The Mechanism Of Apoptosis Induced By EV-D68 And The Protective Effect Of Caspase-3 Inhibitor

Human Enterovirus 68(EV-D68),which is a member of the Enterovirus group D,has been found to be associated with various diseases,mainly respiratory diseases and neurological complications.Over the past decade,the outbreak of EV-D68 has been frequently reported in many countries and regions around the world.At present,the main treatment for patients infected with EV-D68 is supportive treatment.There is no specific treatment for the virus,and there is no vaccine to prevent the outbreak of EV-D68.Enterovirus is closely related to apoptosis,Cysteinyl aspartate specific proteinase(Caspase)containing cysteine is an important protein in the occurrence of apoptosis,and Caspase-3 is the most important terminal shear enzyme in the process of apoptosis.So does EV-D68 induce apoptosis through the Caspase pathway,and what is the mechanism? How is the interaction with the host cell? Can Caspase-3inhibitor(Z-DEVD-FMK)also play an important role in inhibiting virus proliferation?This study is aim to illustrate these issues.Methods First,we need to explore whether the cytopathic effect caused by EV-D68 is apoptosis.RD cells were took as the object to observe the cell status,DNA degradation characteristics and the expression of viral protein vp1 and the activation of caspase-3 in cells through analyzing in cell morphology,Hoechst nucleus staining,PI staining,DNA ladder,Western Blot,etc.Then,we analyze the protective effect of Caspase-3 inhibitor on the host cells infected with EV-D68 as well as to the impact on the production of EV-D68,through performing the analysis of cell morphology,cell counting,flow cytometry,Western Blot and activity measurement at 24 hours after infection;RT-qPCR analysis of intracellular viral genomes level at 2,12,and 24 hours after infection with EV-D68,Western Blot and TCID50 analysis of intracellular viral protein vp1 expression and changes in the number of supernatants and total number of viral particles in cells at 24 h after infection.At the same time,the effect of Caspase-3activator(PAC-1)on the production of EV-D68 was analyzed,RT-qPCR analysis of the expression of viral genome in the cells after 12 h treatment of activator treatment,Western Blot and TCID50 analysis of the expression of viral proteins in the cells and the total number of viral particles in the supernatant and cells change at 24 h after infection.Subsequently,we investigated whether EV-D68 activated Caspase-8 and Caspase-9 which were the upstream of Caspase-3,and observed the changes in the activity values of Caspase-8 and Caspase-9 and the activation of active forms of Caspase-3,8 and 9 in cells at 24 hours after infection by activity assay and Western Blot.Finally,we detected the infection of EV-D68 on the four target cell lines RD,293 T,Vero,and MRC5,which were common target cells of EV71,and analyzed the genomic level and cell number changes of EV-D68 in cells by RT-qPCR and cell count.Results and analysis1.Enterovirus D68(EV-D68)infect RD cells to induce apoptosis:RD cells showed obvious cytopathic effects infected by EV-D68 infection such as shrinkage,roundness and detachment from culture dishes compared with the mock-infected group.The infected group showed the enhanced the fluorescence intensity of nucleus staining with Hoechst,while no PI staining was found,which confirmed that the cytopathic effect induced by EV-D68 was apoptosis.2.Caspase-3 inhibitor(Z-DEVD-FMK)can alleviate EV-D68 induced cytopathic effects,cell cycle arrest in G0/G1 phase,and activation of Caspase-3:The observation of cell morphology showed that the cytopathic effects induced by EV-D68 such as the shrinkage,roundness and detachment of culture dishes were obviously alleviated in EV+In group compared with EV+Con group;The results of cell counting showed that the number of cells in EV+Con group(56.33±11.84)×10~4decreased significantly compared with the Mock+Con group(125.00 ±12.49)×10~4,while the number of cells recovered significantly in the EV+In group(98.00±8.50)×10~4(P <0.01);The flow cytometry showed that the percentage of cells in the G0/G1 phase was increased in EV+Con group compared with Mock+Con(P <0.001),and the phenomenon of cell cycle arrest in the G0/G1 phase was alleviated significantly after treatment with Caspase-3 inhibitor(Z-DEVD-FMK)(P <0.01);The results of Western Blot and activity assay showed that the expression and activity of active Caspase-3 proteins in EV+Con group were higher than those in Mock+Con group,but the increase could be obviously inhibited by Caspase-3 inhibitor.3.Caspase-3 inhibitor(Z-DEVD-FMK)does not affect EV-D68 entering into the host cells and intracellular genome replication,but can reduce mature viral protein expression to decrease the production of EV-D68 viral particles:The results of RT-qPCR showed that at 2 h and 12 h after EV-D68 infection,there was no significant difference in the viral genome levels between the mock-treated cells and Caspase 3 inhibitor(In)-treated cells,while the viral genome levels,vp1 protein expression,and the total number of viral particles in the supernatants and cells in the mock-treated cells were significantly higher than those in the Caspase 3 inhibitor(In)group at 24 h after infection.4.Caspase-3 activator(PAC-1)does not affect EV-D68 replication,but can increase the expression of mature viral proteins and the production of EV-D68 viral particles:The results of RT-qPCR showed that PAC-1 did not affect the replication in cells at 12 h after infection.The results of Western Blot showed that with the increasing of PAC-1 dose(0 ?M?0.2 ?M?0.5 ?M),the expression of intracellular viral protein was also accordingly enhanced at 24 h after infection.The results of TCID50 showed that at 24 h after infection,PAC-1 treatment can increase the number of viral particles from 2.06±0.98×108 to 25.57±3.86×108 compared with the control treatment(P<0.001).5.Caspase-3 are utilized by EV-D68 for their own amplification.Combined with the results of 3 and 4 we can draw this conclusion.6.EV-D68 activate the upstream adaptors,Caspase-8 and Caspase-9,inducing the activation of Caspase-3:The results of Caspases activity assay and Western Blot showed that the expression of the active Caspase-8 and Caspase-9 proteins as well as their activity were increased significantly in the EV+Con group,while the inhibitor treatment(EV+In8/9 group)inhibited their activations.Meanwhile,the activation of Caspase-3 was also inhibited.7.EV-D68 can cause cytopathic effects in RD and 293 T cell lines,but not in Vero and MRC5 cell lines:The results of RT-qPCR and cell counting showed that in 293 T and RD cell lines,compared with Mock-infected cells,in infected cells with EV-D68 at multiplicity of infection(MOI)of 2 there were more viral genome replication in EV-D68 infection group,and the cell number was also decreased accordingly.Whereas EV-D68 with MOI of 5 had no viral repluication in MRC5 and Vero cell lines because the Ct value of viral genome was greater than 30(nonsense)and there was no obvious difference in cell number between mock-infected and EV-D68 groups.Conclusion Caspase-3 plays an important role in the pathogenesis and production of EV-D68.Thus it is a potential target for the treatment and prevention of EV-D68 and caspase-3inhibitor can treat the EV-D68.Innovation1.For the first time,it was discovered that EV-D68 induces cell apoptosis by activating Caspase-3 and leads to cytopathic effects.2.Caspase-3 inhibitor(Z-DEVD-FMK)was firstly found to reduce the production of EV-D68 viral particles by affecting its protein expression in viral maturation stage.3.It is firstly found that Caspase-3 plays an important role in the production of EV-D68.