The Mechanism Of Grass Carp Reovirus Type Ⅰ-induced Apoptosis Of Grass Carp Ovary Cell

Grass carp(Ctenopharyngodon idella)is one of four traditional freshwater fishes in our aquaculture history,and it is popular in our food as it was delicious and nutrient-enriched.However,the grass carp hemorrhagic disease(GCHD)which is a virus disease frequently breaks out throughout the country every year,and the pathogen is grass carp reovirus(GCRV).Incalculable losses in economic occurs with high mortality of grass carp after GCRV infection,and the development of grass carp aquaculture industry is affected seriously.It was reported that virus induced apoptosis to facilitate the proliferation and diffusion of itself during infection.Currently,there are many researches about apoptosis induced by viruses of the orthoreovirus,which structure is similar to GCRV,but few about the regulatory effects of GCRV and its virus protein on apoptosis.In our study,the GCRV-I strain GCRV-HH196 was selected as the research object.The molecular mechanism of GCRV-HH196 and its encoded nonstructural protein NS17 inducing apoptosis of grass carp ovary cells(GCO)was investigated.This research initially revealed the molecular mechanism of apoptosis induced by GCRV-HH196 and the nonstructural protein NS17 encoded by GCRV-HH196,enriched the understanding of the nonstructural protein encoded by GCRV-HH196,provided new insights into the mechanism of virus-host interaction during GCRV-HH196 infection,and also provided new ideas and theoretical support for effective prevention and control of GCHD in the future.The main results are as follows:1.GCO cells apoptosis induced by GCRV-HH196After GCO cells were inoculated with the GCRV-HH196,DNA Ladder,DAPI and TUNEL staining results showed that DNA of cells was broken after infection.Flow cytometry analysed that the percentage of apoptotic cell increased significantly during infection.Real-time fluorescence quantification PCR(qRT-PCR)detected that mRNA expression level of fasl and caspase8 which are apoptotic-releated gene in apoptosis extrinsic pathway both significantly up-regulated after infection,and the mRNA expression level of bax,aif and caspase9 which take part in apoptotic intrinsic pathway all significantly enhanced after infection.The activity of caspase8,caspase9 and caspase3 was significantly up-regulated after infection.These results indicated that the GCRV-HH196 could activate the extrinsic and intrinsic pathway of apoptosis during infection,resulting in cells genomicDNA fragmentation and inducing apoptosis.2.GCRV-HH196 NS17 protein accelerated viral infection by inducing GCO cells fusion and apoptosis(1)Sequence analysis of GCRV-HH196 NS17 proteinThe open reading frame(ORF)of GCRV-HH196 NS17 gene is 471 bp in length,encoding 156 amino acid residues with a calculated molecular mass of 17 kDa and the theoretical isoelectric point of 7.968.Phylogenetic tree revealed that the NS17 protein of GCRV-HH196 formed a cluster with FAST proteins of Aquareovirus C group and with high homology(Bootstrap value is 1),suggesting that NS17 maybe belong to the FAST protein family.Similar to the identified NS16 protein motifs of GCRV-873,NS17 of GCRV-HH196 contains essential features,such as ectodomain,transmembrane domain and endodomain.There has dicysteine closed to the TM,and a polybasic(PB)region,ahydrophobic patch(HP)and a proline-rich motif(PP)in endodomain.Whereas the C terminal of NS17 protein owned ten amino acids more than GCRV-873 NS16 protein.We found the NS17 protein existed not only in cytoplasm but also on cell membrane.(2)Effects of GCRV-HH196 NS17 protein on cell fuison,apoptosis and viral infectionGCO cells were overexpressed the GCRV-HH196 NS17 then infected GCRV-HH196,the wider range of cell-cell fusion was observed by DAPI staining with the confocal microscopy.Meanwhile,virus copies increased by qRT-PCR during infectin and significantly higher than control group,indicating that virus skipped over the process of adsorption and invasion and could replication efficiently in cells as the virus promoted cell-infected fused with cell-unfected.Flow cytometry dectected that percentage of apoptotic cell significantly increased in GCO cells overexpressed with NS17 after 72 hour post transfection(h.p.t.).The positive signal of TUNEL was more numerous and brighter in GCO cells overexpressed with NS17 after 72 h.p.t.,suggesting that NS17 protein could trigger cell’s genomicDNA fragamention and lead to apoptosis.It showed that the level of ROS in cells overexpressed with NS17 was completely opposite to that of the control group,and was significantly higher than that of the control group after48 h.p.t.,indicating that NS17 protein could regulate the expression of ROS and cause intracellular ROS disorder to activate apoptosis.The above results revealed that the role of NS17 protein is mainly to promote cell fusion and apoptosis during GCRV-HH196 infection,strived for greater space,material and energy for virus proliferation and replication,and then induced cell apoptosis and fragmentation to release a large number of virions for further expanding the infection.