Study On The Effect Of Muscovy Duck Reovirus And S-class Proteins On The RhoA/ROCK1/MLC Signaling Pathways In Vero Cells

Muscovy duck reovirus(MDRV)infects Muscovy ducks and mainly causes clinical symptoms of soft foot,diarrhea,hypogenesis,and lesions with liver and spleen swollen and covered with acicular white necrotic foci thus leading to significantly economic losses to the poultry industry.According to the previous reports,apoptosis,autophagy and cell fusion induced by Avian reovirus(ARV)are associated with Rho family proteins.MDRV differs from ARV in the pathogenicity and genomic composition,although it has been reported that MDRV infection can induce apoptosis,autophagy and cell fusion,the molecular mechanisms involved remain unclear.Therefore,the effect of MDRV on the RhoA/ROCK1/MLC signaling pathways in Vero cells was explored in this study.The main contents and results are as follows:1.The effect of MDRV on the RhoA/ROCK1/MLC signaling pathways in Vero cells.In this study,Vero cells were used as a model to detect the expression of GTP-RhoA,RhoA,p-ROCK1,ROCK1,p-MLC and MLC in MDRV-infected Vero cells with Western Blot analysis.The results showed that the expression levels of GTP-RhoA,p-ROCK1 and p-MLC in MDRV-infected Vero cells were significantly increased compared with the negative control(P<0.01),indicating that MDRV infection activates the RhoA/ROCK1/MLC signaling pathway in Vero cells.For further study,we used ROCK-specific inhibitor Y-27632 to pre-treat Vero cells following by MDRV infection,and then detected the expression levels of GTP-RhoA,RhoA,p-ROCK1,ROCK1,p-MLC and MLC using Western Blot analysis.The results showed that the increasing expression levels of p-ROCK1 and p-MLC in MDRV infected Vero cells were significantly(P<0.05)inhibited due to the pretreatment of Y-27632.While the expression of GTP-RhoA was not affected after the pretreatment by Y-27632,indicating that RhoA and MLC proteins were located at the upstream and downstream of ROCK1 molecule,respectively.These results further demonstrate that MDRV infection activate RhoA/ROCK1/MLC signal transduction pathways in Vero cells.2.The effect of MDRV S-class proteins on the RhoA/ROCK1/MLC signaling pathways in Vero cells.In order to preliminarily screen out the viral protein factors that influence the RhoA/ROCK1/MLC signaling pathway in Vero cells,the previously constructed eukaryotic expression plasmids that containing the ?A-,?NS-,?C-and p10.8-encoding genes of MDRV were separately transfected into Vero cells,and the expressions of GTP-RhoA,RhoA,p-ROCK1,ROCK1 as well as p-MLC and MLC were detected by Western Blot analysis.The results showed that all the four proteins were successfully expressed in Vero cells,The expression levels of GTP-RhoA,p-ROCK1 and p-MLC in ?C-and p10.8-transfected Vero cells were significantly(P<0.05)higher than the blank control.?A protein transfection only activated the expression of GTP-RhoA(P<0.05),and could not effectively activate the downstream ROCK1 and MLC factors.In addition,The expression of GTP-RhoA,p-ROCK1 and p-MLC did not change significantly after ?NS transfection in Vero cells.Collectively,the results suggest that MDRV ?C,p10.8 but not ?NS proteins can activate the RhoA/ROCK1/MLC signaling pathway in Vero cells,additionally?A protein only activate the GTP-RhoA,with no direct effects on the activation of ROCK1 and MLC.