Effect And Mechanism Of Transmissible Gastroenteritis Virus Protein 7 On Autophagy In Porcine Intestine Epithelial Cell

Transmissible gastroenteritis(TGE)is an acute,highly contagious infectious disease that causes huge economic losses to the pig industry.The pathogen of TGE is transmissible gastroenteritis virus(TGEV).Autophagy is an evolutionarily conserved process in eukaryotes that degrades intracellular substances and occurs in a number of physiological and pathological processes,including viral infection.It was found that TGEV infection caused autophagy of swine testicular cells(ST cells)and porcine kidney(PK-15).ST cells were infected with TGEV gene 7 mutant strain(r TGEV-Δ7)and TGEV wild strain,respectively,and transcription levels of autophagy-related genes was altered in ST cells infected with r TGEV-Δ7 in compare with TGEV-infected cells,suggesting protein 7 of TGEV involves in regulatng celluar autophage.However,the detailed effect and mechanism of TGEV-P7 on autophagy in intestinal small porcine epithelial cells(IPEC-J2)has not been reported so far.In this paper,to determine the effects of TGEV and TGEV-P7 on autophagy,IPEC-J2 cells were respectively infect with TGEV and r TGEV-Δ7.LC3-II protein level was detected.Then,TGEV-P7-interacted protein was analyzed by immunoprecipitation and mass spectrometry and by GO and KEGG.The autophagy-associated protein was selected and identified by coimmunoprecipitation and indirect immunofluorescence assay.The results are shown below.1.The effects of TGEV-P7 overexpression,r TGEV-Δ7,and TGEV on autophagy were detected by Western blot.The results showed that LC3-II protein level was down-regulated by overexpression of protein 7 and up-regulated by r TGEV-Δ7,indicating that TGEV-P7 inhibited autophagy.2.The TGEV-P7 was overexpressed by pCMV-3Tag-TGEV-P7.TGEV-P7-interacted protein complex was obtained by coimmunoprecipitation.The number of TGEV-P7 interaction proteins were 220.Kyoto encyclopedia of genes and genomes(KEGG)annotation and cluster and Gene ontology(GO)analysis of identified interaction proteins were performed.KEGG enrichment analysis showed that TGEV-P7-interacted protein was mainly enriched in autophagosomes,gap junctions,endocytosis and phosphoinositide metabolism and phosphoinositide signaling system.GO analysis results showed that in Biological Process classification,the interaction proteins were mainly enriched in organelle organization,metabolic processes,vesicle transport,mitochondrial structure and autophagy;in Molecular Function classification enzymatic activity,hydrolase activity,pyrophosphatase activity and hydrolase activity acting on an acid anhydride were mainly enriched;in Cellular Component classification intracellular components was mainly enriched.3.According to reliability and abundance,motif analysis of TGEV-P7 amino acid sequence,GO,and KEGG analysis results,Gamma-aminobutyric acid receptor-associated protein(GABARAP),a number of ATG8 family protein,was selected as a candidate interaction protein of TGEV-P7.The coimmunoprecipitation and indirect immunofluorescence assays were used to identify the interaction between TGEV-P7 and GABARAP.The results showed that TGEV-P7 interacted with GABARAP.4.pCI-His-GABARAP and pCI-neo were respectively transfected into IPEC-J2 and infected with TGEV.The protein level of LC3-II were detected and increased by overexpression of GABARAP.pCI-His-GABARAP and pCMV-3Tag-TGEV-P7 were co-transfected into IPEC-J2.pCI-His-GABARAP and pCMV-3Tag-1 were co-transfected into IPEC-J2.pCI-neo and pCMV-3Tag-TGEV-P7 were co-transfected into IPEC-J2.After transfection,the cells were infected with TGEV.The level of LC3-II of cells co-transfected with pCI-His-GABARAP and pCMV-3Tag-TGEV-P7 was higher than that of co-transfected pCI-neo and pCMV-3Tag-TGEV-P7 and lower than the group co-transfected with pCI-His-GABARAP and pCMV-3Tag-1,indicating that TGEV-P7 inhibits autophagy via interacting with GABARAP.The data of this study indicate that TGEV-P7 inhibits autophagy by interacting with GABARAP,further elucidating the mechanism of TGEV-P7 during TGEV-induced autophagy and laid the foundation for uncovering the pathogenesis of TGEV.