The Role Of Interferon-stimulated Gene GPR171 In Viral Infection

Viral diseases seriously endanger human health.According to the latest statistics from UNAIDS and World Health Organization,AIDS has killed more than 25 million people since it was identified on June 5,1986.Furthermore,as the globalization process accelerates faster and faster,the spread of the virus is much more convenient.Therefore,it is important to understand the infection mechanism of the virus and to develop corresponding treating strategies.As the most important factor in viral infection,interferon is of great significance for curing viral diseases.After viral infection,interferons binds to interferon receptors and induces more than 300 kinds of interferon-stimulated genes(ISGs),which play important roles in viral infection,but the function of most ISGs is still unknown.Therefore,we used human recombinant interferon α and interferon γ to stimulate human peripheral blood mononuclear cells and bone marrow derived macrophages respectively.Screening and evaluating 245000 transcripts by high-throughput RNAseq technology system,the results showed the expression of GPR171 was significantly increased.Meanwhile,the expression of GPR171 was significantly up-regulated in viral infection.In order to investigate the molecular mechanism of interferonstimulated GPR171 expression,we pre-treat the cells with IFNAR antibody and JAK inhibitor and demonstrate that the interferon stimulated GPR171 expression was significantly inhibited.Whereas,STAT1 inhibitor could not inhibit interferonstimulated GPR171 expression,suggested that interferon-stimulated GPR171 expression is little dependent on the classical STAT1 signaling pathway.Subsequently,we used the corresponding inhibitors against the downstream signaling pathways of JAK(JNK,MEK1/2,NF-κB,P38)and found that only NF-κB inhibitor can inhibit interferon-stimulated GPR171 expression significantly.In conclusion,both interferon and virus can induce GPR171 expression,suggesting that GPR171 may play a crucial role in viral infection.To explore the role of GPR171 in viral infection,we overexpressed GPR171 in cells and found that GPR171 promotes viral replication.Biglen,as a GPR171-specific endogenous ligand,activates the intracellular signaling pathway of GPR171 specifically.Which is consistent with the function of GPR171,Biglen promotes the replication of virus in the cells too.At the same time,when knockdown the expression of GPR171,the replication of virus reduced obviously.To further confirm its function in vivo,we constructed GPR171 knockout mouse using CRISPR/cas9 technology.In GPR171 deficient peritoneal-derived macrophages and bone marrow-derived macrophages the replication of virus is decreased obviously.At the same time,peritoneal and olfactory bulb VSV infection model also demonstrate that the expression of VSV in GPR171 knockout live,spleen,lung and olfactory bulb are all reduced significantily.Accordingly,if we treated the GPR171 knockout macrophages with Biglen,the Biglen promoted viral infection is diminished.In order to further explore the mechanism of GPR171 in viral infection,Poly(I:C),LPS and VSV were used to stimulate macrophages.q PCR assay showed that GPR171 knockout had little effect on IFNβ expression,indicating that the GPR171 promoted viral replication is not directly through inhibiting type I interferon production.Subsequently,we examined the expression of the interferon-stimulated gene ISG15 under Poly(I:C),LPS,and VSV stimulation conditions,and found that the expression of ISG15 was significantly increased in GPR171 deficient cells.At the same time,the expression of various typical interferon-stimulated genes(ISG15,OAS1,IFIT1,PKR)were all significantly up-regulated after GPR171 deletion.In summary,GPR171 may promote viral replication by inhibiting the expression of interferon-stimulated genes independent of IFNβ.The mechanism of interferons mediated protection to viral infection is of great significance for understanding the interaction between host and virus in viral diseases.This study takes the advantage of RNA-seq technology to identify GPR171 as a new interferon-stimulated gene which promote the replication of virus by inhibiting the production of ISGs.Our study demonstrate that the interferons activated ISGs not only restrict the viral infection but also balance the over-activated antiviral immune responses through forming a negative feedback regulation of ISGs in virus infection.It provides new understanding of interferon-stimulated genes in antiviral immune responses.