Porcine epidemic diarrhea virus inhibits cellular apoptosis via their accessory protein

Abstract:

[Background] orf3 is located between the s and e genes of porcine epidemic diarrhea virus (PEDV) genome. It is the only accessory gene found in PEDV and encodes the ORF3 protein. Our earlier study showed that the ORF3 protein might has an effect on cell apoptosis induced by PEDV infection. [Objective] To study the virulence mechanism of ORF3 protein in the process of PEDV infection and replication. [Methods] Vero cells were infected with 3 different PEDVs, namely rDR13att-?ORF3 (with orf3 deleted from the genome), DR13-ORF3att (with a N-terminal 92 aa truncated orf3) and rDR13att-ORF3wt (with full length orf3). We observed cell pathogenic effect (CPE). Then different methods such as live cell imager, flow cytometry, TUNEL assay were used for apoptosis analysis of the cells at different time points of infection. Analysis of major apoptosis-related protein such as the cleaved Caspase-3 was also carried out by Western blotting in PEDV-infected cells. Finally, transcriptome sequencing was used to study the differential expression genes within the cells infected by the different viruses. Transcriptome results were further verified by real-time PCR. [Results] rDR13att-?ORF3 induced more CPEs in the infected cells than the other two viruses. 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 rDR13att-?ORF3 than the cells infected by the other two viruses (P<0.05). The flow cytometry also checked the apoptotic difference with the cells infected with the viruses: higher proportion of apoptotic cells among the cells infected with rDR13att-?ORF3. The result was further confirmed by terminal deoxynucleotidyl transferase (TDT)-mediated dUTP nick end labeling (TUNEL) staining. There are more TUNEL staining cells among the cells infected with rDR13att-?ORF3 than the cells infected by the other two viruses. The result of Western blotting showed rDR13att-ORF3wt can inhibit Caspase-3 activation in the infected cells. Transcriptome analysis found there was a significantly higher-level heat shock 70 kD protein 1B (HSP70) transcription in cells infected with rDR13att-ORF3wt than in the cells infected with rDR13att-?ORF3. Real-time PCR showed the expression of HSP70 in cells infected by rDR13att-ORF3wt was higher than that of rDR13att-?ORF3. [Conclusion] 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.