Molecular Mechanism Of The Interaction Between Porcine Epidemic Diarrhea Virus And Endoplasmic Reticulum Stress Marker GRP78

Porcine Epidemic Diarrhea（PED）is an intestinal infectious disease of pigs manifested by diarrhea,vomiting,and dehydration,and characterized by acute and highly contagious transmission.The causative agent of PED is porcine epidemic diarrhea virus（PEDV）.At present,the widespread GⅡgenotype PEDV strain has a lethality rate of nearly 100%in newborn piglets,which has caused great economic losses to the pig industry in our country.Endoplasmic reticulum（ER）stress is a mechanism for cells to process unfolded proteins and plays an important role in maintaining ER homeostasis and determining cell fate.Studies have shown that ER stress can inhibit the replication of human coronaviruses such as severe acute respiratory syndrome coronavirus 2 and Middle East respiratory syndrome coronavirus,which in turn can interfere with the expression of ER stress-related proteins.However,whether this relationship exists between PEDV and ER stress has not yet been clarified.In order to provide a reference for the study of the pathogenic mechanism of PEDV,and provide new targets and new antiviral molecules for the development of anti-PEDV drugs,this project focuses on the interaction between PEDV and ER stress,with the aim of clarifying the effect of endoplasmic reticulum stress on the replication of different genotypes of PEDV,analyzing the mechanism of PEDV antagonizing host endoplasmic reticulum stress,and developing related anti-PEDV molecules.The specific research content of the subject is as follows:1.PEDV replication was inhibited by ER stressTo clarify the effect of ER stress on the replication of PEDV,cells were treated with classic ER stress inducers,tunicamycin（TM）and thapsigargin（Tg）.The drug treatment method adopted in the experiment was to pretreat the cells with TM and Tg for 8 h before virus infection,to exclude the influence of the direct interaction between the drug and PEDV virion or PEDV proteins.The effect of ER stress on the replication of GⅠ-b,GⅡ-a,and GⅡ-b sub-genotype PEDV was tested on Vero cells,and the results showed that the proliferation level of PEDV gradually decreased with the increase of TM and Tg concentrations.The test results on LLC-PK1 cells also showed that TM and Tg pretreatment inhibited PEDV replication in a dose-dependent manner.In addition,it was also found that inhibiting ER stress by 4-phenylbutyric acid can promote the replication of PEDV.These indicate that ER stress can assist host cells to resist the infection of different genotypes PEDV.This project also tested the effect of ER stress on the replication of PEDV on piglets.The administration method was to orally administer TM to piglets 8 hours before PEDV challenge.The results showed that the pigs in the TM treatment group had significantly less intestinal gas and intestinal wall thinning than the PEDV control group pigs,and the load of PEDV in the duodenum,jejunum,ileum,and colon of pigs treated with TM were significantly lower than those of pigs in the PEDV control group,indicating that ER stress can also effectively inhibit PEDV replication at the pig level.We further explored the major unfolded protein response（UPR）that assists ER stress in repressing PEDV,the results showed that only activating protein kinase R like endoplasmic reticulum kinase inhibited PEDV replication.In addition,inhibition of inositol-requiring enzyme 1αwas also found to inhibit PEDV proliferation,suggesting that PEDV replication may depend on this pathway.Finally,the effect of ER stress-related host proteins on PEDV replication was determined,and it was found that only overexpressing 78 k Da glucose-regulated protein（GRP78）could significantly inhibit PEDV replication,suggesting that GRP78 is the main host protein assisting ER stress to suppress PEDV replication.2.The expression of GRP78 protein was inhibited by PEDVIn order to explore whether PEDV can antagonize the antiviral effect of ER stress,this experiment evaluated the effect of PEDV on the expression of GRP78.Quantitative PCR（q PCR）results showed that both GⅠand GⅡgenotype PEDV could promote the transcription of GRP78.However,the results of Western Blot showed that the expression of GRP78 protein did not increase after PEDV infection,but decreased significantly at 36hours after virus infection.Therefore,it is speculated that PEDV may negatively regulate GRP78 at the protein level.The cells were treated with TM and Tg at different time points before and after PEDV infection to further verify the effect of PEDV on GRP78 protein expression.The results showed that when cells were pretreated with TM 8 h before PEDV infection or cells were treated with TM at the same time of PEDV infection,the expression of GRP78 in PEDV/TM group was less than that in TM control group.However,the decrease of GRP78 was not significant,which was presumed to be related to the significant inhibition of PEDV replication under these two conditions.Furthermore,the effect of TM and Tg treatment on the expression of GRP78 protein 24 h after PEDV infection was tested.The results showed that both GI genotype and GII genotype PEDV could significantly inhibit the expression of GRP78 induced by TM and Tg under this condition,and the inhibitory effect was effective in both Vero cells and LLC-PK1 cells,indicating