Studies On Interaction Between Muscovy Duck Reovirus Replication And Autophagy

MDRV is a member of the genus Orthoreovirus in the family Reoviridae.The g-enome including ten segments of double-stranded RNAs(dsRNA)can be divided into three size classes:large(L1?L3),medium(M1?M3)and small(S1?S4)segments,w-hich is not unusual for genic reassortment and viral evolution,thus increasing the dif-ficulties in studies on pathogenesis.Autophagy is an evolutionarily conserved life pro-cess in eukaryotic cells to degrade and generate intracellular substances.More and more studies have showed complex interplay between virus replication and autophagy,some viruses even can exploit autophagy for replication.Studies which including obs-erving morphological structure of autophagosome by transmission electron microsco-pe,determining expression of LC3? and p-mTOR in test and control group using western blotting,and detecting of p10 gene copies with real-time PCR,were perform-ed to explore interaction of MDRV-YB replication with autophagy,which will further facilitate understanding the molecular mechanisms of viral replication.The main con-tents and results are as follows:1?Morphological structure of autophagosome was observed in MDRV-YB-infected DF-1 cells at 24 h post-infection as well as control group by transmission electron microscope.As expected,cellular organelles structure in control group was normal and evenly distributed in the cell,while obvious double membrane vacuoles contained cytoplasm were found in MDRV-YB-infected DF-1 cells,which indicated that MDRV-YB might induce autophagy in DF-1 cells.2?Effects of MDRV-YB on autophagy in DF-1 cells.DF-1 cells were divided into eight groups,which including control group,RAPA group,CQ group,YB group,YB-inactivated group,3-MA+YB group,CQ+YB group and RAPA+YB group.Expr-ession of LC3? and p-mTOR in each group were determined by western blotting.Results showed that the quantity of p-mTOR and mTOR in control group remained stable.Although quantity of mTOR also was unchanged in YB group,expression of p-mTOR displayed a significant decrease and subsequent increase,with a minimum at 24 h;in addition,no LC3? was detected in control group,but expression of LC3?showed a significant increase and subsequent decrease in YB group,with a maximum at 36 h;both of the above results demonstrated MDRV-YB could induce autophagy in DF-1 cells.Expression levels of LC3? in both RAPA and RAPA+YB groups showed identical trend to YB group,while the timepoint of LC3? maximum was brought for-ward in RAPA+YB groups,which suggested that autophagy agonist RAPA can prom-ote MDRV-YB-triggered autophagy in DF-1 cells;As expected,there was a tremend-ous amount of LC3? accumulation in CQ and CQ+YB groups,which declared auto-phagy inhibitors CQ can interdict MDRV-YB triggered autophagy in DF-1 cells;In addition,no LC3? was detected in 3-MA+YB and YB-inactivated groups as well as control group.These phenomena indicated that autophagy inhibitors 3-MA can also interdict the autophagy,and MDRV-YB induced DF-1 cells autophagy was dependent on viral infection ability or viral replication.3?Effects of MDRV-YB on autophagy in MDEF cells.MDEF cells were divided into six groups,which including control group,YB group,RAPA group,CQ group,CQ+YB group and RAPA+YB group.Expression of LC3? and p-mTOR in each group were determined by western blotting.Results showed a similar trend of p-mTOR and LC3? with DF-1 cell,and the most obvious autophagy was obtained at 36 h post-treatment,which suggested MDRV-YB could also induce autophagy in MDEF cells,and RAPA can promote the autophagy,while CQ interdict this process.4?Interaction of MDRV-YB non-structural proteins p10,?NS with autophagy in DF-1 cells.DF-1 cells were divided into five groups,termed as control group,YB group,vector group,p10 transfection group and ?NS transfection group.Results showed that LC3? was not detected in both control group and vector group,while the expression of LC3? displayed a significant increase and subsequent decrease in YB,p10 and ?NS groups.These results demonstrated p10 and ?NS could also induce autophagy in DF-1 cells;besides,expression of p10 in YB group and p10 transfection group,as well as ?NS in YB group and ?NS transfection group kept the same trend with LC3?,which indicated expression of p10 and ?NS were dependent on auto-uphagy.5?Effect of autophagy in DF-1 cells on the relative copies of MDRV-YB p10 gene.DF-1 cells were divided into three groups:YB group,CQ+YB group and RAPA+YB group,and cultured for 12 h?24 h?36 h?48 h and 60 h,respectively.Relative quantitative PCR assays were carried,YB group was regarded as relative reference,p10 as target gene and GAPDH as internal reference.Results showed that relative copies of p10 in varied timepoints were the highest in the RAPA+YB group,while the lowest in CQ+YB group,which meant that RAPA can promote p10 repli-cation,while CQ can suppress p10 replication.6?Effect of autophagy in DF-1 cells on the absolute copies of MDRV-YB p10 gene.The same three groups were cultured for 6h,12h,18h,24h,30h,36h,42h,48h,54h,60h,respectively.Absolute quantitative RT-PCR assays were carried,and p10 recombinant plasmid was regarded as standard and viral p10 as target gene.Results showed that the copies of p10 displayed a first rise and following decrease in the three groups,with a maximum at 48 h;and p10 of all timepoints in RAPA+YB group were the highest,followed by YB group and CQ+YB group,respectively.All above results further confirmed that RAPA can promote the replication of p10,and CQ suppress it.The above results show that the MDRV-YB can induce autophagy of MDEF and DF-1 cells,inactivated MDRV-YB cannot induce cell autophagy,and cell autophagy is advantageous to the replication of MDRV-YB in DF-1 cells.