PMC:7108609 / 5493-8096
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7108609","sourcedb":"PMC","sourceid":"7108609","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7108609","text":"The interaction between the SARS-CoV spike protein and its cellular receptor ACE2 can be studied using a cell-based assay, as described previously (Chou et al. 2005). In this assay, the viral S protein expressed by transfection into Chinese hamster ovary (CHO) cells mediates adhesion to Vero E6 cells that possess ACE2. CHO cells do not express ABH antigens because of the lack of an α1,2-fucosyltransferase activity and of either the A or B histo-blood group enzymes. In order to obtain cells able to express the A antigen, parental CHO cells were stably transfected successively with the rat Fut2 cDNA and a rat A enzyme cDNA. Unlike mock-transfected cells, these double transfectants strongly express cell surface A antigen as detected by flow cytometry. Transfection of the S protein–EGFP fusion construction (S–EGFP) into these cells allowed the expression of the S protein together with the histo-blood group A antigen (Figure 1A). Observation of the triple transfectants by confocal microscopy revealed that, as expected, the A antigen and the S–EGFP fusion protein partially colocalized at the cell surface (Figure 1B). In addition, western blot analysis revealed that among various A antigen positive glycoproteins, a band at the expected size of the S–EGFP fusion protein, between 210—and 230 kDa (Chou et al. 2005), was present in the extract from the triple transfectant Fut2/A/SP but absent from the double Fut2/A transfectant cell extract. It indicated that the S-protein expressed by A-positive CHO cells carried A histo-blood group epitopes. Specificity of the anti-A labeling was ensured since no band was detected in extracts from CHO Fut2 only transfectants (Figure 1C). A stable A antigen and S-EGFP expressing clone showed significantly higher adhesion to Vero cells than either mock transfectants or the A expressing clone devoid of the S protein (Figure 2A and B). Similar results were obtained after transient transfection of the S-EGFP construct (not shown). The presence of the A and/or H antigens on the S protein expressing cells did not affect adhesion since CHO cells only transfected with the S-EGFP construct, as well as CHO cells transfected with both the S-EGFP and Fut2 cDNAs, adhered to Vero cells at a similar level as the A antigen S protein triple transfectants (Figure 2A). In order to control that the cell adhesion was dependent upon the ACE2/S protein interaction, blocking experiments with either an anti-ACE2 or an anti-S protein were performed. Both antibodies significantly inhibited adhesion, although the anti-ACE2 mAb proved more efficient (Figure 2C).","tracks":[{"project":"2_test","denotations":[{"id":"18818423-15582697-45167055","span":{"begin":160,"end":164},"obj":"15582697"},{"id":"18818423-15582697-45167056","span":{"begin":1321,"end":1325},"obj":"15582697"},{"id":"T77007","span":{"begin":160,"end":164},"obj":"15582697"},{"id":"T33542","span":{"begin":1321,"end":1325},"obj":"15582697"}],"attributes":[{"subj":"18818423-15582697-45167055","pred":"source","obj":"2_test"},{"subj":"18818423-15582697-45167056","pred":"source","obj":"2_test"},{"subj":"T77007","pred":"source","obj":"2_test"},{"subj":"T33542","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ecd793","default":true}]}]}}