| Porcine encephalomyocarditis is an infectious disease caused by encephalomyocarditis virus(EMCV),which endangers the health of pigs and can be transmitted among a variety of mammals.It is mainly manifested in encephalitis and myocarditis.It exists widely in pig farms and can cause human infection,which poses a potential threat to pig industry and public health safety.Studies have shown that the nonstructural protein 2A protein of EMCV could inhibit apoptosis,but the relevant evidence is lack and the molecular mechanism is unclear.In this study,we proved that 2A protein can inhibit apoptosis.Combined with pull-down and mass spectrometry,we screened the cellular proteins that interact with 2A protein to inhibit apoptosis.Annexin A2 is closely related to a variety of virus infections and plays an important role in virus attachment,internalization,transportation and regulation of apoptosis.However,there is not research to clarify the molecular pathway of annexin A2 involved in the regulation of apoptosis during virus infection.The study clarified the molecular pathway of annexin A2 involves in 2A protein inhibiting apoptosis.Our research provides a theoretical basis for revealing the pathogenic mechanism of EMCV and designing new antiviral small molecule target drugs.EMCV is a lytic virus that releases virus particles by cleaving cells.In order to promote proliferation and diffusion,EMCV evades the defense mechanism of cells by inhibiting apoptosis.Clarifying the molecular mechanism of 2A protein inhibiting apoptosis is helpful to reveal the molecular pathogenesis of EMCV.We expressed 2A protein in PK15 cells and proved that 2A protein could inhibit apoptosis.Then,2A protein was expressed in prokaryotic cells and purified.The cellular proteins interacting with 2A protein were screened by pull-down and mass spectrometry.After consulting the literature,annexin A2 was selected as the interaction protein to study the inhibition of apoptosis by 2A protein,and the interaction was verified by Co-IP and laser confocal.Next,si RNA interference technology was used to reduce annexin A2 in both PK15 and BHK21 cells,respectively.Compared with the control,the expression of annexin A2 was reduced,and the inhibitory effect of 2A protein on apoptosis was weakened in both cells.The results indicated that annexin A2 plays an important role in inhibiting apoptosis of 2A protein.In the following virus infection test,we found that the EMCV infected the cells reducing annexin A2 could lead to a significant increase in apoptosis and inhibition of proliferation.The above results showed that the interaction between 2A protein and annexin A2 inhibits apoptosis and promotes the proliferation and diffusion of EMCV.In this study,BHK21 cells reducing annexin A2 by si RNA expressed 2A protein.The changes of the pathway molecules such as caspase 3,caspase 8,caspase 9,p53,c-Jun and JNK were detected by q RT-PCR and Western blotting.The results showed that the interaction between 2A protein and annexin A2 inhibited apoptosis through JNK/c-Jun molecular pathway.In order to further verify the interaction between 2A protein and annexin A2,two 2A protein truncates were constructed at the C-end of 2A protein with different lengths.The Co-IP test showed that annexin A2 did not interact with 2A protein truncates.In Western blot,2A protein truncates could not cause the corresponding changes of cell related pathway molecules,the results indicated that the C-end of 2A protein is the key sites of protein interaction.And then,the changes of molecules during EMCV infection were detected by q RT-PCR and Western blot,which proved that EMCV inhibited apoptosis through JNK/c-Jun pathway.In conclusion,by pull-down and mass spectrometry we screened the cell protein annexin A2 interacts with 2A protein.This study clarified 2A protein inhibits apoptosis by interacting with annexin A2 through JNK/c-Jun pathway and revealed the molecular pathogenic mechanism of EMCV inhibiting apoptosis and promoting proliferation. |
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