H2BE Promotes The Replication Of Porcine Epidemic Diarrhea Virus By Inhibiting Endoplasmic Reticulum Stress Cell Apoptosis

Porcine epidemic diarrhea(PED)is an acute contact enteric infectious disease in pigs caused by Porcine epidemic diarrhea virus(PEDV)of the genus Coronavirus in the family Coronaviridae.The main symptoms include diarrhea,vomiting and dehydration,resulting in a high mortality rate of piglets.An in-depth understanding of the interaction between host cell proteins and PEDV is essential for the prevention,control and treatment of this disease.Histone Cluster 2,H2be(HIST2H2BE),a variant of histone H2B,is a major protein component of chromatin and participates in a variety of cellular life processes,including chromatin replication,repair,transcription,and apoptosis.However,it is not clear whether H2BE is associated with PEDV replication.In this study,the interaction protein H2BE with PEDV non-structural protein 9(NSP9)was identified by immunoprecipitation mass spectrometry(IP-MS),and the molecular mechanism of H2BE affecting PEDV replication was studied by overexpression and knockdown experiments.The following results were obtained:1.H2BE interacts with PEDV NSP9.In this study,NSP9-EGFP vector was transfected into Marc-145 cells alone or co-transfected with H2BE-Flag vector.Immunoprecipitation-mass spectrometry(IP-MS),co-immunoprecipitation and laser confocal microscopy showed that H2BE bound and co-localized with PEDV NSP9.In addition,PEDV NSP9 was found to up-regulate the mRNA and protein levels of H2BE by inhibiting IRX1 expression by qPCR and Western blot assay.These results indicate that H2BE is an interacting protein of PEDV NSP9.2.The expression of H2BE was upregulated in PEDV-infected cells.Marc-145 cells were infected with PEDV and collected at different time points after infection.qPCR,Western blot and indirect immunofluorescence assay showed that the fluorescence intensity of H2BE protein was increased in PEDV infected cells.The mRNA and protein levels of H2BE were significantly up-regulated with the increase of PEDV infection time,and the expression level of H2BE was dose-dependent.These results suggest that PEDV can upregulate H2BE expression.3.H2BE promoted PEDV replication.The H2BE overexpression vector H2BE-Flag or H2BE small interfering RNA(si-H2BE)were transfected into Marc-145 cells and infected with PEDV.The results of qPCR,Western blot,indirect immunofluorescence assay and viral titer assay showed that overexpression of H2BE significantly promoted PEDV N mRNA level,protein level,virions and viral titer,while knockdown of H2BE significantly inhibited PEDV replication.These results suggest that H2BE is a positive regulator of PEDV.4.H2BE did not affect the expression of IFNβ.The effects of H2BE overexpression or knockdown on the adsorption,endocytosis,innate immunity and autophagy related proteins of PEDV were examined by qPCR and Western blot.The results showed that H2BE overexpression or knockdown had effects on the adsorption and endocytosis of IFNβ,ISG15,LC3 II and PEDV.This suggests that H2BE does not affect PEDV replication through innate immunity,adsorption,endocytosis,and cell autophagy.5.H2BE inhibited PEDV-induced ER stress.Marc-145 cells were transfected with H2BE-Flag vector and infected with PEDV.Western blot analysis showed that GRP78,phosphorylated PERK(p-PERK),phosphorylated eIF2(p-eIF2),and the expression of endoplasmic reticulum stress-related proteins was detected.The protein levels of phosphorylated IRE1(p-IRE1)and phosphorylated JNK(p-JNK)were significantly decreased,while knockdown of H2BE by si-H2BE significantly promoted the expression of these proteins.These results indicated that H2BE could inhibit PEDV-induced ER stress.6.H2BE inhibited PEDV-induced ER stress-mediated apoptosis.Overexpression or knockdown of H2BE protein in Marc-145 cells,and Western blot assay was used to detect the expression of CHOP and apoptosis-related proteins.The results showed that overexpression of H2BE inhibited PEDV-induced ER stress-apoptosis in Marc-145 cells.Knockdown of H2BE promoted PEDV-induced ER stress-apoptosis,and ER stress inhibitor 4-PBA could effectively alleviate the promoting effect of si-H2BE on apoptosis.These results suggest that H2BE can inhibit PEDV-induced ER stress-apoptosis.7.H2BE N-terminal amino acids 1-28 are essential for PEDV replication.The constructed plasmids H2BE-ΔN-Flag and H2BE-ΔC-Flag were transfected into Marc-145 cells and infected with PEDV.qPCR and Western blot showed that H2BE-ΔN-Flag lost the ability to promote PEDV replication.Further studies showed that the deletion of H2BE N-terminal amino acid sequence 1-28 could not promote PEDV replication.These results suggest that H2BE amino acids 1-28 are the major regions that promote PEDV replication.In conclusion,H2BE could interact with PEDV NSP9 and its expression was upregulated in PEDV-infected Marc-145 cells,which promoted PEDV replication by inhibiting ER stress-induced apoptosis.This study provided new insights into the host-PEDV interaction,which may provide new ideas for the prevention and control of PEDV.