The Mechanism Study On Snakehead Vesiculovirus Multiplication Mediated By The Interaction Between Hsp90 And L Protein

Snakehead vesiculovirus(SHVV)is a kind of rhabdovirus isolated from diseased snakehead fish.At present,the detailed mechanism of SHVV pathogenecity.Polymerase protein(L)and phosphoprotein(P)form a L-P complex,which is responsible for the transcription and replication of viral genome RNA,and it’s the core of virus proliferation.In this study,we found that heat shock protein 90(Hsp90)interacts with SHVV L protein.Inhibition of Hsp90 activity by Hsp90 inhibitor resulted in degradation of L protein and inhibition of SHVV proliferation.L protein was mainly insoluble when expressed alone,but the solubility of L protein was significantly improved when co-expressed with the P protein.Inhibition of Hsp90 activity by Hsp90 inhibitors resulted in the degradation of L proteins in the soluble state by autophagy pathway,and the degradation of L proteins in the insoluble state by proteasome pathway.The results of the study will help to understand the role of Hsp90 in regulating the proliferation of negative-strand RNA viruses.The detailed research results are as follows:1.Preparation of polyclonal antibody of SHVV L proteinTo study its role in virus proliferation,we amplified the first 900 bases of SHVV L gene and inserted it into p ET-32a(+)to construct p ET32a-L recombinant prokaryotic expression plasmid.The plasmid was then transformed into E.coli BL21(DE3)competent cells.Through the preliminary experiment,the induced expression and purification conditions were optimized.After purification,the recombinant His-L protein was fully mixed with a certain amount of adjuvant and immunized into New Zealand white rabbits three times to prepare polyclonal antibodies.Western blot results showed that polyclonal antibodies against L protein could specifically recognize L protein.Indirect immunofluorescence was used to observe the location of L protein in channel catfish ovary(CCO)cells after SHVV infection,L protein located in cytoplasm of CCO cells.The experimental results show that the L protein polyclonal antibody was successfully prepared with good specificity.2.Screening and validation of host proteins interacting with SHVV LIdentifying host protein that interact with viral protein is critical in determining the role of host proteins in virus proliferation.The SHVV-L protein polyclonal antibody and rabbit Ig G were used to perform Co-Immunoprecipitation(Co-IP)experiments on samples of SHVV infected CCO cells at 24 h post infection(hpi),and CCO cells without SHVV infection were used as controls.The samples were analyzed by mass spectrometry and 32 potential interacting proteins were identified.Two heat shock proteins(Heat shock protein 90 and Heat shock protein 70)were selected for Co-IP verification,and the interaction between Hsp90 and L protein was confirmed.The subcellular co-localization experiment proved that Hsp90 can co-localize with L protein in the cytoplasm.3.The effect of Hsp90 on the proliferation of SHVVIn order to explore the effect of Hsp90 on the proliferation of SHVV,si RNA of Hsp90 was transfected into CCO cells and then infected with SHVV.The results showed that knockdown of Hsp90 significantly inhibited SHVV proliferation.Furthermore,during the process of SHVV infection in CCO cells,the cells were treated with Hsp90 inhibitor 17-DMAG or Geldanamycin,DMSO was used as a control.The results indicate that Hsp90 inhibitors inhibit Hsp90 activity and significantly inhibit SHVV proliferation.At the same time,the Hsp90 inhibitor 17-DMAG was added or not during SHVV infection in CCO cells.The G m RNA and genomic RNA of SHVV were detected by quantitative real-time PCR(q RT-PCR),and it was found that the Hsp90 inhibitor 17-DMAG could significantly inhibit the transcription and replication of SHVV.The results show that Hsp90 is necessary for the proliferation of SHVV.4.The effect of Hsp90 activity on SHVV L proteinThe Hsp90 inhibitor 17-DMAG was added or not during SHVV infection in CCO cells,and the samples were detected for the expression levels of L protein and Hsp90 at different time points poi.The results showed that in the absence of 17-DMAG,the expression level of SHVV L protein,but not Hsp90 increased gradually along with the time,while the addition of 17-DMAG significantly decreased the expression level of SHVV L protein.At the same time,293 T cells were transfected with eukaryotic plasmids expressing SHVV N,P,M,G,and L proteins to study the effect of 17-DMAG on the expression levels of SHVV N,P,M,G,or L protein.The results showed that 17-DMAG did not significantly change the levels of N,P,M,or G protein,but significantly reduced the level of L protein,indicating that L protein is the client protein of Hsp90.293T cells were transfected with pCDNA-L alone or co-transfected with pCDNAP,and the cells were treated with 17-DMAG or DMSO,respectively.We found that 17-DMAG significantly degraded L protein,while the co-expression of P protein promoted the degradation of L protein by 17-DMAG.Furthermore,proteasomal pathway inhibitors(MG132),autophagy pathway inhibitor(3-MA),and lysosomal pathway inhibitor(NH4CL)were used to study the degradation pathway of 17-DMAGmediated degradation of SHVV L protein.The results showed that L protein was degraded by the proteasome pathway when expressed alone,and by the autophagy pathway when co-expressed with P.By analyzing the components of soluble and insoluble fractions,it was found that L protein was mainly in insoluble state when expressed alone,and the presence of P protein promoted the solubility of L protein.Further studies showed that soluble L proteins were degraded by autophagy pathway,while insoluble L proteins were degraded by proteasome pathway.