Aquareovirus species vary with respect to pathogenicity,and the nonstructural protein NS80 of aquareoviruses has been implicated in the regulation of viral replication and assembly,which can form viral inclusion bodie...Aquareovirus species vary with respect to pathogenicity,and the nonstructural protein NS80 of aquareoviruses has been implicated in the regulation of viral replication and assembly,which can form viral inclusion bodies(VIBs) and recruit viral proteins to its VIBs in infected cells.NS80 consists of 742 amino acids with a molecular weight of approximately 80 kDa.Interestingly,a short specific fragment of NS80 has also been detected in infected cells.In this study,an approximately58-kDa product of NS80 was confirmed in various infected and transfected cells by immunoblotting analyses using α-NS80 C.Mutational analysis and time course expression assays indicated that the accumulation of the 58-kDa fragment was related to time and infection dose,suggesting that the fragment is not a transient intermediate of protein degradation.Moreover,another smaller fragment with a molecular mass of approximately 22 kDa was observed in transfected and infected cells by immunoblotting with a specific anti-FLAG monoclonal antibody or α-NS80 N,indicating that the 58-kDa polypeptide is derived from a specific cleavage site near the amino terminus of NS80.Additionally,different subcellular localization patterns were observed for the 22-kDa and 58-kDa fragments in an immunofluorescence analysis,implying that the two cleavage fragments of NS80 function differently in the viral life cycle.These results provide a basis for additional studies of the role of NS80 played in replication and particle assembly of the Aquareovirus.展开更多
Grass carp reovirus(GCRV),the genus Aquareovirus in family Reoviridae,is viewed as the most pathogenic aquareovirus.To understand the molecular mechanism of how aquareovirus initiates productive infection,the roles of...Grass carp reovirus(GCRV),the genus Aquareovirus in family Reoviridae,is viewed as the most pathogenic aquareovirus.To understand the molecular mechanism of how aquareovirus initiates productive infection,the roles of endosome and microtubule in cell entry of GCRV are investigated by using quantum dots(QDs)-tracking in combination with biochemical approaches.We found that GCRV infection and viral protein synthesis were significantly inhibited by pretreating host cells with endosome acidification inhibitors NH4Cl,chloroquine and bafilomycin A1(Bafi).Confocal images indicated that GCRV particles could colocalize with Rab5,Rab7 and lysosomes in host cells.Further ultrastructural examination validated that viral particle was found in late endosomes.Moreover,disruption of microtubules with nocodazole clearly blocked GCRV entry,while no inhibitory effects were observed with cytochalasin D treated cells in viral infection,hinting that intracellular transportation of endocytic uptake in GCRV infected cells is via microtubules but not actin filament.Notably,viral particles were observed to transport along microtubules by using QD-labeled GCRV.Altogether,our results suggest that GCRV can use endosomes and microtubules to initiate productive infection.展开更多
基金supported by funding from the National Natural Science Foundation of China (NO.31372565,31402340,31400139 and 31370190)
文摘Aquareovirus species vary with respect to pathogenicity,and the nonstructural protein NS80 of aquareoviruses has been implicated in the regulation of viral replication and assembly,which can form viral inclusion bodies(VIBs) and recruit viral proteins to its VIBs in infected cells.NS80 consists of 742 amino acids with a molecular weight of approximately 80 kDa.Interestingly,a short specific fragment of NS80 has also been detected in infected cells.In this study,an approximately58-kDa product of NS80 was confirmed in various infected and transfected cells by immunoblotting analyses using α-NS80 C.Mutational analysis and time course expression assays indicated that the accumulation of the 58-kDa fragment was related to time and infection dose,suggesting that the fragment is not a transient intermediate of protein degradation.Moreover,another smaller fragment with a molecular mass of approximately 22 kDa was observed in transfected and infected cells by immunoblotting with a specific anti-FLAG monoclonal antibody or α-NS80 N,indicating that the 58-kDa polypeptide is derived from a specific cleavage site near the amino terminus of NS80.Additionally,different subcellular localization patterns were observed for the 22-kDa and 58-kDa fragments in an immunofluorescence analysis,implying that the two cleavage fragments of NS80 function differently in the viral life cycle.These results provide a basis for additional studies of the role of NS80 played in replication and particle assembly of the Aquareovirus.
基金This work is supported in part by grants from the National Natural Science Foundation of China(31672693,31972838 and 31400139,31372565).
文摘Grass carp reovirus(GCRV),the genus Aquareovirus in family Reoviridae,is viewed as the most pathogenic aquareovirus.To understand the molecular mechanism of how aquareovirus initiates productive infection,the roles of endosome and microtubule in cell entry of GCRV are investigated by using quantum dots(QDs)-tracking in combination with biochemical approaches.We found that GCRV infection and viral protein synthesis were significantly inhibited by pretreating host cells with endosome acidification inhibitors NH4Cl,chloroquine and bafilomycin A1(Bafi).Confocal images indicated that GCRV particles could colocalize with Rab5,Rab7 and lysosomes in host cells.Further ultrastructural examination validated that viral particle was found in late endosomes.Moreover,disruption of microtubules with nocodazole clearly blocked GCRV entry,while no inhibitory effects were observed with cytochalasin D treated cells in viral infection,hinting that intracellular transportation of endocytic uptake in GCRV infected cells is via microtubules but not actin filament.Notably,viral particles were observed to transport along microtubules by using QD-labeled GCRV.Altogether,our results suggest that GCRV can use endosomes and microtubules to initiate productive infection.
