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LitCovid-PD-FMA-UBERON

LitCovid_AGAC

{"project":"LitCovid_AGAC","denotations":[{"id":"p43953s18","span":{"begin":409,"end":417},"obj":"PosReg"},{"id":"p43953s20","span":{"begin":421,"end":431},"obj":"Enzyme"},{"id":"p43953s21","span":{"begin":432,"end":442},"obj":"MPA"},{"id":"p43954s13","span":{"begin":576,"end":586},"obj":"Enzyme"},{"id":"p43954s14","span":{"begin":587,"end":597},"obj":"MPA"}],"text":"Next, we determined whether SARS-CoV-2 S pseudovirions were able to transduce human, monkey, and bat cells. VSV-G pseudovirons were used as a positive control, whereas bald particles with no spike proteins (mock) served as a negative control. As expected, all cell types were effectively transduced by VSV-G pseudovirons (Fig. 2a). Compared to mock control, SARS-CoV-2 S pseudovirions showed an over 500-fold increase in luciferase activities in Calu3 cells, at a level similar to SARS-CoV S pseudovirions (Fig. 2a). Huh7 and Vero 81 cells also gave about 10-fold increase in luciferase activities when transduced by SARS-CoV-2. Transduction of LLCMK2 cells was higher with SARS-CoV S pseudovirions than with SARS-CoV-2 S pseudovirions (Fig. 2a), suggesting that there might be some differences on virus entry on LLCMK2 cells mediated by S proteins between SARS-CoV-2 and SARS-CoV. We then investigated whether any known CoV receptors might be used by SARS-CoV-2 S protein as entry receptor. The SARS-CoV-2 S pseudovirons were used to transduce BHK cells stably expressing human aminopeptidase N (hAPN), the receptor for human CoV 229E, 293 cells stably expressing hACE2 (293/hACE2), the receptor for SARS-CoV, and HeLa cells stably expressing hDPP4 (HeLa/hDPP4), the receptor for MERS-CoV. While BHK/hAPN and HeLa/hDPP4 cells were not susceptible for the transduction of SARS-CoV-2 S pseudovirions, 293/hACE2 cells were highly transduced by SARS-CoV-2 S pseudovirions, consistent with hACE2 as the receptor for SARS-CoV-213. We then determined whether SARS-CoV-2 S protein could directly bind to hACE2 protein. HEK 293T cells transiently expressing SARS-CoV-2 S protein were incubated with soluble hACE2 and analyzed by flow cytometry. As shown in Fig. 2c, SARS-CoV-2 S protein bound to soluble hACE2 at a level similar to SARS-CoV S protein, although the mean fluorescence intensity (MFI) for SARS-CoV-2 S protein was slightly lower than SARS-CoV S protein. To further investigate if hACE2 is the receptor for SARS-CoV-2, we performed inhibition experiments using soluble hACE2. Soluble hACE2 proteins were pre-incubated with SARS-CoV-2 S pseudovirons on ice for 1 h, then virus-protein mixture was added onto 293/hACE2 cells. Entry of SARS-CoV-2 S pseudovirions was significantly prevented by pre-incubation of soluble hACE2 at both 10 μg/ml and 50 μg/ml (Fig. 2d), further supporting the notion that hACE2 is the receptor and soluble hACE2 might be used as a therapeutic inhibitor against SARS-CoV-2 infection.\nFig. 2 Entry and receptor of SARS-CoV-2 S pseudovirons.\na, b Entry of SARS-CoV-2 S pseudovirions on indicated cell lines. Cells from human and animal origin were inoculated with SARS-CoV-2 S (red), SARS-CoV S (blue), or VSV-G (gray) pseudovirions. At 48 h post inoculation, transduction efficiency was measured according to luciferase activities. RS, Rhinolophus sinicus bat embryonic fibroblast; BHK/hAPN, BHK cells stably expressing hAPN, the hCoV-229E receptor; 293/hACE2, 293 cells stably expressing hACE2, the SARS-CoV receptor; HeLa/hDPP4, HeLa cells stably expressing hDPP4, the MERS-CoV receptor. Experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM. c Binding of SARS-CoV S and SARS-CoV-2 S proteins to soluble hACE2. HEK293T cells transiently expressing SARS-CoV and SARS-CoV-2 S proteins were incubated with the soluble hACE2 on ice, followed by polyclonal goat anti-hACE2 antibody. Cells were analyzed by flow cytometry. The experiments were repeated at least three times. d Inhibition of SARS-CoV-2 S pseudovirion entry by soluble hACE2. SARS-CoV S, SARS-CoV-2 S, or VSV-G pseudovirions were pre-incubated with soluble hACE2, then mixture were added to 293/hACE2 cells. Cells were lysed 40 h later and pseudoviral transduction was measured. Experiments were done twice and one representative is shown. Error bars indicate SEM of technical triplicates. Source data are provided as a Source Data file."}

