We investigated the ability of the rabbit hemorrhagic disease virus(RHDV) capsid protein(VP60) to interact specifically with the minor structural protein VP10,using an in vivo cell-based CheckMate Mammalian Two-Hybrid...We investigated the ability of the rabbit hemorrhagic disease virus(RHDV) capsid protein(VP60) to interact specifically with the minor structural protein VP10,using an in vivo cell-based CheckMate Mammalian Two-Hybrid System.RHDV VP60 protein interacted specifically with VP10.Immunofluorescence analysis and co-immunoprecipitation with specific antibodies revealed the existence of biologically important VP60/VP10 complexes.However,when VP60 was divided into two fragments,the interaction between VP60 and VP10 was impaired dramatically.These results will be helpful for further investigating the mechanism of RHDV particle assembly.展开更多
Rabbit hemorrhagic disease virus(RHDV)is a member of the Caliciviridae family and cannot be propagated in vitro,which has impeded the progress of investigating its replication mechanism.Construction of an RHDV replico...Rabbit hemorrhagic disease virus(RHDV)is a member of the Caliciviridae family and cannot be propagated in vitro,which has impeded the progress of investigating its replication mechanism.Construction of an RHDV replicon system has recently provided a platform for exploring RHDV replication in host cells.Here,aided by this replicon system and using two-step affinity purification,we purified the RHDV replicase and identified its associated host factors.We identified rabbit nucleolin(NCL)as a physical link,which mediating the interaction between other RNA-dependent RNA polymerase(Rd Rp)-related host proteins and the viral replicase Rd Rp.We found that the overexpression or knockdown of NCL significantly increased or severely impaired RHDV replication in RK-13 cells,respectively.NCL was identified to directly interact with RHDV Rd Rp,p16,and p23.Furthermore,NCL knockdown severely impaired the binding of Rd Rp to Rd Rp-related host factors.Collectively,these results indicate that the host protein NCL is essential for RHDV replication and acts as a physical link between viral replicase and host proteins.展开更多
Based on the infectious full-length cDNA clone of rabbit hemorrhagic disease virus (RHDV), the in vitro transcripts are introduced into RK13 cells. 12 h later, CPE could be observed clearly, and virual antigen could a...Based on the infectious full-length cDNA clone of rabbit hemorrhagic disease virus (RHDV), the in vitro transcripts are introduced into RK13 cells. 12 h later, CPE could be observed clearly, and virual antigen could also be detected by IFA. The titre of the recovered virus is 104.6/mL. Immune electron micro-scopic observation of the virus particles revealed that the particles were rotund with a diameter of about 30 nm. Besides, virus titre quantification obtained by qRT-PCR showed a correlation between time from infection and virus titre. All these results showed that we have recovered RHDV from RK13 cells by re-verse genetics technology successfully, and this would be very useful in studies of the antigenicity, virulence, pathogenesis, maturation and new type vaccines of RHDV.展开更多
基金supported by the National Natural Science Foundation of China (30870114)the Zhejiang Natural Science Foundation (3100396)the Fundamental Research Funds for the Central Institutes Program(2012JB08)
文摘We investigated the ability of the rabbit hemorrhagic disease virus(RHDV) capsid protein(VP60) to interact specifically with the minor structural protein VP10,using an in vivo cell-based CheckMate Mammalian Two-Hybrid System.RHDV VP60 protein interacted specifically with VP10.Immunofluorescence analysis and co-immunoprecipitation with specific antibodies revealed the existence of biologically important VP60/VP10 complexes.However,when VP60 was divided into two fragments,the interaction between VP60 and VP10 was impaired dramatically.These results will be helpful for further investigating the mechanism of RHDV particle assembly.
基金sponsored by Shanghai Sailing Program(20YF1457700)the National Natural Science Foundation of China(32000109 and 31672572)the China Postdoctoral Science Foundation(2019M660885 and 2021T140718)
文摘Rabbit hemorrhagic disease virus(RHDV)is a member of the Caliciviridae family and cannot be propagated in vitro,which has impeded the progress of investigating its replication mechanism.Construction of an RHDV replicon system has recently provided a platform for exploring RHDV replication in host cells.Here,aided by this replicon system and using two-step affinity purification,we purified the RHDV replicase and identified its associated host factors.We identified rabbit nucleolin(NCL)as a physical link,which mediating the interaction between other RNA-dependent RNA polymerase(Rd Rp)-related host proteins and the viral replicase Rd Rp.We found that the overexpression or knockdown of NCL significantly increased or severely impaired RHDV replication in RK-13 cells,respectively.NCL was identified to directly interact with RHDV Rd Rp,p16,and p23.Furthermore,NCL knockdown severely impaired the binding of Rd Rp to Rd Rp-related host factors.Collectively,these results indicate that the host protein NCL is essential for RHDV replication and acts as a physical link between viral replicase and host proteins.
基金This work was supported by the Natural Science Foundation of Zhejiang Province(Grant No.Y305047).
文摘Based on the infectious full-length cDNA clone of rabbit hemorrhagic disease virus (RHDV), the in vitro transcripts are introduced into RK13 cells. 12 h later, CPE could be observed clearly, and virual antigen could also be detected by IFA. The titre of the recovered virus is 104.6/mL. Immune electron micro-scopic observation of the virus particles revealed that the particles were rotund with a diameter of about 30 nm. Besides, virus titre quantification obtained by qRT-PCR showed a correlation between time from infection and virus titre. All these results showed that we have recovered RHDV from RK13 cells by re-verse genetics technology successfully, and this would be very useful in studies of the antigenicity, virulence, pathogenesis, maturation and new type vaccines of RHDV.
