Screening And Functional Study Of Baculovirus AcMNPV PK-1 Interaction Protein

Baculovirus is widely used in disease and insect control,baculovirus expression vector system,Bionanotechnology,genetic therapy,etc.,and are harmless to humans and animals.At present,the research on the infection mechanism of baculovirus has made great progress.There are more than 30 core genes of baculovirus known to act in various stages of virus replication,assembly,infection and so on.Although the very late stage of baculovirus infection does not affect the replication of the virus,a large number of proteins related to the assembly of viral inclusion bodies are synthesized at this stage.Therefore,it is very important to reveal the mechanism of overexpression of baculovirus gene in the very late stage for further modification of baculovirus expression vector system.PK-1 is necessary for baculovirus replication,and also involved in the overexpression of the virus's extremely late genes.This article conducts a series of studies on PK-1 and its interacting virus-encoded proteins.Firstly,the protein interacting with PK-1 was screened by yeast two-hybrid system.The quality of the constructed cDNA library of AcMNPV-infected Sf9 cells was identified,and the bait vector pGBKT7-Y-PK-1 was constructed to screen from the cDNA library and successfully screened 5 proteins due to PK-1 interaction.Four virus-encoded proteins were selected from these proteins,namely protein kinase interacting protein(AC24),zinc finger protein(AC88),capsid protein(AC87)and the major capsid protein(AC89),and the PK-1 was further verified by co-immunoprecipitation interaction with the selected protein,the results confirmed that AC24,AC87,AC88 and PK-1 all interact,AC89 and PK-1 have the possibility of interaction.Then,subcellular localization analysis of PK-1 and its interacting protein AC24 in the cell was carried out,It was found that both of them had the same localization in the cell,initially existed in the whole cell,and gradually accumulated in the nucleus and cytoplasm with the passage of infection time,which was related to the function of PK-1 and AC24.It is speculated that the two are involved in the phosphorylation of proteins in the nucleus and the overexpression of very late genes,and may be related to the movement and budding of the nucleocapsid in the cytoplasm.Finally,the function of ac24 in the process of baculovirus infection was preliminarily explored by gene knockout,through the construction of ac24 deletion,rescue and wild-type viruses,the viral genome was transfected and infected with insect cells,and the infectivity of the recombinant virus was analyzed.It was found that the deletion of ac24 gene only had a small impact on the proliferation rate of baculovirus particles,and did not affect the production and infectivity of virus particles.In addition,by constructing the deletion virus of PK-1 fusion eGFP expression frame and using the non deletion virus as the control,and observing after transfection and infecting the cells,it was found that the deletion of ac24 would reduce the expression of PK-1.In conclusion,ac24 is a baculovirus non-essential gene,and its function is to enhance the expression of PK-1 in the host,but the specific mechanism needs further study.