: CEACAM6, ACE2, APN, DDP4 and TMPRSS2 are shown with the dot plot indicating both average and per cent expression.
Article Snippet: Publicly available single-cell RNA-sequencing data from the Human Protein Atlas were downloaded ( https://www.proteinatlas.org/humanproteome/single+cell/single+cell+type/data#datasets ).
Techniques: Produced, Glycoproteomics, Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Recombinant, Titration, Expressing, Bioprocessing, Negative Control, Infection, Western Blot, Control, shRNA, Knockdown, Single Cell, Comparison
