Coxsackievirus A6 Induces Necroptosis For Viral Production

Background:Traditionally,typical hand-foot-and-mouth disease(HFMD)is usually caused by Enterovirus 71(EV71)or Coxsackievirus A16(A16),while atypical hand-foot-and-mouth disease is usually caused by Coxsackievirus A6(CA6).In recent years,the incidence of atypical hand-foot-and-mouth disease associated with CA6 has been increased,but the understanding of its pathogenesis is still very limited.Necroptosis(also known as programmed necrosis)is a cell death pathway different from necrosis or apoptosis.In previous studies,we found that infection with CA6 could promote cell death,which was dissimilar to infection with EV71.We speculate that CA6 may induce host cell death through Necroptosis.This study explores the specific pathogenic mechanism of CA6,and provides new therapeutic strategies and drug treatment targets for related diseases caused by CA6.Methods:Human rhabdomyosarcoma cells(RD)sensitive to CA6 were used as host cells.In the first step,host cells were infected with CA6 or EV71.The differences in host cell lesions induced by CA6 or EV71 were compared by cell counting,morphological observation,and staining with propionium iodide(PI).In the second step,necrostatin-1 or apoptotic caspase-3 inhibitor(Z-DEVD-FMK)was used to treat CA6 or EV71-infected cells,respectively.And then PI staining and morphological observation were used to further detect the effects of them on CA6-induced cell damage to preliminary identification of the causative mechanism of CA6.In the third step,Necrostatin-1 or Z-DEVD-FMK was used to treat cells infected by CA6 or EV71.Cell survival rate was measured by cell counting,Western Blot and protein immunofluorescence.Changes in expression levels of necroptosis pathway proteins(RIPK3,MLKL and P-MLKL),apoptotic pathway proteins(Pro-Caspase-3 and Caspase-3),and viral structural protein VP1 were detected to further compare the difference between CA6 and EV71 in inducing cell death,and determine the pathogenic pathway of CA6.In the fourth step,the effect of small molecule interfering RNA(SHRNA)encoding the necroptosis pathway protein RIPK3 and overexpression plasmid of RIPK3 on cell death induced by CA6 was studied.In the fifth step,study the effect of necroptosis on CA6 virus replication(VP1 protein level,m RNA level,virus virulence,virus release),and the way that Necrostatin-1 inhibits virus proliferation.In the sixth step,the specific mechanism of CA6 virus-induced cell death was studied.Firstly,ROS levels were detected by flow cytometry.Then,ROS scavengers(NAC,N-acetyl-L-cysteine)were applied to CA6 infected cells,and explored the therapeutic effects of ROS scavengers on CA6 infected cells.Next,cells were transfected with 3D and 3C plasmids of CA6,and the expression level of RIPK3 protein and its interactions and colocalization with 3D and 3C protein molecules were detected to explore the role of 3D protein or 3C protein in CA6-induced cell death.Results and analysis:1.CA6 induced cytopathology is different from EV71 which induces apoptotic pathway.2.Necrostatin-1,a necroptosis inhibitor,can reverse cell death induced by CA6,while Z-DEVD-FMK,an apoptosis inhibitor,has no such effect.3.The expression of RIPK3,a marker of cell necroptosis,was time-dependently up-regulated in CA6 infection,and the trend of up-regulation was similar to the expression of viral structural protein VP1,while the expression of downstream proteins RIPK3 P-MLKL and MLKL did not change after CA6 infection.And the expression levels of Pro-caspase-3 and Caspase-3,the marker proteins of the apoptotic pathway,did not change,too.However,necrostatin-1,a necroptosis inhibitor,can reverse CA6-induced up-regulation of RIPK3 and VP1 protein expression.4.SHRNA encoding RIPK3 reduces RIPK3 expression level and inhibits CA6 VP1 protein expression;and overexpression of RIPK3 promotes CA6 proliferation.5.CA6-induced necroptosis which affects virus proliferation.(1)Necrostatin-1 and Necrostatin-2 which are both necroptosis inhibitors decreased the expression level of VP1 protein of CA6 virus in a dose-dependent manner.