| Reticuloendotheliosis virus(REV)is a single-stranded,positive-sense RNA virus,belonging to the Retroviridae family,subfamily Orthoretrovirinae and the genus Gammaretrovirus.The virus can cause a range of pathological syndromes with different symptoms including immunosuppression,dwarfism syndrome,acute lethal reticulocytoma and chronic tumors of lymphoid and other tissues.Since the first isolation of REV from broiler chicken in 1957,it has been widely spread all over the world.In our country,the virus presents wide prevalence,high infection rate and easy mixed infection,which has caused huge economic losses to the poultry industry.Autophagy is a process in which eukaryotic cells degrade their own cytoplasmic proteins and damaged organelles by lysosomes under the regulation of autophagy-related genes,it is the basic physiological activity and stress protection mechanism for them to maintain cellular environment homeostasis and genomic stability.Many studies have demonstrated that autophagy plays an important role in the process of viral infection and replication.Many viruses are able to regulate viral replication by affecting autophagy through their own structural proteins.However,the specific interactions between the infection of REV and autophagy are not clear.In order to explore the effect of REV infection on autophagy,we first explored the protein expression levels of LC3 and p62 by western blotting after REV infection.The results showed that REV infection led to up-regulation of LC3-II protein expression level and down-regulation of p62 protein expression level,indicating that REV infection could induce autophagy.In order to further determine the key protein of REV induced autophagy,the genes of three structural proteins of REV(nucleocapsid protein Gag,polymerase protein Pol and envelope protein Env)were cloned into p EX3-HA vector and transfected into DF-1 cells.Through western blot experiment,it was found that the expression level of LC3-II protein was significantly up-regulated in the cells expressing Env protein.Through transmission electron microscope observation,it was found that the number of autophagy-like vesicles increased significantly in REV infection group and Env protein expression group.The above results showed that Env protein induced autophagy.In order to explore whether the autophagy flow induced by Env protein is unobstructed,different doses of Env protein were transfected and detected by western blotting,the results showed that with the increase of transfection dose,the protein expression level of LC3-Ⅱ increased,while that of p62 decreased.Subsequently,adenovirus Ad-m Cherry-GFP-LC3 B expressing fusion protein was infected and observed under confocal microscope.It was found that the number of red fluorescence spots increased,green fluorescence spots decreased and yellow fluorescence gathered in REV infection group and Env protein transfection group,which proved that Env protein could induce complete autophagy flow.In order to clarify the signal pathway of autophagy induced by Env protein,we used SC79,a specific activator of AKT,to activate the signal pathway mediated by AKT.It was found that the level of autophagy induced by Env protein decreased,indicating that Env protein induced autophagy by inhibiting AKT phosphorylation.In order to explore the effect of autophagy on virus replication,autophagy inducer rapamycin(Rapa)and autophagy inhibitor 3-methyladenine(3-MA)were used to treat cells and then infected REV,respectively.Through western blotting and fluorescence quantitative PCR experiments,it was found that inducing autophagy could promote virus replication,while inhibition of autophagy significantly reduced virus replication.In order to explore the effect of autophagy induced by Env protein on virus replication,samples were collected at different time points after infection with REV and transfection with Env protein,fluorescence quantitative PCR detection showed that the level of REV replication was significantly increased in the cells expressing Env protein.3-MA,an autophagy inhibitor,was used to rescue autophagy induced by Env protein.Compared with the group expressing Env protein alone,the level of REV replication was significantly decreased.The above results showed that autophagy induced by Env protein also promoted REV replication.In summary,the Env protein of REV can induce autophagy and promote virus replication,and autophagy may be induced by inhibiting the phosphorylation of AKT.The above results laid a foundation for further study of the relationship between REV and autophagy and revealed autophagic pathway for REV replication. |
