Screening And Verification Of Basic Function Of The Host Protein Interacting With The Structural Protein VP1 Of Chinese Sacbrood Virus

ObjectiveUsing the membrane protein yeast two-hybrid system to screen out the host cellular proteins that interact with Chinese sacbrood bee virus(CSBV)structural protein VP1,and verify the interactions between these two proteins and the launch a preliminary research on the role of them in virus replication.This study will lay a theoretical foundation for the pathogenic mechanism of CSBV.MethodsThe CSBV structural protein VP1 gene was cloned into the vector p BT3-STE to construct the bait palsmid p BT3-STE-VP1,which then was transferred into yeast competent cells NMY32 with the control plasmid to detect its function and self-activating ability.On this basis,the bait plasmid p BT3-STE-VP1 and the Apis cerana larvaetissue membrane protein yeast c DNA library plasmid were co-transformed into yeast NMY32 and cultivated on SD-TL,SD-TLH + 60 m M 3AT media to initially screen out the positive clones.The positive clones were amplified by PCR using p PR3 N specific primers and sequenced.The 23 host-related proteins were obtained after the sequences were analyzed by BLAST.According to the previous studies and reference,thehot shock protein 70(Hsp70)was selected for furtherstudy.The interaction between CSBV VP1 and Hsp70 protein was verified by glutathione S-transferase(GST)pull down and co-immunoprecipitation(Co-IP).The eukaryotic recombinant plasmids p EGFP-C2-Hsp70-Flag were constructed,and p TT5-VP1-His and p EGFP-C2-Hsp70-Flag were co-transfected into HEK293 T cells,co-localization of Hsp70 and VP1 in the cell,after which using a laser confocal microscope to observe their location.The expression of Hsp70 in healthy larvae and CSBV-infected larvae were detected using Real-time polymerase chain reaction(RT-PCR)and western blot to analyze the changes of Hsp70 expression.On this basis,we designed three pairs of si RNAs for Hsp70,selected si RNAs that had a better silencing effect on Hsp70,and conducted subsequent interference tests to analyze the role of Hsp70 in CSBV infection.ResultsThe bait plasmid p BT3-STE-VP1 was successfully constructed and transferred into yeast competent cells NMY32.Self-activation and functional verification showed that the bait plasmid p BT3-STE-VP1 has no self-activation ability and can be normally expressed in yeast cells.These host proteins were screened out through the interaction between the p BT3-STE-VP1 and the c DNA library of Apis cerana larvae.These proteins playimportant roles in the process of metabolism,such as stress response,protein phosphorylation,signal transduction,protein target,cytoskeletal tissue,DNA repair,protein regulation,ribosomal metabolism,and so on.The previous studies display that Hsp70 plays an important role in virus replication and antiviral immunity.Therefore,we chose Hsp70 for further study.The interaction between Hsp70 and VP1 wasvertified by GST-pull down and Co-IP technology.The colocalization of Hsp70 and VP1 proteins in the nucleus and cytoplasm was detected by Laser confocal microscopy.The expression of Hsp70 was significantly increased in the larvae that infected CSBV by real-time polymerase chain reaction(RT-PCR)and western blot.On this basis,when Hsp70 was silenced by si RNA interference experiments,the copy number of CSBV was significantly reduced.Conclusions1.CSBV VP1 interacts with Hsp70,and VP1 and Hsp70 co-localize in the cytoplasm and nucleus.2.The expression of Hsp70 was significantly increased in the larvae that infected CSBV.When Hsp70 was silenced by si RNA interference experiments,the copy number of CSBV was significantly reduced.