Study On The Mechanism Of Enterovirus 71 Structural Protein VP1 Regulating Viral Replication

Enterovirus is the main pathogen of hand foot and mouth disease(HFMD),which is often found in infants under 5 years old.It mainly causes herpes in the hands,feet and mouth of children,often with self limitation.However,severe HFMD is accompanied by pulmonary edema,cerebrospinal meningitis and other serious central nervous system complications.Enterovirus 71(EV71)is the main pathogen of HFMD,and there is no specific antiviral drug.Although the vaccine has been on the market and has good immune effect,its neurotoxicity has not been clarified.EV71 gene encodes four structural proteins,vpl-vp3 is outside the nucleocapsid,VP4 is inside.VP1 is not only the basis of EV71 typing,but also the main antigenic determinant.Most studies have proved that VP1 of EV71 is related to the virulence of virus,and several virulence sites have been screened out.However,the mechanism of VP regulating virulence is not clear.Autophagy is an important process that degrades proteins and damaged organelles and participates in maintaining cell homeostasis.A large number of studies have shown that autophagy can affect virus replication,and virus can also regulate autophagy flow to promote its own replication.However,the mechanism of autophagy affecting virus replication has not been elucidated.In this study,the recombinant strain SDLY107-VP1 was obtained by replacing the VP1 coding region of EV71 with that of the corresponding weak strain.On this basis,the effect of VP1 protein mutation on the replication level of the virus was studied,and its mechanism was preliminarily explored.The results showed that VP1 protein could change the replication ability of the virus by affecting the autophagy ability of neural cell lines,and further revealed that mTOR signaling molecules may participate in the pathway of VP 1 affecting autophagy.The results provide a theoretical basis for further elaboration of neurotoxicity mechanism of HFMD and development of antiviral drugs.Objective1.To explore the mechanism of VP1 affecting the replication of EV71 in nerve cells.2.Study the effect of autophagy on EV71 replication.3.Explore the mechanism by which EV71 induces autophagy.Methods1.The replication ability of SDLY 107,SDLY 107-VP1 and SDJN2015-01 in RD cells and SH-SY5Y cells were detected.2.RD cells and SH-SY5Y cells were infected with recombinant strain SDLY 107-VP1 and parent strains SDLY 107 and SDJN2015-01 respectively.The changes of autophagy related proteins were detected and the effect of VP1 protein on autophagy was analyzed.3.Cells were treated with 3-mA,CQ and rapamycin to change the autophagy state and study the effect of autophagy on virus replication and cell damage.4.The mechanism of autophagy induced by EV71 was studied by SH-SY5Y cells and RD cells.Results1.SDLY 107,SDLY107-VP1 and SDJN2015-01 can induce autophagy in RD cells,and there is no significant difference in the replication ability of the three viruses in RD cells.However,the replication ability and autophagy induction ability of the recombinant virus SDLY107-VP1 in SH-SY5Y cells were significantly weaker than that of the parent strain SDLY 107,similar to that of the weak strain SDJN2015-01.2.After 3-MA inhibited the early stage of autophagy,it had no significant effect on the content of VP1 protein in the cell,but the virus titer in the supernatant of cell culture decreased;after CQ inhibited the degradation of autophagy lysosome,the content of VP1 protein in the cell increased significantly,and the virus titer in the supernatant of cell culture increased;after rapamycin promoted autophagy,the content of VP1 in the cell decreased,and at the same time,the culture increased The titer of the virus was decreased.3.The inhibition of 3-MA on the early stage of autophagy had no significant effect on the degree of cell damage;CQ inhibited autophagy lysosomal degradation leading to increased cell damage;rapamycin promoted autophagy and decreased cell damage.4.After EV71 infected cells,the level of p-mTOR in the cells decreased,and rapamycin inhibited mTOR,resulting in decreased intracellular viral VP1 content and viral RNA synthesis.Conclusions1.EV71 can induce RD cells to produce incomplete autophagy,and its structural protein VP1 is related to the neurovirulence of the virus.2.The formation and accumulation of autophagy is conducive to EV71 replication,while the degradation of autophagy is conducive to pathogen clearance.The replication of EV71 benefits from the stability of autophagy.VP1 protein regulates the replication of virus in nerve cells by affecting the ability of virus to induce autophagy in nerve cells.3.The autophagy induced by EV71 is related to mTOR signal molecule,which regulates EV71 replication at RNA level.VP1 affects virus replication by mTOR regulated autophagy.