The Mechanism Study Of Interaction Between Hsp90 And VP35 In Regulating The Replication Of GCRV-Ⅱ

Grass carp(Ctenopharyngodon idella)is one of the most important freshwater fish in China,but the hemorrhagic disease caused by grass carp reovirus(GCRV)seriously affects the development of grass carp aquaculture,and its pathogenesis is still unclear.GCRV-Ⅱ is the main current strain,and its gene fragment S11 encodes the outer capsid protein VP35.Heat shock protein 90(Hsp90)functions at multiple stages of the life cycle of a variety of viruses.We demonstrated that overexpression of VP35 promoted the replication of GCRV-Ⅱ and that VP35 could interact with Hsp90.The activity of Hsp90 is crucial for the replication of GCRV-Ⅱ.On the one hand,Hsp90 is localized on the cell membrane.It act as a receptor for GCRV-Ⅱ in the process of viral adhesion and invasion;on the other hand,Hsp90 is localized in the cytoplasm and acts as a VP35 chaperone protein,inhibition of Hsp90 results in the degradation of VP35 through the proteasomal pathway.The main findings of this study are as follows:1.VP35 promoted the replication of GCRV-Ⅱ.In order to explore the role of VP35 protein in virus replication,the S11 gene was amplified and inserted into the vector p CMV-3×Flag-14 to construct the S11 plasmid,which was transfected into Ctenopharyngodon idella kidney(CIK)cells,and then infected with GCRV-Ⅱ.After infection the supernatant and cells were collected.First,real-time fluorescent quantitative PCR was used to detect S6 m RNA in cell samples.The results showed that overexpression of VP35 promoted the replication of GCRV-Ⅱ at the level of viral m RNA about two folds.Then the S6 gene was amplified and inserted into the vector p ET32a(+).The protein His-VP4 was purified and polyclonal antibodies was prepared to determine the effects of VP35 on GCRV-Ⅱ at viral protein level.The results showed that overexpression of VP35 promoted the replication of GCRV-Ⅱ at the level of viral protein.Finally,the p ET32a-VP4 plasmid was used as a positive standard plasmid to establish an absolute quantitative detection method for the gene copy number of GCRV-Ⅱ.The p ET32a-VP4 plasmid was used as a template,q PCR was used to detect the expression of the S6 gene,and a standard curve was drawn.The quantitative method was used to determine the effects of VP35 on GCRV-Ⅱ particles in supernatants.The results showed that overexpression of VP35 increased the copy number of GCRV-Ⅱ.This study suggests from three dimensions that overexpression of VP35 protein promotes GCRV-Ⅱ replication.2.Hsp90 interacts with VP35 and Hsp90 promotes GCRV-Ⅱ replication.Further identification of host proteins interacting with the viral outer capsid protein-VP35.The interacting proteins were screened by Co-IP,separated by SDS-PAGE and stained with silver.Mass spectrometry analysis was then performed.Among them,Hsp90 functions in multiple stages of the life cycle of various viruses.Co-IP experiments showed that Hsp90 interacted with VP35.Subcellular localization experiments showed that Hsp90 and VP35 co-localized in cells.Purification of His-Hsp90 recombinant protein.The interaction between Hsp90 and VP35 was further confirmed by His-Pull down.Knockdown of Hsp90 significantly inhibited GCRV-Ⅱ replication.Inhibition of Hsp90 activity with 17-DMAG and Geldanamycin also significantly inhibited viral replication.It indicated that Hsp90 played an important function in the replication process of GCRV-Ⅱ and played a role in promoting virus replication.3.Hsp90 acts as the receptor and chaperone of VP35 to promote the replication of GCRV-Ⅱ.The initial step of the viral infection cycle is the specific binding of the viral outer capsid protein to the receptor protein expressed on the cell surface.To explore the function of Hsp90 in the invasion of host cells by GCRV-Ⅱ,non-permeabilized CIK cells were observed by indirect immunofluorescence.The cell membrane is an elongated triangle with branches,which is the shape of CIK cells.There are punctate distribution of Hsp90 on the cell membrane.Flow cytometry accurately quantified its distribution,and the results showed that 9.24% of cells showed positive signals.It is proved that Hsp90 is not only localized in the CIK cytoplasm,but also localized on the CIK cell membrane.The inhibition of Hsp90 by 17-DMAG significantly reduced the adsorption and entry of GCRV-Ⅱ to CIK cells,demonstrating that Hsp90 activity is crucial for the invasion process of GCRV-Ⅱ to CIK cells.The binding of GCRV-Ⅱ to CIK cells was inhibited by blocking the surface-localized Hsp90 of CIK cells with Hsp90 antibody or by pretreating the virus with the His-Hsp90 fusion protein.Overexpression of Hsp90 facilitates viral entry.Taken together,it is indicated that Hsp90 is the cellular receptor of GCRV-Ⅱ VP35.Inhibition of Hsp90 activity significantly reduced the expression of VP35,indicating that Hsp90 acts as a chaperone of VP35,and inhibiting Hsp90 activity will degrade VP35 through the proteasome pathway.This study confirmed that Hsp90 is the receptor of GCRV-Ⅱ.Hsp90 plays a role in virus replication.Our data not only provide a theoretical basis for further exploring the pathogenic mechanism of GCRV-Ⅱ,but also provide a new antiviral strategy to deal with viral infection.