Inhibition Function Of ORF3 Protein On Cellular Apoptosis Induced By Porcine Epidemic Diarrhea Virus Infection

Porcine Epidemic Diarrhea Virus?PEDV?is one of the major enteric pathogens currently threatening the pig industry worldwide.PEDV infection,especially in newborn piglets,can cause severe diarrhea,dehydration,and death.Beginning from2010,PED broke out in China on a large scale,causing very serious economic losses.PEDV belongs to genus alpha Coronavirus of family Coronaviridae,which owns a single-strand positive sense RNA genome of about 28 kb.Its genome encodes the virus replicase polyproteins?pp?la and1ab,the spike?S?,accessory protein?ORF3?,envelope?E?,membrane?M?and nucleocapsid?N?proteins.Orf3 is located between the s gene and the e gene in the PEDV genome.It encodes a protein of about 224 amino acids,with a molecular weight about 25 k D.In the cell-adaptive PEDV,the ORF3 gene often has a deletion of 49 nucleotides.The model representative strain carrying this gene is the attenuated DR13 strain(JQ023162,designated in the study DR13^att).More research on the function of the protein is needed.It has been reported that PEDV infection can induce apoptosis of infected cells.In our early studies we found viruses without orf3 gene caused heavier cell necrosis than the viruses carrying orf3.We supposed ORF3 protein affected the cellular apoptosis induced by PEDV infection.In this study three different PEDVs carrying whole length(r DR13-ORF3^wt),92 aa truncated(DR13^att)and with the orf3 deleted completely(r DR13^att-?ORF3),were used to study the relationship between the protein and virus-induced apoptosis by employing a variety of methods,such as live cell imaging,flow cytometry,TUNEL and transcriptome analysis.The findings and content of this study are as follows:?1?Cytopathies detection after infection with three PEDV viruses.Cells were infected with the three viruses at an MOI of 0.5.After 24 hours,checking cell pathogenic effect by microscope.r DR13^att-?ORF3 induced more CPEs in the infected cells than the other two viruses.?2?Immunofluorescence analysis?IFA?of S protein and ORF3 protein expression with three viruses.Cells were infected with the three viruses at an MOI of 1 and 0.5.After 24 hours,then Immunofluorescence was done.Expression of S protein and ORF3 protein were detected by Immunofluorescence.The results show that the S protein was expressed24 hours after infection by the three PEDVs.ORF3 proteins was expressed in infected cells with DR13-ORF3^wtand DR13^att,but not with r DR13^att-?ORF3.?3?ORF3 protein's role in cellular apoptosis induced by PEDV infectionMethod 1:Cell imager to detect cellular apoptosis induced by different virusesCells were infected with the three viruses at an MOI of 0.1.The data were analyzed for the degree of apoptosis?early apoptosis,late apoptosis and necrotic cells?when live cells were stained at different time points after infection.Dynamic observation results of live cell imager showed all the three viruses induced obvious apoptosis in the infected cells.However,higher apoptotic level was detected in the cells infected with r DR13^att-?ORF3 than the cells infected by the other two viruses?P<0.05?.Method 2:Flow cytometry to detect cellular apoptosis induced by different virusesCells were infected with the three viruses at an MOI of 5.The infection was terminated at 18 hours and 24 hours respectively.Apoptotic state of cells was detected by flow cytometry.Higher proportion of apoptotic cells was detected in the cells infected with r DR13^att-?ORF3.Method 3:TUNEL method.TUNEL method is a classic experimental method for the detection of apoptosis.Cells were infected with the three viruses at an MOI of 3.There are more TUNEL staining cells among the cells infected with r DR13^att-?ORF3 than the cells infected by the other two viruses.Method 4:Caspase-3 activation detection with Western blotCells were infected with the three viruses at an MOI of 0.1.The infection was terminated at 48 hours.The cells were lysed to make protein samples,and SDS-PAGE and Western blot were employed to detect activation of Caspase-3.The result showed that r DR13^att-ORF3^wtcould inhibit Caspase-3 activation in the infected cells.?4?Transcriptome sequencing and real-time PCR to check the differential transcription of genomes in the cells infected with different virusesCells were infected with the three viruses at an MOI of 5.The infection was terminated at 12 hours.Samples are processed according to the Easep?Super Total RNA Extraction Kit instructions.RNA was extracted for sequencing.Transcriptome analysis found there was a significantly higher level HSP70 transcription in cells infected with r DR13^att-ORF3^wtthan in the cells infected with r DR13^att-?ORF3.Transcriptome results were further verified by real-time PCR.Real-time PCR showed the expression of HSP70 in cells infected by r DR13^att-ORF3^wtwas higher than that of r DR13^att-?ORF3.The above experimental results confirmed ORF3 protein inhibited cellular apoptosis induced by porcine epidemic diarrhea virus infection.The effect may be through inhibition of Caspase-3 activation?cleavage?or promotion of HSP70 expression.