Preliminary Study On Inhibition Of PEDV E Protein On Host Protein Expression Through Translation Initiation-related Factors

Porcine epidemic diarrhea(PED)is an acute and highly infectious intestinal infection caused by porcine epidemic diarrhea virus(PEDV).PEDV spreads rapidly.Spread widely,to the pig industry has brought immeasurable damage.All viruses rely on the resources of the host cell to replicate the proteins necessary for their own life.PEDV E protein is a small envelope protein of virus,which can participate in the formation of virus envelope and virus-like particles.In the earlier stage of this study,PEDV E protein was found to inhibit host protein translation,but its specific mechanism of inhibiting translation remains unclear.In this study,the inhibitory effect of PEDV E protein on host protein translation was first determined by purinamycin labeling method,and the key protein factors involved in inhibiting translation were further screened by Western blot,RT-PCR,indirect immunofluorescence,immunoprecipitation and other methods.Eukaryotic translation initiation factor eIF2α serine 51 phosphorylation blocks translation initiation,which is a protective strategy of cells against various environmental stimuli.In order to further analyze the specific mechanism of PEDV E protein inhibiting protein translation,this study started with eIF2α phosphorylation,and found that when E protein was expressed in cells,the expression level of eIF2α phosphorylated protein could be significantly up-regulated.There are four common eIF2α phosphorylated kinases found in host cells: PKR,PERK,HRI,and GCN2.In this study,we found that PEDV E protein can activate eIF2α phosphorylation by up-regulating expression levels of PKR and PERK phosphorylated proteins,thus inhibiting protein translation in host cells.In the process of protein translation,the initial stage is very important as the rate-limiting step.The translation initiation complex eIF4 F is composed of eIF4 E,eIF4 G and eIF4 A.eIF4 B and PABP also play important roles in the assembly of the translation-initiation complex.In order to explore the effects of PEDV E protein on translation initiation complex eIF4 F,eIF4 B and PABP,we transfected p CMV-Myc-E into IPEC-J2 cells with dose gradient,and detected the protein expression levels of eIF4 E,eIF4 G,eIF4 A,eIF4 B and PABP using western blot technology.The results showed that PEDV E protein had no effect on the protein expression levels of the above translation initiation factors eIF4 E,eIF4 G and eIF4 A,but with the increase of the transfection dose of E protein,the protein expression levels of eIF4 B and PABP were significantly inhibited,and the transcription levels of eIF4 B and PABP were not affected.Co-IP experiment showed that PEDV E protein did not interact with eIF4 B and PABP,but PEDV E protein expression reduced the interaction between eIF4 B and eIF4 A.eIF4 B,as a phosphorylated protein,can be activated by a variety of kinases.Further studies have found that PEDV E protein down-regulates eIF4 B phosphorylation in a dose-dependent manner.Protein translation is a complex process regulated by multiple signaling pathways.In this study,we found that PEDV E protein can dose-dependently down-regulate the expression level of m TOR phosphorylated protein and inhibit protein translation in cells by inhibiting m TOR signaling pathway.Different studies have shown that eIF4 B has different effects on translation in vitro.This study found that PEDV E protein does not degrade eIF4 B through proteasome or lysosome pathways,and the inhibition effect of PEDV E protein on host cell protein translation can be partially restored when eIF4 B is overexpressed in vitro.In summary,this study found that PEDV E protein activated eIF2α phosphorylation by activating PERK and PKR phosphorylation,thereby inhibiting host protein synthesis.PEDV E protein inhibited the translation of host cell proteins by inhibiting the expression of translation initiation factors PABP and eIF4 B,and could inhibit the m TOR signaling pathway.This study enriched the molecular mechanism of PEDV E protein inhibiting host cell protein translation,provided a new direction for the analysis of PEDV E protein pathogenesis,and may provide a new theoretical basis for the design of novel antagonistic drugs against PEDV infection.