Effects Of Bombyx Mori Molecular Chaperone BmHsp90 And Its Chaperone BmCdc37 On The Proliferation Of BmNPV

Bombyx mori nuclear polyhedrosis virus,BmNPV,is a double stranded DNA virus whose genome is about 128 kb,and its blood pus disease causes harm to the sericulture is extremely serious,not only reduces the yield and quality of silk,but also not benefit for the stability and development of China's silk industry.In recent years,with the deepening of the research of BmNPV,we found that part of the host protein was involved in the virus proliferation and replication.Hsp90 is a member of the heat shock protein family,as a molecular chaperone,plays an important role in intracellular protein conformation maturation,and is also an important participant in many viral protein synthesis.Cdc37,as co chaperone,can raises the protein specifically to bind to Hsp90 to form a molecular chaperone complex,involved in the maintenance of protein stability and normal function.In order to clarify the Hsp90 and Cdc37 played in the BmNPV in the proliferation process.The results of the study are as follows: 1.Identification and expression pattern analysis of Bm Hsp90 and BmCdc37 genesThe cloned BmHsp90 gene is 2148 bp,encoding a protein of 716 amino acid residues;full-length 1116 bp for BmCdc37 gene,encoding a protein of 372 amino acid residues.The protein sequence alignment analysis showed that BmHsp90 gene contains N terminal ATP binding domain,domains which function as link and dimerization;BmCdc37 contains N client protein binding domain,intermediate domain and 10.5kDa C domain.Phylogenetic tree analysis showed that silkworm Hsp90 is classified into Hsp90 family,silkworm Cdc37 belongs to Lepidoptera Papilio xuthus Cdc37 relationship.Immunofluorescence experiments observed that both proteins are located in the the cytoplasm and uniform distribution.The experiment of heat shock induced BmHsp90 found that in a certain temperature range,the expression level of the gene with the increase of heat shock temperature rises,consistent with biological characteristics of shock protein.Model analysis found that both high expression in testis and ovary,and the expression trend in different tissues are similar;expression analysis showed that the expression of both genes is similar.All the above experiments implied that they have a certain correlation in function.2.Identification of interaction between BmHsp90 and BmCdc37Recombinant protein overexpression plasmid pIZ/V5-His-BmHsp90-Ha was constructed,pIZ/V5-His-BmCdc37-Flag,pBi FC-BmHsp90-VN,p BiFC-BmCdc37-VC.immunofluorescence co-localization,bimolecular fluorescence Bi FC results showed the interaction between BmHsp90 protein and Bm Cdc37 protein.BmHsp90 protein and BmCdc37 protein interaction bait,further verified BmHsp90 interacts with Bm Cdc37 in the silkworm cells by immunoprecipitation.3.Effects of BmHsp90 and BmCdc37 on the proliferation of BmNPVBmHsp90,BmCdc37 over expression vectors were transfected into cells and transfected by two gene expression plasmid.After 48 h,green fluorescent labeled BmNPV were infected,24 h and 48 h later infection rates were tested by cells flow cytometry.The experimental results showed that over expression of silkworm Hsp90 Cdc37 can promote the proliferation of BmNPV in cells.At the same time,BmCdc37 protein can enhance the ability of BmHsp90 to promote the proliferation of virus.At the transcriptional level,virus gene VP39 expression was detected by the relative expression of fluorescent quantitative.The experimental results also showed that silkworm Hsp90,Cdc37 can promote the proliferation of BmNPV in cells,while BmCdc37 protein can enhance the ability of Bm Hsp90 to promote the proliferation of the virus results.At the protein level,VP39 protein was detected by western blot,the conclusion is same.Then the use of CRISPR/Cas9 technology in B.mori cells at In addition to BmHsp90 and BmCdc37,respectively,from the cellular level of BmNPV proliferation of the expression level of gene transcription and protein were studied.A series of experimental results showed that knockout of silkworm Hsp90,Cdc37 can inhibit BmNPV in the cell proliferation by the CRISPR/Cas9,and inhibition of BmCdc37 protein could enhance the effect of the BmHsp90 virus proliferation.4.Identification of BmHsp90 interacting proteinsBmHsp90 protein was put as bait protein by immunoprecipitation method and identified a molecular weight of 70 k Da,BmGolga5 protein and the molecular weight of 61 kDa BmTbce protein.We found that the BmGolga5 protein in the cells was located in the cytoplasm and showed punctate distribution by immunofluorescence;BmTbce protein in the cells was located in the cytoplasm.Finally through immunofluorescence and immunoprecipitation experiments we confirmed two proteins have interaction with BmHsp90.According to the research of the protein of Tbce and Golga5 related: silkworm Cdc37 raised Tbce,Golga5 protein to Hsp90 and was modified to maintain its steady state,so as to promote microtubule polymerization,reverse transport of Golgi bodies.The virus made viral proteins newly synthesized lysosomal degradation escape fate by using reverse transportation.At the same time the virus protein with the help of the escape track is transported into the nucleus and microtubule assembly of viral nucleic acid,so as to promote the proliferation of the virus.To sum up,BmHsp90,Bm Cdc37 can promote the proliferation of BmNPV,and BmCdc37 can enhance the effect of BmHsp90 to promote the proliferation of the virus.BmHsp90 and BmCdc37 may stabilize and activate Bombyx mori Tbce,Golga5 protein in the form of molecular complexes.These two proteins are involved in the escape process of BmNPV protein,and thus promote the proliferation of the virus.