Mechanism Study Of GPR17 Restricting Rabies Virus Replication

Rabies,caused by rabies virus(RABV),is a zoonotic disease with severe neurological damage affecting human and animal health throughout the world.Although some developed countries have declared complete elimination of RABV,rabies still poses a huge threat to developing countries.Vaccination is sufficient to hinder the development of rabies post exposure.The fatality rate is nearly 100% once RABV enters host brain.however,no effective drug has yet been developed to treat rabies.A key factor that contributes to the consequence is the incomplete understanding of RABV pathogenesis.Therefore,elucidating the pathogenic mechanism of RABV will provide theoretical support for the clinical treatment of rabies.G protein-coupled receptors(GPCRs)are a large family of plasma membrane localized receptors and contain seven transmembrane regions.GPCRs are widely involved in physiological processes including metabolism,neurotransmission,immune regulation,cell proliferation,differentiation and apoptosis,and about 82 GPCRs have been identified as drug targets for the treatment of diseases.A growing number of studies suggest that GPCRs also play an important role in regulating viral infection.As an important member of GPCRs family,GPR17 has a broad distribution at the level of the central nervous system(CNS)and shows abundant expression in various CNS cells.GPR17 is involved in the pathological process of various brain injury diseases and is regarded as an important target in brain repair therapy.As a classical neurotropic virus,RABV infection causes brain inflammation and nerve damage.However,it is still unclear whether GPR17 participates in the regulation of RABV infection.Therefore,this study explored the function of GPR17 in the process of RABV infection and further investigated the underlining mechanism.1.RABV promotes its proliferation by downregulating GPR17.The expression of GPR17 was downregulated in RABV-infected mice and nerve cells including neurons,astrocytes and microglia.We found that upregulation or activation of GPR17 can reduce the virus titer;conversely,the silence or inactivation of GPR17 led to increased RABV replication in N2 a cells.The recombinant RABV expressing GPR17(r RABV-GPR17)showed reduced replication capacity compared to the parent virus r RABV.In addition,mice were infected intramuscularly(i.m.),intracranially(i.c.)or intranasally(i.n.)with r RABV or r RABV-GPR17,results showed that r RABV-GPR17 exhibited attenuated pathogenicity and the viral loads of r RABV-GPR17 were significantly lower than those of r RABV post i.n.infection.2.GPR17 modulates neuronal apoptosis though intrinsic pathway.We further explored the mechanism of GPR17 inhibiting RABV proliferation.Overexpression of GPR17 in N2 a cells reduced cell viability and promoted cell apoptosis.Besides,r RABV-GPR17 caused more severe neuronal apoptosis than r RABV under the same viral loads in mice.Western blotting showed that GPR17 promoted the activation of caspase-3 and Caspase-9 signaling,suggesting that GPR17 may regulate intrinsic apoptosis.Decreased intracellular ATP content,increased calcium ions,increased ROS and mitochondrial cytochrome C leakage were detected in GPR17 overexpressed and activated cells,further demonstrating that GPR17 can modulate intrinsic apoptosis.3.GPR17 induces neuronal apoptosis via the NF-κB-BAK pathway.Subsequently,the intrinsic apoptosis-related genes were examined and we found that GPR17 could significantly upregulate the expression of the pro-apoptotic gene BCL2 associated K protein(BAK).Silence of BAK significantly attenuated the pro-apoptotic effect mediated by GPR17,thereby promoting RABV replication.We further explored the molecular mechanism of GPR17 regulating BAK.Overexpression and activation of GPR17 reduced the levels of c AMP and p-PKA,which led to the activation of NF-κB signaling pathway.The binding sites of transcription factor p65 were predicted on the BAK promoter,and the binding of p65 to the BAK promoter was confirmed by luciferase reporter assay.Furthermore,inhibition of the NF-κB pathway significantly blocked GPR17-induced upregulation of BAK.The above results indicate that GPR17 upregulates the expression of BAK through the c AMP-PKA-NF-κB pathway,thereby mediating neuronal apoptosis and ultimately inhibiting viral replication.Our findings uncover an unappreciated role of GPR17 in the antiviral response to RABV infection.GPR17 restricts RABV replication via BAK-mediated apoptosis,while RABV promotes its proliferation by downregulating GPR17 expression.This study provided insight into the pathogenic mechanism of RABV,and suggested potential therapeutic targets for clinical treatment of rabies.