Study On The Interaction Mechanism Of Hsp90 Between Bombyx Mori And Bombyx Mori Nucleopolyhedrovirus

Bombyx mori is an important economic insect and has made great contributions to the development of our national economy.It is one of the good model organisms for studying the interaction between insects and viruses because often challenged by various pathogenic microorganisms during reproduction.Bombyx mori nucleopolyhedrovirus(Bm NPV)is one of the main pathogens that endanger the silkworm industry.Once infected,the silkworm will cause a large number of deaths,seriously affecting the sustainable development of the silkworm industry.In order to control Bm NPV infection,develop disease-resistant silkworm varieties,and use costeffective bioreactors safely,researchers have carried out a large number of studies in an attempt to reveal the interaction mechanism between silkworm and Bm NPV,but so far,the mechanism is still unknown.Heat shock protein 90(Hsp90)is a highly conserved molecular chaperone responsible for the correct folding and maturation of key proteins and plays an important role in a variety of life processes.In recent years,more and more evidences have shown that Hsp90 plays a crucial role in the replication of many DNA viruses or RNA viruses,but there are few studies on the role of Hsp90 in silkworm virus infection.In this study,Geldanamycin(GA),a Hsp90 protein activity inhibitor,was used to inhibit the activity of Hsp90 protein in silkworm,and the proliferation of Bm NPV in silkworm cells of GA treatment group and control group was analyzed.The role of Hsp90 in Bm NPV infection was further analyzed by knockdown in vitro and overexpression of Hsp90 gene in silkworms.In addition,in order to explore the interaction mechanism between Hsp90 and Bm NPV,we also compared the differences of protein expression between cells infected with Bm NPV in GA-treated group and control group by quantitative proteomic analysis based on tandem mass spectrometry(TMT).The main findings are as follows: 1.Determination of suitable GA concentration and adding methodAfter treatment of Bm N cells with different concentrations of GA,MTT analysis showed that there was no significant adverse effect on the survival rate of Bm N cells treated with GA of 2.5 μM or less.Bm N cells were treated with different combinations of GA(1 μM and 2.5 μM)and Bm NPV containing EGFP marker gene.The results of virus titer test showed that the virus titer of GA treated silkworm cells was two orders of magnitude lower than that of the control group.In terms of virus inhibition effect and operability,it is the best way to treat Bm N cells by adding 2.5 μM GA and Bm NPV virus suspension at the same time,so this method is used in subsequent experiments.2.Effect of inhibiting Hsp90 protein activity by GA on Bm NPV proliferationThe DNA copies of ie-1,lef-3 and gp64 were analyzed by absolute q RT-PCR at different time points.The results showed that the DNA copies of each virus gene increased with the increase of time.In the same time point,the copy numbers of virus gene in the control group were significantly higher than those in the GA treatment group.Western blot showed that the expression of Bm NPV nucleocapsid protein 39(vp39)in GA treated group was significantly lower than that in control group.The results showed that inhibition of Hsp90 protein activity could significantly inhibit the proliferation of Bm NPV.In addition,the expression levels of nine genes related to immune pathway were also detected.The results of q RT-PCR showed that the gene expression levels related to Toll and IMD pathway had no significant change,while the gene expression levels related to JAK/STAT pathway had significant change,stat gene expression was up-regulated,socs6 and socs2 gene expression was down regulated;The expression level of two antiapoptotic genes,Bm NPV inhibited iap1 and bmiap2,was significantly lower than that of the control group.It is speculated that inhibition of Hsp90 may affect the expression of key genes in JAK/STAT signaling pathway and anti apoptosis related genes,and then affect the proliferation of Bm NPV.3.The effect of knockdown Hsp90 gene on the proliferation of Bm NPVThe Hsp90 gene was knockdown in silkworm cells by RNA interference.q RTPCR was used to analyze the transcription level of Hsp90 after si RNA interference.The results showed that the expression of Hsp90 gene in si RNA interference group was significantly lower than that in the control group,indicating that the knockdown was successful.Furthermore,the DNA copies of virus genome in the experimental group and the control group was compared and analyzed.The results showed that the DNA copies of virus genome in the control group was significantly higher than that in the experimental group,indicating that knockdown of Hsp90 could significantly inhibit the proliferation of Bm NPV.4.The effect of overexpression of Hsp90 on the proliferation of Bm NPVThe Hsp90 gene was amplified with silkworm c DNA as template and cloned.The fusion expression vector p Fast Bac1-e GFP-Bm Hsp90-3×Flag and the control vector p Fast Bac1-e GFP-3×Flag were constructed.The recombinant vectors were transformed into Bm DH10 bac sensitive cells respectively to obtain the recombinant baculovirus.After inoculating the silkworm,Hsp90 was overexpressed in the silkworm.The results of q RT-PCR showed that the relative expression of Hsp90 in the overexpression group was significantly higher than that in the control group,indicating that Hsp90 was overexpressed successfully in the silkworm;the results of Western blot further showed that Hsp90 protein was successfully expressed in the silkworm.Then,TCID50 method was used to detect the replication level of Bm NPV after overexpression of Hsp90.The results showed that the TCID50 value of overexpression group was significantly higher than that of control group.q RT-PCR was used to detect the change of copy number of virus genome DNA.It was found that when Hsp90 gene was overexpressed and the content of virus genome DNA was significantly higher than that of the control group at 24 hours after infection,which indicated that overexpression of Hsp90 could promote the replication of Bm NPV,especially in the early stage of infection.In addition,the study also found that the expression level of Bm NPV inhibitor of apoptosis protein 1(iap1)and bmiap2 in the silkworm was significantly higher than that in the control group after overexpression of Hsp90,which was contrary to that in the GA treated group,indicating that the loss of Hsp90 activity may lead to the degradation of Bmiap and Bm NPV-iap,thus promoting the apoptosis of infected cells and inhibiting the proliferation of Bm NPV.5.The effect of inhibition of Hsp90 protein activity on the host protein related to Bm NPV infectionQuantitative proteomic analysis was used to identify the role of Hsp90 in the interaction between silkworm and Bm NPV.A total of 195 differentially expressed proteins were identified,including 136 up-regulated proteins and 59 down regulated proteins,based on the screening criteria of multiple variation greater than 1.3 and P value less than 0.05.KEGG pathway analysis showed that the differentially expressed proteins were involved in the key pathways,such as longevity regulatory pathway,m TOR signaling pathway,Fox O signaling pathway and Toll and IMD signaling pathway.After inhibition of Hsp90,the expression levels of apolipoprotein III and Bm IBP in Bm NPV infected cells increased,and 11 zinc finger proteins were up regulated in Bm NPV infected cells treated with GA.It is suggested that inhibition of Hsp90 activity may directly or indirectly induce the expression of zinc finger protein.The expression level of PARP and XRCC6 increased,indicating that inhibition of Hsp90 can trigger DNA damage repair pathway.In addition,we also found that four small heat shock proteins related to longevity regulation pathway were up-regulated,and the expression levels of immune related proteins and cell DNA repair related proteins were significantly increased.The expression level of 11 protein kinases decreased significantly.The results showed that inhibition of Hsp90 activity could result in significant changes of protein expression profile in Bm N cells.The above results confirmed the positive role of Hsp90 in Bm NPV infection,and explored the potential role of Hsp90 in host immune response to Bm NPV infection.The results are helpful to understand the mechanism of Hsp90 in Bm NPV infection,and may provide a new clue to elucidate the molecular mechanism of the interaction between silkworm and virus,and a new research direction for Bm Hsp90 candidate customer proteins involved in Bm NPV infection in Bm N cells.