Fig 2: 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.

Fig 3: mGluR2 is required for internalization of SARS-CoV-2 but not for cell binding.a, b SARS-CoV-2 binding (a) and internalization (b) assays were performed in mGluR2-silenced Vero-E6 cells and Caco-2 cells. Viral binding or internalization was quantified by normalization to the respective scrambled siRNA-transfected cells. c Vero-E6 cells were treated as described in a, except they were not treated with acid buffer/trypsin. Cell nuclei (blue), SARS-CoV-2 S protein (green). Left: representative images are shown, and the dashed box is magnified at the indicated location of the same image. Right: the fluorescence signal of SARS-CoV-2 particles was quantified by immunofluorescence staining using ZEN software. The relative fluorescence of cell-bound HRB25 under permeabilized or unpermeabilized conditions was quantified by normalization to the corresponding scrambled siRNA-transfected cells. The circles represent individual data points. n, the number of quantified cells. Data represent the sum of three independent experiments. d Surface expression level of mGluR2 was detected by using flow cytometry after infection with HRB25 at 37 °C for 30 min under unpermeabilized conditions in Vero-E6 cells. e, f Multiplex immunofluorescence was performed in Vero-E6 cells (e) and HPAE cells (f). Colocalization of mGluR2 (green), SARS-CoV-2 N protein (red), and clathrin (purple) was observed and quantified. The yellow arrows indicate the representative colocalization of mGluR2 (green), SARS-CoV-2 N protein (red), and clathrin (purple), shown in three dimensions. The data shown are representative results from three independent experiments (a, b, d n = 3), means ± SD, Student’s t-test, ns, not significant, *P < 0.05, ***P < 0.001.

Fig 4: mGluR2 is important for SARS-CoV-2 infection of mice.a Multiplex immunofluorescence staining for the detection of mGluR2-positive cells in olfactory epithelium sections of young mouse infected with SARS-CoV-2 on day 3 p.i.. SARS-CoV-2 N protein (red), mGluR2 (green), Ace2 (magenta), GAP43 (white), CK5 (gold), CK8 (yellow), and OMP (cyan). The yellow and green areas are shown adjacently at a higher magnification, respectively. The dashed box indicates the individual cell boundary. b Multiplex immunofluorescence staining for the detection of mGluR2-positive cells in lung sections of young mouse infected with SARS-CoV-2 on day 3 p.i.. SARS-CoV-2 N protein (red), mGluR2 (green), Ace2 (magenta), Foxj1 (white), SPC (gold), Tubb4 (yellow), and CC10 (cyan). The yellow and green areas are shown adjacently at a higher magnification, respectively. The red arrows indicate a mGluR2+/SARS-CoV-2+/Ace2+/CC10+ cell, and the green arrows indicate a mGluR2+/SARS-CoV-2+/Ace2+ cell. c The 3D-rendered image was generated by using Imaris software and the co-localization of mGluR2 and SARS-CoV-2 from the two single fluorescence channels is showed. d–g mGluR2 gene knockout (mGluR2-/-) (n = 13) and WT (n = 12) mice were infected with HRB26M (150 PFU/mouse), a mouse-adapted SARS-CoV-2 strain, via intranasal inoculation. At 3 days p.i., viral RNA copies and virus titers in the nasal turbinates (d, e) and lungs (f, g) were determined by use of qPCR (d, f) and plaque assays (e, g), respectively. The horizontal dashed line indicates the limit of detection. Data represent the sum of three independent experiments (d–g), means ± SD, Student’s t-test, ***P < 0.001.

Fig 5: mGluR2 is expressed in the respiratory system.a, b Multiplex immunofluorescence staining for the detection of mGluR2-positive cells in a normal human lung section. mGluR2 (green), ACE2 (magenta), Tubb4 (cyan), and SPC (red). The yellow areas are shown adjacently at a higher magnification in the alveoli (a) or bronchia (b). c Multiplex immunofluorescence staining for the detection of mGluR2-positive cells in olfactory epithelium sections of young mouse. mGluR2 (green), Ace2 (magenta), GAP43 (white), CK5 (gold), CK8 (yellow), and OMP (cyan). d Multiplex immunofluorescence staining for the detection of mGluR2-positive cells in lung sections of young mouse. mGluR2 (green), Ace2 (magenta), Foxj1 (white), SPC (gold), Tubb4 (yellow), and CC10 (cyan).