Muscovy Duck Reovirus Promotes Virus Replication By Inhibiting Autophagy-lysosomal Degradation Pathway

Muscovy duck reovirus infection can cause immunosuppressive infectious diseases of Muscovy ducklings and semi-muscovy ducks,mainly causing severe damage to the digestive tract and respiratory tract.The pathogen of this infectious disease is Muscovy duck reovirus.In our laboratory’s research on the interaction between the virus and host cells,it was found that MDRV could induce autophagy in infected cells,but its specific role and mechanism were not clear.This study focuses on the interaction between MDRV and cell autophagy,and analyzesthe effects of MDRV on cell autophagy and itself.The main research contents include the following aspects:1.MDRV infection caused autophagy in DF-1 cellsTransmission electron microscopy,immunofluorescence microscopy and Western Blotting were used to determine the autophagy level of MDRV-infected DF-1 cells.Compared with the diffusely distributed LC3 protein in uninfected cells,LC3 in MDRV-infected showed clear punctiform aggregations.Western Blot detected that the level of LC3-II of MDRV group significantly higher than the MOCK group,and increased with virus replication.The above results indicated that MDRV infection could induce autophagy in DF-1 cells and suggested that a positive correlation between autophagy levels and viral replication.2.Muscovy duck reovirus promotes virus replication by inhibiting autophagy-lysosomal degradation pathway（1）By treating DF-1 cells with autophagy regulators before infecting MDRV,the expression of viral structural protein and the TCID50 of the virus were detected and analyzed.The results showed that the autophagy inducer Rapamycin could increase the level of autophagy in DF-1 cells,the expression of sigma A protein increased significantly,and the TCID₅₀of virus was also markedly increased.On the contrary,the autophagy inhibitor 3-MA reduced the level of autophagy in DF-1cells,the expression of the viral protein was significantly reduced,and the viral titer also declined significantly.These results revealed that autophagy could facilitate the replication of MDRV in DF-1 cells.（2）Based on the relationship between cell autophagy and virus replication,the relationship between autophagosomes and viruses caused by MDRV further studied.The structural proteinσA and non-structural proteinσNS,which plays an important role in the MDRV replication process,were found to be co-localized with the autophagosome marker molecule LC3-II by fluorescence immunoassay,further indicating that the autophagosome structure provides membrane support for MDRV replication.（3）Related autophagosomes and lysosomal degradation blockers were selected to study the interaction between autophagy and virus replication.The results showed that blocking normal fusion of autophagosomes and lysosomes could inhibit MDRV replication.By transfection of the double fluorescent plasmid mcherry-EGFP-LC3,it was found that autophagosomes were more likely to fuse with acidic vesicles after MDRV infection compared with the blank group.In addition,p EGFP-LC3B fluorescence showed punctate aggregation in MDRV infected cells.After co-localization with LAMP1,yellow fluorescence that coincided with red and green appeared,which indicated that MDRV infection promoted the fusion of autophagosomes and lysosomes.At the same time,Western Blot analysis found that endogenous p62 did not appear a normal downward trend.The above results revealed that the the autophagic lysosome formed in the end did not effectively degraded.（4）The lysosomal related proteins were analyzed by Western Blot in the experiment,the specific mechanism of MDRV blocking autophagolysosomal degradation was further studied.It was found that the expression of LAMP1 in MDRV-infected cells would be abnormally accumulated.Late endosomes can fuse with transport vesicles containing lysosomal enzymes to form prelysosomes that have not yet started to perform the degradation function.As a marker protein of late endosomes/early lysosomes,LAMP1 expression was increased,indicating an increase in the number of immature lysosomes.（5）This study examined the maturation of lysosomal acid hydrolase Cathepsin D in cells,and further confirmed the effect of MDRV on lysosomal maturation.Cathepsin D needs to undergo a series of post-expression modifications to form a 33KDa mature Cathepsin D.Western blot found that the expression of the mature form of Cathepsin D,33 k Da in size,was significantly reduced after MDRV infection,suggesting that MDRV inhibited lysosomal maturation.In summary,Muscovy duck reovirus induces autophagy in infected DF-1 cells,promotes the fusion of autophagosomes and lysosomes,inhibits the degradation activity of autophagolysosomes,thereby promoting virus replication.