The Role Of Interferon Stimulated Gene P2Y₁₄in Viral Infection

Recently,the outbreak of influenza A,Ebola and Zika infection has brought great panic to people all over the world.Sustained epidemic situation making the prevention and treatment of viral diseases become urgent.Thus,the understanding of mechanism involved in viral infection and antiviral immune responses is important for us to explore the antiviral drugs.After being infected by viruses,bacteria,parasites and fungal,type I interferon is released from host cells and plays important roles in both innate and adaptive immune responses through direct or indirect ways.Especially,type I interferon is now known as the most important anti-viral molecule.Studies have shown that interferons play antiviral activity through inducing the expression of hundreds of interferon stimulated genes?ISGs?.But till to now,the antiviral role of most ISGs still need to be further explored.In this study,we will mainly focus on ISGs to screen host key regulators involved in viral infection and explore the molecular mechanism.First,we used recombinant human interferon a to stimulate human peripheral blood mononuclear cells for high-throughput RNAseq.Then,we identified the ISGs from 245000 transcripts and found that the expression of purinergic receptor P2Y₁₄ is significantly increased.Meanwhile,we validated the potential of P2Y₁₄ as an interferon inducible gene in murine macrophages by real-time fluorescence quantitative PCR.In addition,TLR3 knockout and the inhibition of STAT1 can significantly reduce the Poly?I:C?and IFN? induced P2Y₁₄ expression.Furthermore,expression of P2Y₁₄ and the release of its endogenous ligand UDPN are both increased in viral infection.Taken together,all these data suggested that P2Y₁₄ and its related signaling pathways mayplay important roles in viral infection.In order to further clarify the important role of P2Y₁₄ in viral infection,we used P2Y₁₄ specific ligand UDPN to pretreat virus infected cells and mice,and we found the inhibitory effect of UDPN in virus infection by QPCR and Immunofluorescence.To further explore the antiviral mechanism of UDPN,we used CRISPR/cas9 technology to construct P2Y₁₄ knockout mice,and found that the virus infection is promoted significantly both in vivo and in vitro after P2Y₁₄ deletion.In addition,UDPN mediated protection to HSV-1 infection is eliminated in P2Y₁₄ knockou cells,indicated that the antiviral effect of UDPN is mediated by P2Y₁₄.In order to further explore the antiviral mechanism of UDPN/P2Y₁₄,we found that UDPN and P2Y₁₄ had no significant effect on the expression of IFN by QPCR,indicating that the antiviral effect of UDPN/P2Y₁₄ is independent of type ? interferon.And VSV infection significantly promotes the expression of cAMP and the downstream signaling protein EPAC1 by ELISA?QPCR?Western blot assay.Whereas,UDPN inhibits the Forskolin activated cAMP level obviously.In addition,we used QPCR? crystal violet staining technique to confirm that blocking cAMP and EPAC1 signaling pathway significantly inhibits the replication of the virus.At the same time,we found that blocking of EPAC1 could significantly inhibit the antiviral function of UDPN in EPAC1 knockdown Hela cells.These results suggested that UDPN/P2Y₁₄ may play an important role in antiviral function by inhibiting cAMP/EPAC1 signaling pathway.With the deepening study of extracellular signal and purinergic receptors,more and more attention has been paid to the immune role of extracellular nucleotides.In this study,we demonstrated P2 Y14 as an ISG through high-through RNA-seq systematically and evaluated the antiviral functions and mechanism of UDPN/P2Y₁₄ both in vitro and invivo.The research aims to provide a theoretical basis for understanding the antiviral mechanism of extracellular nucleotides and their receptors which shows great potential of them in antiviral drugs research and development.