Mechanism Of PEDV Nsp14 Protein Inhibiting Host Protein Synthesis By Regulating Translation Initiation Factors

Porcine epidemic diarrhea(PED)is an acute,highly contagious intestinal infection caused by porcine epidemic diarrhea virus(PEDV).In recent years,the global aquaculture industry has brought huge economic losses.Almost all known viruses rely entirely on the host cell’s protein synthesis mechanism to produce the proteins that viruses need to carry out their life activities.Therefore,viruses are usually able to influence the protein translation system of cells,circumvent cellular defenses that limit viral transmission,inhibit the antiviral responses of cells such as apoptosis,inflammation and immunity,and ensure the normal translation of their m RNA.PEDV Nsp14 protein is a protein with dual enzyme function.Its C-terminal has exonuclease activity and N-terminal has methyltransferase activity,which is a part of the replication-transcription complex and plays an important role in the process of viral replication.We first verified that PEDV Nsp14 protein can inhibit protein synthesis through Western Blot experiment,but the specific mechanism of its inhibition of protein synthesis remains unclear.Since the synthesis of GFP protein is mainly synthesized through cap-dependent translation,we then co-transfected PRK5-Myc-Nsp14 recombinant plasmid with p EGFP-N1 plasmid to detect the expression of GFP protein.To determine whether PEDV Nsp14 protein might inhibit host cell protein synthesis by acting on cap-dependent protein translation.The results showed that PEDV Nsp14 protein could also inhibit the expression of GFP protein in a dose-gradient dependent manner.At the beginning of protein synthesis,protein translation initiation complex 4F(EIF4F)plays an important role.Composed of e IF4 G,e IF4 E and e IF4 A,it plays an important role in the translation process.Therefore,next,we explored the influence of PEDV Nsp14 protein on translation-related proteins,and Western Blot results showed that PEDV Nsp14 had inhibitory effects on the expression of both e IF4 G and e IF4 E proteins.In order to explore how PEDV Nsp14 protein reduces the expression levels of e IF4 G and e IF4 E,we first started from the protein degradation pathway and treated cells transfected with PRK5-Myc-Nsp14 recombinant plasmid for 24 h with proteasome inhibitors and lysosome inhibitors,respectively.Western Blot detection showed that the expression level of e IF4 E protein was basically recovered,but the expression level of e IF4 G protein was only partially recovered.Correspondingly,the synthesis of total proteins in the cells was not completely recovered.This suggests that PEDV Nsp14 protein may have other ways to inhibit protein expression in host cells.As one of the measures taken by cells to resist virus invasion is endoplasmic reticulum stress to reduce protein synthesis of cells,we speculated that PEDV Nsp14 protein could affect protein synthesis of cells through this pathway.Therefore,Western Blot detection showed that PEDV Nsp14 protein could up-regulate the expression level of P-e IF2α protein,and the up-regulation of P-e IF2α protein indirectly led to translation inhibition.Next,we continued to explore the role of PEDV Nsp14 mutant with deletion of methyltransferase activity in the process of inhibiting protein synthesis.Since PEDV Nsp14 mutant recombinant plasmid with deletion of methyltransferase activity has been obtained in previous experiments,we transfected it into cells.It was found that PEDV Nsp14 mutant did not inhibit host cell translation and could no longer up-regulate the expression level of P-e IF2αprotein.In summary,in this study,it was found that PEDV Nsp14 protein could inhibit the expression of e IF4 G and e IF4 E proteins in protein translation initiation complex through lysosome pathway,thus inhibiting the protein synthesis of host cells.Methyltransferase activity of PEDV Nsp14 protein plays an important role in virus replication and antiviral response to escape from the host.This study preliminarily elucidated the molecular mechanism of PEDV Nsp14 protein inhibiting host cell protein synthesis,providing new ideas and potential targets for the development of PEDV drugs and vaccines.