| Muscovy duck reovirus(MDRV)belongs to the family Reoviridae,Orthoreovirus,and it can cause muscovy duckings sick and showed as soft-foot,the liver,spleen,gray white necrotic spots from the age of 4 to 45 days,which is a kind of acute infectious diseases with high morbidity and high mortality,has caused major economic losses to the Muscovy duck breeding industry.Cellular autophagy is a type of conserved activity that degrades intracellular substrates in eukaryotic cells,which can participate in a variety of cellular physiology processes and maintain cell homeostasis.At present,research on chicken reovirus(CRV)is more in depth.It has been reported that CRV p17 protein is a positive regulator of cellular autophagy and can initiate autophagy through the activation of the AMPK signaling pathway to enhance the replication of the virus.Previous studies in our team showed that MDRV-σNS proteins can also induce autophagy and promote virus proliferation.MDRV and CRV belong to Avian reovirus(ARV),but there is a certain difference between the two encoded proteins.MDRV S group protein does not contain p17 encoding gene,its exact mechanism to induce autophagy and promote virus replication may be different from CRV,and there is no relevant research report.For this purpose,this subject through the electron microscopy technology,siRNA gene silencing technology and Western Blot method to explore the regulating effect of MDRV and σNS protein induced Vero cell autophagy by AMPK signaling pathway,clear the regulatory mechanism.The main experiment contents and results are as follows:1.Transmission electron microscope to observe autophagyBlank Vero cells and MDRV-infected Vero cells were collected after cultivated for 36 hours,and GlutARal dehyed 2.5%water solution by fixing solution to make ultrathin sections.The morphology of the autophagosomes was observed under a transmission electron microscope.The results showed that compared with the blank cells,typical double-membrane structure of cytoplasmic components was found in MDRV-infected cells,and it was confirmed from the morphological point that MDRV could induce Vero cells to ouucr autophagy.2.Effect on AMPK/ULK1/Beclinl signaling pathway of Vero cells by MDRVIn this study,taken Vero cells as a model,using Western Blot to detect AMPK,p-AMPK,mTOR,p-mTOR,ULK1,p-ULK1,Beclinl,LC3II and other autophagy and AMPK/ULK1/Beclinl pathway-related changes in protein expression levels in Vero cells after MDRV infection 36h.The test results showed that:(1)Compared with the control group,the MDRV-σNS protein was detected in the MDRV-infected group,indicating that the virus entered Vero cells and replicated;(2)Compared with the blank control group,the LC3II expression level was significantly higher in the MDRV-infected group,indicating that MDRV infection can induce autophagy in Vero cells;(3)Compared with the blank control group,the expression levels of total AMPK,mTOR,and ULK1 proteins in the MDRV-infection group were basically invariable,while the expression levels of p-AMPK and Beclinl protein were increased,p-ULK1 and p-mTOR protein expression levels were down-regulated.The preliminary experiments showed that MDRV could induce autophagy in Vero cells,and the AMPK/ULK1/Beclinl pathway was involved.3.Effect on AMPK/ULK1/Beclinl pathway of Vero cells induced by MDRVTo further investigate the effect of MDRV-infected Vero cells on the AMPK signaling pathway,we used siRNA silencing techniques to silence and transfect the AMPK,ULK1,and Beclinl genes following pretreatment of Vero cells with the AMPK activator AICAR,then infected by virus;The effect of autophagy and AMPK pathway on the expression levels of the autophagy and AMPK pathway in the samples infected with MDRV was detected by Western Blot at 36 hours after infection.The results showed that:(1)After transfected with sh-AMPK,sh-AMPK group compared with blank group,AMPK and p-AMPK expression levels were significantly down-regulated,mTOR and p-mTOR expression levels did not change significantly,indicating that the transfection of sh-AMPK can effectively inhibit the expression of intracellular AMPK protein;Compared with MDRV infection group,the expression of total AMPK protein in MDRV+sh-AMPK group was down-regulated,mTOR was not changed significantly,and the expression levels of p-AMPK,LC3II,and p-mTOR were decreased,which indicated that after AMPK was silenced,the AMPK signaling pathway was negatively feedbacked and inhibited autophagy.