that PEDV could inhibit the expression of GRP78 at the protein level.In addition,PEDV was also found to inhibit plasmid-mediated overexpression of GRP78.Since PEDV promotes the transcription of GRP78,the mechanism of PEDV inhibiting the expression of GRP78protein was explored from two aspects of m RNA translation and protein degradation.The results showed that PEDV infection can reduce the overall translation level of cells,while interfering with the host protein degradation pathway could not alleviate the inhibition of GRP78 protein expression by PEDV,indicating that PEDV negatively regulates GRP78protein expression mainly by inhibiting translation.3.The expression of GRP78 was inhibited by PEDV nonstructural protein 14In order to further explore the mechanism by which PEDV inhibits the expression of GRP78,this subject identified the main viral protein that assists PEDV to inhibit the expression of GRP78.We tested the effects of all PEDV proteins except for non-structural protein（nsp）2,nsp3,and nsp11 on the expression of GRP78,and found that only nsp14had the potential to inhibit the expression of GRP78.The effect of PEDV nsp14 on the expression of GRP78 was further measured on HEK293t,Vero,and LLC-PK1 cells.The results showed that PEDV nsp14 could effectively inhibit the expression of GRP78 induced by TM and Tg at the protein level,indicating that PEDV nsp14 could negatively regulate the expression of GRP78 protein.In order to confirm that the inhibition of GRP78expression by nsp14 is a common feature of all PEDV strains,the amino acid sequences of nsp14 of 83 PEDV strains were compared.It was found that there are 24 amino acid sequence types in PEDV nsp14.Since the representative strains of these sequence types cover all PEDV genotypes,these 24 sequence types were considered to broadly represent the characteristics of the amino acid sequence of PEDV nsp14.The results of the homology analysis showed that the average homology between these sequence types reached 99.25%.Sequence comparison analysis showed that there were no insertions and deletions of amino acids in these 24 sequence types,and there were no mutations in each functional site.These indicate that the amino acid sequence and function of PEDV nsp14 are conserved.Expression vectors of PEDV nsp14 with silenced exonuclease domain or silenced N7methyltransferase domain were constructed respectively,and these vectors were transfected into HEK293t cells to evaluate the effect of each nsp14 mutant on the expression of GRP78protein.The results showed that only the point mutation of the functional site in the N7methyltransferase domain completely silenced the ability of PEDV nsp14 to inhibit the expression of GRP78 protein,indicating that nsp14 mainly depends on the N7methyltransferase domain to inhibit the expression of GRP78 protein.The host factors interacting with PEDV nsp14 were analyzed by immunoprecipitation combined with liquid chromatography-mass spectrometry,and a total of 153 specific proteins were identified.GO annotation and signaling pathway clustering analysis showed that PEDV nsp14 mainly interacted with translation-related host proteins,indicating that PEDV nsp14 can affect cellular translation.The puromycin labeling test confirmed that PEDV nsp14 can inhibit cell translation,speculating that PEDV nsp14 negatively regulated the expression of GRP78 protein by inhibiting translation.The cell localization of PEDV nsp14 was analyzed,and it was found that nsp14 could enter the nucleus.Given that PEDV nsp14 can inhibit the transcription of GRP78,it was speculated that nsp14 may interfere with the initiation of GRP78 transcription in the nucleus.The effect of PEDV nsp14 on the GRP78 promoter was further identified,and it was found that PEDV nsp14 inhibited its transcription by interfering with the activity of the GRP78 promoter.4.Screening of anti-PEDV molecules targeting endoplasmic reticulum stressIn view of the fact that ER stress is an effective host anti-PEDV mechanism,we further screened anti-PEDV molecules targeting endoplasmic reticulum stress and demonstrated for the first time that LA has anti-PEDV activity.The effect of LA on the replication of PEDV was tested on Vero and LLC-PK1 cells.The results of TCID₅₀,immunofluorescence,and q PCR tests all showed that LA inhibited the replication of GⅠand GⅡgenotype PEDV in a dose-dependent manner,indicating that LA has the potential to be developed as an anti-PEDV drug.In order to explore the effect of LA on each replication process of PEDV,cells were treated with LA at different time points after PEDV infection.The results indicated that LA mainly limited the adsorption and invasion of PEDV.The relationship between the inhibitory activity of LA against PEDV and ER stress was further verified.It was found that LA can promote the transcription of endoplasmic reticulum stress-related genes,such as ATF4,CHOP,GRP78,and the expression of GRP78 protein while inhibiting ER stress with 4-phenylbutyric acid could antagonize the inhibition of PEDV replication by LA,suggesting that LA exerts an anti-PEDV effect dependent on ER stress.In addition,it was also found that inhibition of oxidative stress which is the upstream response of ER stress by NAC can antagonize the inhibition of PEDV replication by LA,indicating that the anti-PEDV effect of LA depends on the oxidative stress-ER stress pathway.