LitCovid-PD-MONDO

LitCovid-PD-CLO

LitCovid-PD-CHEBI

LitCovid-PD-GO-BP

{"project":"LitCovid-PD-GO-BP","denotations":[{"id":"T9","span":{"begin":629,"end":641},"obj":"http://purl.obolibrary.org/obo/GO_0009293"},{"id":"T10","span":{"begin":1356,"end":1368},"obj":"http://purl.obolibrary.org/obo/GO_0009293"},{"id":"T11","span":{"begin":2790,"end":2802},"obj":"http://purl.obolibrary.org/obo/GO_0009293"},{"id":"T12","span":{"begin":3821,"end":3833},"obj":"http://purl.obolibrary.org/obo/GO_0009293"}],"text":"Next, we determined whether SARS-CoV-2 S pseudovirions were able to transduce human, monkey, and bat cells. VSV-G pseudovirons were used as a positive control, whereas bald particles with no spike proteins (mock) served as a negative control. As expected, all cell types were effectively transduced by VSV-G pseudovirons (Fig. 2a). Compared to mock control, SARS-CoV-2 S pseudovirions showed an over 500-fold increase in luciferase activities in Calu3 cells, at a level similar to SARS-CoV S pseudovirions (Fig. 2a). Huh7 and Vero 81 cells also gave about 10-fold increase in luciferase activities when transduced by SARS-CoV-2. Transduction of LLCMK2 cells was higher with SARS-CoV S pseudovirions than with SARS-CoV-2 S pseudovirions (Fig. 2a), suggesting that there might be some differences on virus entry on LLCMK2 cells mediated by S proteins between SARS-CoV-2 and SARS-CoV. We then investigated whether any known CoV receptors might be used by SARS-CoV-2 S protein as entry receptor. The SARS-CoV-2 S pseudovirons were used to transduce BHK cells stably expressing human aminopeptidase N (hAPN), the receptor for human CoV 229E, 293 cells stably expressing hACE2 (293/hACE2), the receptor for SARS-CoV, and HeLa cells stably expressing hDPP4 (HeLa/hDPP4), the receptor for MERS-CoV. While BHK/hAPN and HeLa/hDPP4 cells were not susceptible for the transduction of SARS-CoV-2 S pseudovirions, 293/hACE2 cells were highly transduced by SARS-CoV-2 S pseudovirions, consistent with hACE2 as the receptor for SARS-CoV-213. We then determined whether SARS-CoV-2 S protein could directly bind to hACE2 protein. HEK 293T cells transiently expressing SARS-CoV-2 S protein were incubated with soluble hACE2 and analyzed by flow cytometry. As shown in Fig. 2c, SARS-CoV-2 S protein bound to soluble hACE2 at a level similar to SARS-CoV S protein, although the mean fluorescence intensity (MFI) for SARS-CoV-2 S protein was slightly lower than SARS-CoV S protein. To further investigate if hACE2 is the receptor for SARS-CoV-2, we performed inhibition experiments using soluble hACE2. Soluble hACE2 proteins were pre-incubated with SARS-CoV-2 S pseudovirons on ice for 1 h, then virus-protein mixture was added onto 293/hACE2 cells. Entry of SARS-CoV-2 S pseudovirions was significantly prevented by pre-incubation of soluble hACE2 at both 10 μg/ml and 50 μg/ml (Fig. 2d), further supporting the notion that hACE2 is the receptor and soluble hACE2 might be used as a therapeutic inhibitor against SARS-CoV-2 infection.\nFig. 2 Entry and receptor of SARS-CoV-2 S pseudovirons.\na, b Entry of SARS-CoV-2 S pseudovirions on indicated cell lines. Cells from human and animal origin were inoculated with SARS-CoV-2 S (red), SARS-CoV S (blue), or VSV-G (gray) pseudovirions. At 48 h post inoculation, transduction efficiency was measured according to luciferase activities. RS, Rhinolophus sinicus bat embryonic fibroblast; BHK/hAPN, BHK cells stably expressing hAPN, the hCoV-229E receptor; 293/hACE2, 293 cells stably expressing hACE2, the SARS-CoV receptor; HeLa/hDPP4, HeLa cells stably expressing hDPP4, the MERS-CoV receptor. Experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM. c Binding of SARS-CoV S and SARS-CoV-2 S proteins to soluble hACE2. HEK293T cells transiently expressing SARS-CoV and SARS-CoV-2 S proteins were incubated with the soluble hACE2 on ice, followed by polyclonal goat anti-hACE2 antibody. Cells were analyzed by flow cytometry. The experiments were repeated at least three times. d Inhibition of SARS-CoV-2 S pseudovirion entry by soluble hACE2. SARS-CoV S, SARS-CoV-2 S, or VSV-G pseudovirions were pre-incubated with soluble hACE2, then mixture were added to 293/hACE2 cells. Cells were lysed 40 h later and pseudoviral transduction was measured. Experiments were done twice and one representative is shown. Error bars indicate SEM of technical triplicates. Source data are provided as a Source Data file."}

LitCovid-sentences

LitCovid-PubTator

PMC:7100515 / 8695-12701 JSONTXT

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PMC:7100515 / 8695-12701 JSON TXT