| Bombyx mori is an important silking economic insect.It has a long history of domestication in China.However,due to lack of specific immune system,the insect itself is susceptible to pathogenic microorganisms.The resulting economic losses are very serious in China by silkworm disease,among which Bombyx mori nuclear polyhedrosis virus is the main pathogen should be prevented and controlled in sericulture industry.Hsp60 is an important member of heat shock proteins.It not only can participate in the correct folding of proteins,but also responds to high temperature,hypoxia,hunger,etc.,and it is also an essential factor for the growth,development and innate immunity of insects.Some scholars have found that Hsp60 is one of the proteins directly interacting with BmNPV through virus coverage assays.Our previous study also demonstrated that Hsp60 was hijacked by LEF-11,a late expression factor of BmNPV,to promote virus proliferation.However,the specific mechanism of Hsp60 promoting BmNPV proliferation remains unknown.In this study,Hsp60 interacting proteins of the silkworm were caught and their effects on BmNPV proliferation and cellular ATP levels were identified.The regulatory relationship between them was preliminarily explored,which was benefit for investigating the the regulatory networks between the BmNPV and the host,and the following results were obtained: 1.The identification of Interaction proteins between BmHsp60First,it was verified that BmHsp60 can promote the proliferation of BmNPV at the cellular level,and was upregulated after BmNPV infection.Then by immunoprecipitation-mass spectrometry analysis,the candidate proteins interacting with it were retrieved.Finally,the interaction between BmANT1,BmHsp90 and BmHsp60 was identified.The expression level was down-regulated by BmNPV,and its location in the mitochondria and protein structure predicted to have three transmembrane domains,suggesting that BmANT1 is anchored on the mitochondrial membrane and may be involved in the process of cellular energy metabolism and apoptosis,all of which might be associated with BmNPV infestation.2.Effect of BmHsp60 and BmANT1 on proliferation of BmNPVAt the cellular level,48 h after transfection of the BmANT1 overexpression vectors,the fluorescent cells were observed after infection with BmNPV,and the proportion of infected cells was analyzed by flow cytometry.The results showed that after overexpression BmANT1,viral proliferation was inhibited.The level of gene transcription and protein,tested by RT-PCR and western blotting,showed the same results.Furthermore,knockout efficiency of the CRISPR/Cas9 knockout vector for detection of BmANT1 was more than 20%,and it was found that knocking out BmANT1 can also inhibit BmNPV proliferation.3.Co-effects of BmHsp60 and BmANT1 on BmNPV proliferationThe BmHsp60 overexpression vector was transfected with the BmANT1 overexpression vector and the knockout vector respectively in the silkworm cells.48 hours later,the cells was infected of BmNPV.The infected cells were observed under fluorescence microscope.The proportion of infected cells was analyzed by flow cytometry.RT-PCR and western blotting were used to testing the expretion of vp39.The results showed that overexpression of Hsp60 could promote the proliferation of BmNPV,but after overexpression or knockout of BmANT1,the proliferation of the virus was inhibited.In contrast,while overexpression of BmANT1 and BmHsp60,inhibition of proliferation of the virus is attenuated,otherwise,knockout of BmHsp60 at the same time,the inhibition was more deep.In addition,the detection of ATP levels showed that infection with BmNPV could increase the ATP level of the host cells,and after overexpression or knockout of BmANT1,it had little effect on cellular ATP levels;But overexpression of BmHsp60,the cellular ATP level was increased,on the contrary,the level of ATP was down-fegulated.4.BmHsp60 regulates BmANT1 mechanismFrom the above results,it is speculated that ATP levels are very important for BmNPV proliferation,but BmANT1 may not be the most important for host ATP production,and BmNPV proliferation may be dependent on other functions,which may be independent of ADP/ATP transport function of BmANT1.BmHsp60 can promote virus proliferation by increasing the host ATP level.On the one hand,it may increase ATP levels through other pathways.On the other hand,BmHsp60 may be regulated through other effects of BmANT1.Therefore,the regulatory relationship between them must be explored.After overexpression of BmHsp60,the expression of BmANT1 was decreased by RT-PCR,whereas BmANT1 expression was increased after BmHsp60 was knocked out or overexpressed,indicating that BmHsp60 can inhibit the expression of BmANT1.Overexpression or knockout of BmANT1,BmHsp60 expression decreased,which may be due to the complex feedback regulation mechanism of BmANT1 on BmHsp60.The expression of TLR4,an intrinsic immune recognition transmembrane protein widely found in various organisms,detected in silkworm cells by RT-PCR revealed that BmNPV infection as well as overexpression of BmHsp60 induced TLR4 expression and activated TLRs pathway.After treated with PDTC,an inhibitor of NF-κB,a key downstream transcription factor of TLR4,the proliferation of BmNPV was inhibited,demonstrating that the TLRs pathway participates in the proliferation of BmNPV.In further experiments,the expression of BmHsp60 and BmTLR4 were not been affected by PDTC,demonstrating that BmHsp60 is upstream of the TLRs pathway.In addition,after PDTC pretreatment,infection with BmNPV or overexpression BmHsp60,the expression of BmANT1 was not inhibited anymore.In summary,BmHsp60 can promote the proliferation of BmNPV,and the effect of BmANT1 is opposite.After the virus infects the host,it can stimulate the host cell to generate energy for its replication and proliferation.BmHsp60 promotes virus proliferation by providing the necessary energy and activating downstream pathways.BmANT1 does not play the most critical role in the production of host ATP,so its inhibition fuction of virus proliferation does not occur via energy levels,but may be achieved through other pathways.In conclusion,after BmNPV infects host cells,virus enhances the expression of BmHsp60,which may activate the host TLR4 pathway and NF-κB,inhibits the expression of BmANT1,and provides favorable conditions for virus proliferation. |
