The Role Of 3C Protein In Apoptosis Induced By EV71 And The Protective Effect Of Caspase-3 Inhibitor

Hand-foot-and-mouth disease(HFMD)is generally a rash and fever illness in children under 5,with the symptoms of vesicles in palms and soles,buccal mucosa,tongue and buttocks,and the main transmitted pathogens is enterovirus71(EV71).Studies have shown that the mechanism between apoptosis and viral replication is complex.It is known that EV71 virions infect host cells and induce apoptosis.Therefore,it is crucial to elucidate the mechanism of apoptosis in the replication of EV71 virus for applying apoptosis-related drug against hand and,foot and mouth disease.The protease caspase-3 is an important factor involved in apoptosis,and the effect of apoptosis on viral replication is mainly achieved through the non-structural proteins.Viral non-structural protein 3C not only plays a key role in the maturation of viral proteins,but studies have also shown that EV71 activates caspase-3 to induce apoptosis in host cells via the non-structural viral protein 3C.Until now it is unclear how 3C activates caspases and how activation of caspase affects viral production.Firstly,we explored the relationship between 3C and caspase protein activation.The research object was human hepatocellular carcinoma cell Hep G2.We studied the cytopathic effect of Hep G2 transfected 3C and the protection of of caspase-3 inhibitor to Hep G2 transfection 3C through cytomorphology and nuclear staining;The activation of caspase-3,8 and 9 when transfected with 3C and 3C mutants was detected by immunoblotting,co-immunoprecipitation,and activation detection method and other biochemical tests and the mechanism of action of 3C protein and caspases protein was further studied.Secondly,we fully examined the protective effect of capsase-3 inhibitors on host cells infected with EV71(MOI = 1),the study object were human rhabdomyosarcoma cells RD,set up different experimental treatment groups: control group(Mock+Con),EV71 infection Group(EV+Con),inhibitor group(Mock+In),and the EV71-infected group protected by inhibitors(EV+In)to observe the changes of cell cycle,release of inflammatory factors,expression of viral protein,virulence of the virus and others after virus was infected for 24 hours by flow cytometry,Elisa,cell counting,real-time PCR,TCID50,and other methods.The results of the relationship between 3C and Caspase protein activation show that Caspase-3 inhibitors can inhibit the cytopathic effect caused by 3C transfection,and also have a protective effect on cell DNA fragmentation;transfection of 3C can increase the activation of caspase-3,8,9 in Hep G2 cells and increase expression at the same time(P<0.001);Co-immunoprecipitation showed that 3C was able to bind Caspase-8,9 but not Caspase-3;The activity of Caspase-3 was also decreased after the activity of Caspase-8 or Caspase-9 in cells transfected with 3C was inhibited(P<0.001).Compared with the transfected 3C mutant,the activity of Caspase-3,8,9 was lower than that of transfected 3C(P<0.001);immunoprecipitation Western Blot showed that showed that both 3C and 3C mutants can bind to caspase-8 but not to caspase-3.The results of studies on the protective effect of caspase-3 inhibitors on host cells show that,first,the caspase-3 inhibitor protects the host cells from apoptosis induced by EV71 infection.Western Blot showed that the amount of caspase-3 protein in the EV+In group was significantly lower than that in the EV+Con group(P<0.001),and the number of cells in the EV+Con group was decreased(0.57±0.01×104)(P <0.001)compared with Mock+Con(1.53±0.03×104),but the number of cells in the EV + In group significantly recovered(1.06 ± 0.06 × 104);ELISA results showed that EV + In group decreased inflammatory factor IL-6 levels that significantly increased in EV + Con group(P<0.001);Mod Fit LT analyzed that cell cycle EV+In group reversed S phase arrest in EV+Con group(P<0.001);RTPCR experiments showed VP1 gene expression in EV+Con and EV+In groups has no difference with 5’UTR m RNA levels and related Luciferase activity,but Western Blot showed that EV+In reduced the amount of viral protein VP1 in the intracellular and supernatant(P<0.001);after treatment with caspase-3 inhibitors The real-time PCR analysis of the supernatant virus confirmed that the viral genome level decreased from 1.00 ± 0.39 to 0.08 ± 0.003(P <0.001),meanwhile the TCID50 / m L decreased after the inhibitor treatment.Conclusion:1.EV71-encoded non-structural protease 3C induces apoptosis of host cells through the Caspase pathway.3C binds to Caspase-8 and Caspase-9,but does not bind to Caspase-3,and caspase activation depends on the proteolytic activity of 3C;2.Caspase-3 inhibitors can protect host cells against apoptosis induced by 3C proteins;3.Caspase-3 inhibitors can reduce cell damage caused by EV71 infection,and reduce the content of EV71 virions by inhibiting S-phase arrest of host cells;4.Caspase-3 inhibitors do not affect the entry,replication and translation of EV71,but can reduce the production of EV71 virions and virus virulence;5.Caspase-3 of host cells is manipulated by EV71 for reproduction and amplification of the virus itself.