Screening And Verification Of Basic Function Of The Host Protein Interacting With The Structural Protein VP2 Of Deform Wing Virus

ObjectiveThe membrane protein yeast two-hybrid system was applied to screen the potential binding protein partners of Deform wing virus(DWV)structural protein VP2.After confirmed the interaction between VP2 and host protein,we preliminary exploration their role in virus replication.Our study could provide a theoretical foundation for the pathogensis of DWV.MethodsThe DWV structural protein VP2 gene was cloned into the p BT3-STE carrier to construct the bait palsmid p BT3-STE-VP2,then co-transferred it and control plasmids into yeast competent cells NMY32 to detect its function and self-activating ability.On this basis,the bait plasmid p BT3-STE-VP2 and the Apis cerana larvae tissue membrane protein yeast c DNA library plasmid was co-transformed into the yeast NMY32 and cultivated on a series of selective medium to obtain the potentially positive clones.The plasmids were extracted and transformed into E.coli DH5α,then amplified by PCR using p PR3 N specific primers and sequenced.Total 20 host-related proteins were obtained,after the sequences were analyzed by BLAST.Among them,the heat shock protein 10(Hsp10)was found associated with many biochemical processes and might play a significant role in virus infection.Therefore,we took Hsp10 for further research.To further confirmed the interaction between DWV VP2 and Hsp10,we conducted glutathione S-transferase(GST)pull-down and co-immunoprecipitation(Co-IP).Then we used Confocal microscopy to observe the localization of VP2 and Hsp10 in BHK cells.Meanwhile,VP2 and Hsp10 were co-transfected into cells to investigate whether VP2 regulate the expression of Hsp10.In addition,Real-time polymerase chain reaction(RT-PCR)was used to analyze the expression of Hsp10 in healthy bee and DWV-infected bee.ResultsThe bait plasmid p BT3-STE-VP2 was successfully constructed.Autoactivation and function detection reveald that the bait plasmid p BT3-STE-VP2 constructed in this study had function and no self-activation ability in yeast cell.Using the p BT3-STE-VP2 bait screened the c DNA library showed that 38 positive clones were initially screened deficient medium,the results of PCR showed that 24 of them were amplified 500~2000 bp of bands.Sequence analysis of these positive plasmids indicated that these plasmids represented 20 host proteins.These proteins are mainly involved in physiologica metabolism,biochemical reaction of honey bees,host antiviral mechanism and virus replication.Among them,we chose Hsp10 for further study.The glutathione S-transferase(GST)pull down and co-immunoprecipitation(Co-IP)showed that there was obviously interaction between DWV VP2 and Hsp10.Laser confocal microscopy revealed that VP2 and Hsp10 completely overlapped in the cells and were primarily localized in the cytoplasm.Overexpression test confirmed that VP2 could repress the expression of Hsp10 in cells.The real-time polymerase chain reaction(RT-PCR)showed that the expression of Hsp10 was significantly decreased in the bee that infected DWV.Conclusions1.DWV VP2 interacts with Hsp10,and VP2 co-localize with Hsp10 in the cytoplasm.2.The expression of Hsp10 was significantly decreased in the bee that infected DWV.VP2 overexpressed in cell could significantly reduced the expression of Hsp10.