(2)Both Necrostatin-1 and Necrostatin-2 reduced the genomic level of CA6 virus. Necrostatin-1(150 μM)intervention group(Nec-1)reduced VP1 m RNA levels to 0.41 ± 0.02(P <0.001),Necrostatin-2(180 μM)intervention group(Nec-2)reduced VP1 m RNA levels to 0.05 ± 0.00(P <0.001).(3)Necroptotic inhibitors reduced CA6 virus virulenceThe TCID50 results showed that the TCID50 / ml of the Necrostatin-1(150 μM)intervention group(Nec-1)was 4.66 ± 0.81 × 10~5,which was ten times lower than that of the control group(Con)TCID50 / ml 45.27 ± 16.16 × 10~5(P <0.05).);Necrostatin-2(180 μM)intervention group(Nec-2)TCID50 / ml 3.68 ± 0.37 ×105 was also significantly lower than the control group(Con)TCID50 / ml 31.09 ±9.26 × 10~5.(4)Necroptotic inhibitors affectd CA6 virus release and inhibit virus proliferation.Intervention of CA6 infected cells with Necrostatin-1(150 μM).The results showed that both in the VP1 m RNA level and TCID50 / ml,the extracellular non-treatment group was much higher than the Necrostatin-1 treatment group(123times,P <0.001;229 times,P <0.001),while the intracellular difference was not so obvious.Therefore,Necrostatin-1 mainly inhibits virus proliferation by inhibiting virus release.6.3D protein encoded by CA6 directly hijacked RIPK3 to induce necroptosis,replacing the traditional ROS-dependent necroptotic pathway.(1)CA6-induced necroptosis was independent of ROS. Flow cytometry results showed that ROS levels did not increase in the CA6 infection group compared with the control group.Morphological features,PI staining,VP1 protein expression,and TCID50 results showed that NAC did not affect CA6-induced cell death.(2)3D protein encoded by CA6 directly hijacked RIPK3 protein to induce necroptotic.Protein immunoprecipitation analysis showed that: RIPK3 protein antibodies can pull down 3D-HA protein,but not HA the label protein;while RIPK3 protein antibodies cannot pull down 3C-HA after transfecting 293 T cells with 3D and 3C plasmids for CA6 36 hours.Western blot and protein immunofluorescence results showed that compared with the empty vector control group,the expression of RIPK3 protein in the 3D group was significantly up-regulated,and RIPK3 co-localized with3D-HA,but not co-localized with HA.RD cells were transfected with 3D,and the expression of RIPK3 protein was correspondingly increased.The number of necroptotic cells was positively correlated with the 3D plasmid transfection dose.Conclusions:1.CA6 can up-regulate the expression of RIPK3 protein in host cells and induce necroptosis.The expression levels of traditional downstream necroptotic execution protein MLKL and P-MLKL protein are not affected by CA6.2.Necroptosis induced by CA6 was not related to ROS,but the non-structural protein3 D encoded by CA6 directly hijacked RIPK3 to induce necroptosis,and 3D protein can be up-regulated by RIPK3.In addition,the 3C protein does not bind to RIPK3.3.Necrostatin can inhibit the up-regulation of RIPK3 expression induced by CA6 and inhibit necroptosis.4.Necroptosis induced by CA6 can promote virus release,and then promote virus proliferation.Necrostatin-1 can inhibit necroptosis induced by CA6,inhibit virus release,and inhibit virus proliferation.Innovation:1.It was discovered for the first time that the pathogenic mechanism of CA6 was to induce necroptosis in host cells,which provided new ideas for finding therapeutic targets for atypical hand-foot-mouth disease drugs.2.It was discovered for the first time that CA6 induced necroptosis by directly hijacking the necroptosis-dependent factor RIPK3 by 3D protein,which was a new mechanism of inducing necroptosis independent of ROS.3.Necrostatin-1 can inhibit the up-regulation of RIPK3 protein expression induced by CA6 to inhibit necroptosis of host cells induced by CA6.4.It was discovered for the first time that CA6-induced cell necroptosis could promote virus release and then promote virus proliferation.Necrostatin-1 can inhibit necroptosis,thereby inhibiting virus release and inhibiting virus proliferation.This suggests that Necrostatin-1 could be used as a treatment for atypical hand,foot and mouth disease.