(2)After preconditioning with AMPK activator AICAR,compared with the blank control group,the levels of total AMPK,p-AMPK,and LC3II in the AICAR group were significantly up-regulated,whereas the expression levels of mTOR were not significantly changed,indicating that AICAR can Effectively promote the expression of AMPK and p-AMPK;Compared with MDRV control group,the expression levels of total AMPK,p-AMPK,and LC3II in MDRV+AICAR group were relatively higher,the total mTOR expression level had no obvious change,and the expression level of p-mTOR was down-regulated obviously.The decrease indicated that the AMPK/ULK1/Beclinl signaling pathway is positively regulated after activation of AMPK and autophagy occured.(3)After transfected with sh-ULK1,the expression levels of ULK1 and p-ULK1 were significantly down-regulated and the expression levels of Beclinl and LC3Ⅱ were up-regulated in sh-ULK1 group compared with the blank group,indicated that the transfection of sh-ULK1 can effectively inhibit ULK1 espressed in cells;In the MDRV+sh-ULK1 group,the total ULK1 and p-ULK1 protein expression levels were down-regulated compared to the MDRV-infected group,whereas the Beclinl and LC3II protein expression levels were relatively higher,indicated that after the ULK1 was silenced,AMPK/ULK1/Beclinl signaling pathway is positively regulated and autophagy occured.(4)After transfected with sh-Beclinl:Compared with the blank control group,the expression levels of Beclinl in sh-Beclinl group was significantly down-regulated,indicating that transfected with sh-Beclinl can effectively inhibit the expression of Beclinl protein;Compared with the MDRV group,the expression levels of Beclin1 and LC3II in cells were down-regulated in MDRV+siBeclinl group,indicating that after the silencing of Beclinl,the AMPK/ULK1/Beclinl signaling pathway was negatively feedbacked and autophagy was inhibited.The above results further confirmed that it was under the influence of AMPK/ULK1/Beclinl pathway that autophagy occurred in MDRV infected Vero cells.4.Effect on AMPK/ULK1/Beclinl pathway of Vero cells induced by MDRVOur group has confirmed that MDRV-σNS protein can induce autophagy in avian cells(results had published in Virology Journal),in order to further explore the relationship between MDRV-σNS protein induced autophagy and the AMPK signaling pathway,this study transfected the eukaryotic expression plasmid pCI-flag-σNS of the MDRV-σNS protein gene(previously constructed by laboratory)into Vero cells,cells were harvested 36 hours later,and autophagy was observed by transmission electron microscopy;then,siRNA silencing technique was used,after the preconditoning of AMPK activator AICAR,AMPK,ULK1 and Beclinl genes were silenced and transfected respectively,and then transfected with pCI-flag-σNS plasmids.After transfection and cultured for 36 h,detected the expression levels of autophagy and AMPK pathway-relatived protein in each transfection group by Western Blot respectively The results showed that:(1)Transmission electron microscopy revealed that the transmembrane pCI-flag-σNS showed a double-layered membrane structure in the cytoplasm,but the blank group was not found.Morphologically confirmed that the non-structural protein σNS of MDRV could induce Vero cell autophagy;(2)pCI-flag-σNS was transfected,compared with the blank control group,the expression levels of p-AMPK,Beclin1 and LC3Ⅱ protein were up-regulated,the expression levels of p-mTOR and p-ULK1 protein were down-regulated,and the MDRV non-structural protein σNS was confirmed from the molecular level.It can induce autophagy in Vero cells,and it is also initially shown to be related to AMPK/ULK1/Beclin1 pathway;(3)Compared with MDRV-σNS group,the expression of LC3Ⅱ protein is decreased after transfected sh-AMPK and sh-Beclinl,indicating that the transfection of sh-AMPK and sh-Beclinl reduced the level of autophagy;In pretreatment with sh-ULKl and AICAR,the expression of LC3Ⅱ protein was up-regulated relative to MDRV-σNS group,indicating that transfected with sh-ulkl and AICAR pretreatment promoted autophagy.The above results further confirmed that autophagy occurred in Vero cells transfected with MDRV non-structural protein σNS under the influence of AMPK/ULK1/Beclinl pathway.5.ConclusionMDRV and its non-structural protein σNS can induce autophagy in Vero cells,and regulate AMPK/ULK1/Beclin1 signaling pathway to induce Vero cell autophagy by promoting phosphorylation of AMPK protein and inhibiting ULK1 protein phosphorylation and up-regulating Beclinl protein. |
