ALV-J Inhibits Autophagy Through GADD45β/MEKK4/p38MAPK Signaling Pathway

Avian leucosis Subgroup J,a neoplastic disease,seriously harms the poultry industry.Due to chickens with different genetic backgrounds have different susceptibility to ALV-J,some breeds of which show strong genetic resistance to ALV-J.In our previous studies,GADD45β,an important disease resistance gene was screened,which is mainly involved in regulating cell proliferation,cell cycle,apoptosis,DNA damage repair and cell autophagy,etc.Studies have found that ALV-J infection and overexpression of GADD45βcan affect both autophagy and apoptosis.What’s more,the expression of GADD45β is closely related to ALV-J virus replication.Therefore,this study will further study the regulatory mechanism of GADD45β on ALV-J to inhibit cell autophagy and promote apoptosis.1.In this study,we detect the expression of GADD45β on DF-1 cells at different time periods after ALV-J challenge by q-PCR,Western Blot,and laser confocal immunofluorescence,The results indicated that ALV-J infection promoted the expression of endogenous GADD45β.2.In our research,we constructed the eukaryotic expression vector of GADD45β.The expression of key autophagic proteins LC3,p62 / SQSTM and ATG5 protein were analyzed by western blot,the number of GFP-LC3 aggregation points were detected by using laser confocal immunofluorescence technology,the co-localization of GFP-LC3 with lysosomes and LAMP proteins,and the number of autophagosomes of the dual fluorescence system GFP-m Cherry-LC3 were examined followed.All data showed that ALV-J infection and overexpression of GADD45β inhibit the early stage of autophagy and block the binding of autophagosomes and lysosomes,the same conclusion was obtained under the effect of Rapa stimulation.3.To further investigate the relationship between GADD45β,ALV-J and autophagy,the Gadd45β gene was knocked down by Gadd45β-specific si RNA,the level of LC3 II,SQSTM1 and ATG5 significantly changed in ALV-J-infected cells treated with si Gadd45βin comparison with ALV-J-infected cells without si RNA,Subsequently,the p38 MAPK and JNK pathway was activated after ALV-J infection.Collectively,these observations indicate that GADD45β is an upstream regulator of p38 MAPK and JNK by ALV-J-mediated incomplete autophagy,and that ALV-J infection does not directly affect the phosphorylation of p38 MAPK and JNK.4.To explore the mechanism that ALV-J inhibits cell autophagy through GADD45β,we verified the interaction between GADD45β and MEKK4 proteins in DF-1 cells,and analyzed the change of autophagy related proteins by interfering with MEKK4,these results proved that ALV-J can inhibit autophagy and activate p38 MAPK and JNK phosphorylation through GADD45β binding to MEKK4.5.In this study,SB203580 and SP600125,as common p38 MAPK and JNK inhibitor separately,and we choose the optional concentration to inhibit the phosphorylation of p-p38 and p-JNK,and subsequently infected with ALV-J.The expression of autophagy related proteins LC3,p62 / SQSTM,and ATG5 were used to demonstrate that ALV-J can inhibit autophagy through GADD45β-MEKK4-p38 MAPK signaling pathway instead of JNK signaling pathway.6.To prove the effect of ALV-J infection and overexpression of GADD45β on autophagy and apoptosis,we detect the levels of autophagy-related proteins,apoptotic proteins and the phosphorylation of p38 and JNK at different time periods after ALV-J infection or overexpression of GADD45β,the autophagy-related proteins LC3,SQSTM1,and ATG5 were significantly changed after infection with ALV-J or overexpression of GADD45β for 4 h,and the activation of p38 MAPK was also detected,inhibition of autophagy and activation of p38 MAPK all began with 4 h after ALV-J infection or overexpression of GADD45β and persisted for 24 h.However,when ALV-J was infected or overexpressed with GADD45β for 6 h,caspase-3 and caspase-8 were definitely activated.Unexpectedly,JNK was also activated at the same time,and continuously activated up to24 h.On the other hand,autophagic compartments but without apoptotic features occurred at 4 h after ALV-J infection and overexpression of GADD45β and the number of autophagosomes or autophagosomes were less than that of the normal group,nevertheless,both autophagic compartments and apoptotic features were occurred in some DF-1 cells at12 h after ALV-J infection and overexpression of GADD45β,with the nucleus ruptured into several segments and no intact nuclear morphology.Binding data demonstrate that ALV-J infection or overexpression of GADD45β results inhibit autophagy faster than induce apoptosis in DF-1 cells.In general,ALV-J infection significantly promotes the expression of GADD45β and inhibits autophagy by regulating GADD45β / MEKK4 / p38 MAPK signaling pathway,and confirms that ALV-J infection affects autophagy proceed apoptosis.Our study provides new insights in control the balance of the autophagy and apoptosis which affected by ALV-J Infection.