Fig 1.
siRNA silencing and overexpression of mGluR2 affect RABV infection of cells.
(A) Schematic for mGluR2 protein. Transfection with siRNA s6195 resulted in the downregulation of mGluR2 mRNA in transfected HEK293 cells (B), SK cells (C), and N2a cells (D). Silencing of mGluR2 inhibited ERA-eGFP infection of HEK293 cells (E), SK cells (F), and N2a cells (G). NT, non-targeting siRNA. (H) Overexpression of mGluR2 enhanced the growth of ERA-eGFP at a multiplicity of infection (MOI) of five in transfected HEK293 cells; virus titers in the cell culture supernatant were determined as focus forming units (FFU) in BSR-T7/5 cells. A one-way ANOVA was used for the statistical analysis. *, p<0.05. **, p<0.01.
Fig 2.
Interactions between RABV G and mGluR2.
The mGluR2-Flag interacted with ERAG-Myc (A), CVSG-Myc (B), GX/09G-Myc (C), and WCBVG-Myc but not VSVG-Myc (D) in co-IP assays with plasmid-transfected HEK293 cell lysates. Purified mGluR2-GST pulled down purified ERAG-His (E). Purified mGluR2-GST pulled down ERAG-Myc, CVSG-Myc, and GX/09G-Myc but not VSVG-Myc from lysates of transfected HEK293 cells (F).
Fig 3.
Antibodies to mGluR2 block RABV infection of cells in a dose-dependent manner.
The monoclonal antibody (mAb) or polyclonal antibody (pAb) against mGluR2 blocked ERA-eGFP infection of HEK293 cells (A), SK cells (B), N2a cells (C), and mPN cells (D). The mAb against mGluR2 also blocked VSV∆G-ERAG-eGFP infection (E) but failed to block VSV-eGFP infection (F) of HEK293 cells. The mAb or pAb against mGluR2 decreased the replication of ERA-eGFP in HEK293 cells (G) and mPN cells (H); virus titers in the cell culture supernatant were determined as FFU in BSR/T7-5 cells. The isotypes IgG2a and IgG at the highest concentration were used as controls for the mAb and pAb, respectively. A one-way ANOVA was used for the statistical analysis. *, p<0.05. **, p<0.01. ***, p<0.001.
Fig 4.
The mGluR2 ectodomain soluble protein (mGluR2-GST) neutralizes the infectivity of RABV in a dose-dependent manner.
mGluR2-GST neutralized ERA-eGFP infection of HEK293 cells (A), SK cells (B), N2a cells (C), and mPN cells (D), and neutralized VSV∆G-ERAG-eGFP infection of HEK293 cells (E) but failed to neutralize VSV-eGFP infection of HEK293 cells (F). A one-way ANOVA was used for the statistical analysis. *, p<0.05. **, p<0.01. ***, p<0.001.
Fig 5.
The mGluR2 ectodomain soluble protein (mGluR2-GST) protects mice from lethal challenges in a dose-dependent manner.
mGluR2-GST neutralized the infectivity of GX/09 street virus and protected mice from lethal virus challenges via intramuscular (A) or intracerebral (B) inoculation. The Log-rank (Mantel-Cox) test was used to analyze the statistical difference between the survival rates of the challenged mice. *, p<0.05. **, p<0.01.
Fig 6.
RABV binding results in the downregulation of cell surface mGluR2.
Flow cytometry detected cell surface mGluR2 on RABV ERA-infected HEK293 cells (ERA), which was compared to uninfected HEK293 cells by staining with anti-mGluR2 monoclonal antibody (mGluR2) or isotype antibody control (IgG2a) at 4°C or 37°C (A). The flow cytometry results were analyzed with a one-way ANOVA (B). *, p<0.05. **, p<0.01.
Fig 7.
RABV and mGluR2 are internalized into cells and transported together in early and late endosomes.
N2a Cells were stained by using the Tyramide Signal Amplification immunofluorescent method. Absence of co-localization of tubulin (green), Rab7 (red), and Tomm20 (purple) served as a negative control for co-localization (A and B). Significant co-localization of the mitochondrial marker AIF (green) and Tomm20 (red) served as a positive control for co-localization (C and D). N2a cells infected with ERA-N-mCherry for 20 minutes at 37°C were used to perform immunofluorescence staining for RABV antigen (red), mGluR2 (green), Rab5 or Rab7 (purple), and the cell nuclei (blue). Co-localization of the RABV-mGluR2 complex with Rab5 (E, F) or Rab7 (H, I) was observed and counted. The images, comprising three single fluorescence channels (G, J), represent amplified random co-localization spots in the merged image within the small white box (G from E, F from J). The 3D-rendered images were generated by using Imaris software (G, J) and the co-localization of the RABV-mGluR2 complex with Rab5 (G) or Rab7 (J) from the three single fluorescence channels is indicated with the white arrowhead.
Fig 8.
The interaction between RABV G and mGluR2 is retained under lower pH conditions.
mGluR2-Flag interacted with ERAG-Myc in co-IP assays with plasmid-transfected HEK293 cell lysates at pH 5.5 and pH 7.2 (A). Purified mGluR2-GST pulled-down ERAG-Myc from the lysates of transfected HEK293 cells at pH 5.5 and pH 7.2 (B).