PHB2 Is Involved In Enterovirus 71 Infection Via Autophagy

Background and ObjectsIn recent years,large-scale outbreaks of hand,foot and mouth disease(HFMD)have occurred in the world,especially in the Asia-Pacific region.HFMD caused by enterovirus 71(EV71)seriously endangers children's health.EV71 is the main pathogen of HFMD,and it is also the main cause of severe and fatal HFMD cases.HFMD caused by EV71 can develop into a severe neurological disease,which progresses rapidly and has a high mortality rate.It is unclear whether the marketed EV71 inactivated vaccine can prevent severe HFMD.The pathogenic mechanism of EV71 infection and severe HFMD still needs to be clarified.Autophagy is a intracellular degradation process which is ubiquitous in eukaryotes.Autophagy is an important antiviral defense mechanism,but many studies have shown that RNA viruses can use autophagy to promote self-replication.EV71 can induce and utilize autophagy to promote self infection.VP1 is a structural protein of EV71 and has major neutralizing epitopes,which is of great significance for research.Previous studies have found that EV71 VP1 can induce autophagy,but the mechanism is unclear and needs further research.VP1 binds to cell surface receptors and interacts with many host proteins.Our group discovered that there may be an interaction between EV71 VP1 and the host protein PHB2.PHB2 is an autophagy-related protein and may be involved in EV71 infection via autophagy.The interaction needs to be further verified,and the effect of PHB2 on EV71 infection and autophagy needs to be further explored.Methods1.Screen and verify host proteins that interact with EV71 VP1 and regulate autophagy.Mass spectrometry were used to obtain proteins that may interact with EV71 VP1.We screened the candidate proteins to get the protein of our interest,PHB2.Then this interaction was verified by co-immunoprecipitation.The co-localization was detected by immunofluorescence.2.The role of the host protein PHB2 in EV71 infection and autophagy.RD cells were transfected with siRNA targeting PHB2(SiPHB2)and then infected with EV71,and CCK-8 was performed to detect cell survival rate.At different time points after infection,qPCR was used to detect intracellular viral RNA,TCID50 was used to detect the virus titers of the cell supernatant,and Western Blot was used to detect viral proteins and autophagy proteins.3.EV71 infection in autolysosomes.RD cells were pretreated with lysosomal acidification inhibitor Bafilomycin A1(Baf-Al)before EV71 infection.At different time points after infection,qPCR was used to detect intracellular viral RNA,TCID50 was used to detect viral titers in the cell supernatant.The ATP6AP2 gene was used to knock down v-ATPase(the lysosomal proton pump).RD cells were transfected with SiATP6AP2 and then infected with EV71,and CCK-8 was performed to detect cell survival rate.At different time points after infection,qPCR was used to detect intracellular viral RNA,TCID50 was used to detect the virus titers of the cell supernatant,and Western Blot was used to detect viral proteins and autophagy proteins.Results1.Mass spectrometry analysis suggested that prohibitin 2(PHB2)interacts with VP1 protein.The interaction between VP1 and PHB2 was confirmed by co-immunoprecipitation.Immunofluorescence results showed the colocalization of VP1 and PHB2 at 12h,24h,48h after EV71 infection.2.At 48h after EV71 infection,PHB2 knockdown significantly increased cell survival rate.At 12h,24h,48 h after EV71 infection,PHB2 knockdown significantly decreased viral replication,release and VP1 protein.At the same time,LC3-II/LC3-I was decreased,indicating that autophagy was inhibited.Moreover,PHB2 knockdown also inhibited autophagy induced by rapamycin treatment and the following VP1 expression.4.Baf-A1 treatment significantly decreased viral replication and release.At 48h after EV71 infection,ATP6AP2 knockdown significantly increased cell survival rate.At 12h,24h,48h after EV71 infection,ATP6AP2 knockdown significantly decreased viral replication,release and VP1 protein.At the same time,lysosomal pH,LC3-II/LC3-I and P62 expression were increased,indicating that the acid degradation environment of lysosome was damaged.Conclusion1.EV71 VP1 protein interacts with the host protein PHB2.2.PHB2 is involved in EV71 infection via autophagy.3.EV71 infection is partly dependent on the acidic environment of autolysosomes.