Function And Mechanism Study Of Extracellular ADP In Viral Infection

Recently, bird flu, SARS, Ebola and other viruses bring panic to people all around the world, but also bring big challenges to modern health care system. Therefore, how to prevent and control the spread of viral diseases has become an urgent issue. And studying the routes of infection and antiviral mechanism is the key to solve these problems.It is well known that interferons are the most important cytokines, which resist viruses by inducing 300 kinds of ISGs (Interferon Stimulated Genes). And screening ISGs has become an important way of antiviral gene discovery. Therefore we screen 245,000 transcripts in IFN-a 2a stimulated PBMC through GeneChip(?) Human Transcriptome Array 2.0 (HTA2.0) from Affymetrix. And we find the expression of purinergic receptors P2Y12 and P2Y13 increase significantly, which shows the typical characteristics of ISGs. What’s more, we analysis database of NCBI and find P2Y12 and P2Y13 are up regulated in hepatitis B virus infected patients. Then we further clarify P2Y13 up regulated in virus infected cells. All of these suggest that P2Y13 and P2Y13 may play an important role in viral infection.Purine receptors are a kind of membrane receptors, whose ligands are extracellular nucleotides. Purine receptors play an important role in nervous, cardiovascular and immune function. To our surprise, P2Y12 and P2Y13 have the same ligand ADP, though they are different purine receptors. So we detect the concentration of extracellular ADP when viruses infect. The result shows that the extracellular ADP was released obviously in viral infection. This feature further confirmed the potential role of ADP and its receptors in viral infection.Then we evaluate the function of ADP and its receptors in different process. First, the cells are treated by ADP before and when viruses infect, but little influence was observed in ADP treated cells when compared with control. Whereas, if we kept treating the cells with ADP for a long time, the quantity of VSV decreased significantly, which points out that ADP may inhibit the replication of VSV. And we get the same results with NDV, HSV and Lentivirus. After that, we verify the antiviral function of ADP in mice model.To further clarify the receptors involved in ADP mediated antiviral function, we screen the major receptors associated with ADP. ADP has three receptors:P2Y1, P2Y12 and P2Y13 P2Y1 mainly activates PLC-β signaling. P2Y12 and P2Y13 inhibit the activation of cAMP. First, we use Forskolin (the activator of cAMP) and U73122 (the inhibitor of PLC-p) to value the function of P2Y1, P2Y12 and P2Y13. The results show that Forskolin can limit the function of ADP, however U73122 can not. It suggests that the major receptors may be P2Y12 and P2Y13. Meanwhile, we use CRISPRs to generate the P2Y1, P2Y12 and P2Y13 knockout mice model. When BMDM of WT, P2Y1-KO, P2Y12-KO and P2Y13-KO are treated with same quantity of VSV for 12h, there is little difference between WT BMDM and P2Y12-KO BMDM. However, there are much more VSV in P2Y13-KO BMDM than WT BMDM. And in P2Y12-KO BMDM P2Y13 is up regulated. So there are less VSV in P2Y12-KO BMDM than WT BMDM. Therefore, P2Y13 is a major receptor.It is well-known that intracellular cAMP plays an important role in the regulation of physiological functions, however, it has little reports about its antiviral function. So we want to do more about it. First, we detect the expression of IFN in ADP stimulated cells by QPCR. The result shows that ADP can not induce the expression of IFN. Second, we test the influence on cell viability by ADP. And the results point out that ADP also can not increase cell viability significantly.Interestingly, Xinrong Tao indicated the key role of Epac1 (induced by cAMP) in promoting virus replication in 2014. And at the same time, our study also find ESI09, a kind of inhibitor of Epacl, can limit the replication of viruses as ADP does. And cAMP/Epacl could be increased in viruses infection to facilitate virus’ replication. It suggests that ADP and its receptors may reduce viruses infection by thrgouh cAMP/Epac1 signaling.Taking into account all these analyses I have made above, it is conspicuously to say that there are much ADP release and increased P2Y13 expression when the viruses infection. After ADP activating to its receptors, the function of cAMP and Epac1 will be limited, which will inhibit virus’s replication in the host cells. Our study enrich the understanding of extracellular ADP and its receptors in viral infection, which suggest a potential therapeutic significance of them in viral diseases.