Rabies Virus Uses Metabotropic Glutamate Receptor Subtype 2 As An Entry Receptor

Rabies is a neurotropic zoonosis caused by rabies virus(RABV)and is characterized by infection of central nervous system(CNS)and fatal neurological symptoms The mortality of rabies can be as high as 100% once symptomatic.Rabies kills nearly 60,000 human deaths every year and the majority of them occurred in India and China.The only way to prevent rabies is by immunization and no effective therapeutic methods are available currently.Even though some host factors such as nAchR,NCAM and p75 NTR have been identified as candidate receptors for RABV,however none of them are conclusive for RABV infection,suggesting that RABV entry is a multistep process that uses multiple host molecules and still more receptors remain unknown.Our Previous work has identified the host factors that necessary for RABV infection in HEK-293 cells by human genome-wide RNAi screening.The mGluR2 was chosen by further bioinformatics analysis.The mGluR2,which belongs to family C GPCR,group II metabotropic glutamate receptors,expresses at a relatively high level in CNS,and has been linked to multiple neurodegenerative diseases.To date,mGluR2's role in RABV infection process is still unclear.In order to identify mGluR2 as a receptor of RABV and explore its role in RABV's endocytic pathway,a series of experiments have been carried out.Through membrane proteins extraction,the mGluR2 was found located on membrane of HEK-293,N2 a and SK-N-SH cells.Knocking down mGluR2 in HEK-293,SK-N-SH and N2 a cells significantly inhibited RABV infection.On the other hand,overexpression of mGluR2 in HEK-293 promoted RABV infectivity.By co-IP and pulldown technology,the results showed that the ectodomain of mGluR2(mGluR2-GST)directly interacts with the ectodomain RABV glycoprotein(RABVG-His).It was proved that by breaking interaction between RABV and mGluR2,mGluR2 antibodies and mGluR2 soluble protein could efficiently inhibited RABV infection in vitro or in vivo in a dose-dependent manner.The flow cytometry results showed that binding of RABV promoted the endocytosis of mGluR2.By multiplex immunofluorescence staining and super-resolution microscopy,it was proved that RABV and mGluR2 were internalized into cells and transported to early and late endosome together.In addition,co-IP results showed that the interaction of RABV G and mGluR2 remained unaffected under pH 5.5.Next by knockdown of ?-arrestin2 in HEK-293,it was proved that RABV invasion did not utilize the ?-arrestin2 signal pathway.Further,the mGluR2/3 agonist LY379268 inhibited RABV replication in HEK-293 in a competitive manner,while the forskolin-stimulated intracellular cAMP level was unaffected by infection of RABV.In conclusion,these results demonstrated that mGluR2 is an RABV entry receptor and has the potential of becoming therapeutic for rabies.Our research would be conducive to the RABV pathogenesis studies and the elimination of rabies.