Fig. 1: mGluR2 is required for SARS-CoV-2 infection and directly interacts with the S protein. | Cell Discovery

Fig. 1: mGluR2 is required for SARS-CoV-2 infection and directly interacts with the S protein.

From: SARS-CoV-2 uses metabotropic glutamate receptor subtype 2 as an internalization factor to infect cells

Fig. 1: mGluR2 is required for SARS-CoV-2 infection and directly interacts with the S protein.

a Knockdown of mGluR2 was measured by use of qPCR in Vero-E6 cells, Caco-2 cells, and HPAE cells. simGluR2#1 and simGluR2#2, siRNAs specific for mGluR2 mRNA from monkey and human, respectively. siControl, scrambled RNA. b, c mGluR2-silenced Vero-E6 cells (b), Caco-2 cells (b) or HPAE cells (c) were infected with HRB25. At 24 h p.i., virus in the culture supernatant or in the cell lysate (HPAE cells) was detected by use of plaque assays or qPCR. d, e mGluR2-transfected cells were infected with HRB25. At 24 h p.i., virus in the culture supernatant was detected by use of plaque assays (d). The expression of exogenous mGluR2 in HEK293 cells and HEK293-ACE2 cells was confirmed by western blotting assay (e). f–i mGluR2-Flag and SARS-CoV-2 S-Myc (f), SARS-CoV-2 S1-Myc (g), SARS-CoV-2 RBD-Myc (h), or SARS-CoV-2 S1-∆RBD-Myc (i) were co-transfected into HEK293 cells and then immunoprecipitated by using anti-Flag agarose beads. j–l Purified recombinant GST-tagged mGluR2 ectodomain (mGluR2-GST) was pooled with lysate from SARS-CoV-2 S-Myc- (j), SARS-CoV-2 S1-Myc- (k) or SARS-CoV-2 RBD-Myc- (l) transfected HEK293 cells and then pulled down by using anti-GST beads. m, n mGluR2-GST was pooled with cell lysate from SARS-CoV-2-infected Vero-E6 cells (m), or purified SARS-CoV-2 S1 subdomain protein (SARS-CoV-2 S1-His) (n) and then pulled down by using anti-GST beads. o, p Vero-E6 cells (o), Caco-2 cells (o), and HPAE cells (p) were treated with mGluR2-Ab at different concentrations or IgG2a (20 μg/mL for Vero-E6 cells and Caco-2 cells; 15 μg/mL for HPAE cells) for 1 h at 4 °C, and then infected with HRB25. Virus in the culture supernatant or in the cell lysate (HPAE cells) was detected by use of plaque assays or qPCR at 48 h p.i.. q HRB25 was pooled with different concentrations of mGluR2-GST or GST (200 μg/mL), and then Vero-E6 cells were infected by mixing for 1 h at 37 °C. Virus in the culture supernatant was detected by use of plaque assays at 24 h p.i.. The data shown are representative results from three independent experiments (a–d, o–q n = 3), means ± SD, Student’s t-test, ns, not significant, **P < 0.01, ***P < 0.001.

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