The objective of the study was to explore whether autophagy was induced by swine acute diarrhea syndrome coronavirus(SADS-CoV)infection and its impact on virus replication during autophagy.Vero E6 cells were infected with SADS-CoV at a MOI of 0.1 and then observed by transmission electron microscope.The results showed that the SADS-CoV infected cells produced an autophagosome like double-layer membrane containing cytoplasm,proving that SADS-CoV could induce autophagy in Vero E6 cells.A eukaryotic expression plasmid of pEGFP-LC3 was constructed and identified by enzyme digestion analysis and sequencing,the resulting plasmid was transfected into Vero E6 cells after infection with SADS-CoV,and then observed by confocal laser microscope.Compared with the negative control group,the SADS-CoV infected cells showed a dot aggregation of green fluorescence.Western blot analysis of the autophagy related proteins LC3-Ⅱ and p62 in SADS-CoV-infected Vero E6 cells showed an increase in LC3-Ⅱ band intensity,an elevated LC3-Ⅱ/GAPDH ratio,and a significant decrease in p62 protein expression(P<0.05),indicating a robust autophagic response to SADS-CoV infection in Vero E6 cells.To explore the effect of autophagy on viral replication,we used autophagy inhibitor 3-MA and autophagy inducer Rapamycin to detect the expression level of SADS-CoV N protein by western blot and the viral titers(TCID50).The results showed that compared with the control group infected only with SADS-CoV,the expression level of SADS-CoV N protein and the viral titers were significantly decreased after Vero E6 cells were treated with 3-MA significantly decreased(P<0.05).After treatment with Rapamycin,the expression level of SADS-CoV N protein and the viral titers were significantly increased(P<0.05).Additionally,three specific ATG5 siRNAs(siATG5 siRNA-1/2/3)were designed for ATG5 gene.The siATG5-1,with higher knockdown efficiency,was transfected into Vero E6 cells and then infected with SADS-CoV to detect the change of the expression level of SADS-CoV N protein.The findings revealed a significant decrease in the expression level of SADS-CoV N protein and the viral titers(P<0.05)compared to 3 control group cells,indicating the knockdown of ATG5 reduced viral replication.This study shows for the first time that SADS-CoV infection can induce autophagy and promote virus replication,which has positive significance for further study of SADS-CoV infection and its pathogenic mechanism as well as prevention and control of disease.
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