Research On The Function Of LEF-10 And Prion Status Release

Autographa californica Multiple Nucleopolyhedrovirus?Ac MNPV?is the model species of baculovirus.The late expression factor LEF-10 is necessary for virus replication,but its function and regulatory mechanism are not yet clear.Previous studies have shown that LEF-10 protein has prion properties and is a prion encoded by baculovirus.When the amino acid residue at position 21 of LEF-10 was mutated from a leucine residue to an alanine residue,it significantly affected its prion status and upregulated the expression of late viral genes.Previous yeast two-hybrid experiments confirmed the interaction between LEF-10 and LEF-6,LEF-12 and P47.This study is divided into two parts.The first part uses fluorescence microscopy and flow cytometry to detect the effect of a series of LEF-10 mutants on LEF-10function and prion status;the second part uses AC-Seq,Pulldown and Bi FC Technology to explore the function of LEF-10.In the first part,we first constructed the lef-10^L21X Bacmid,a recombinant virus of the ectopic insertion of mutant LEF-10,in which the amino acid residue 21 of the LEF-10 protein was mutated to 19 other amino acid residues.After the recombinant virus is transfected into insect cells,only wild type,L21I,L21V,L21F,L21C,L21M,L21A can produce infectious progeny virus particles.At the same time,in this study,a recombinant virus Bacmid lef-10^L21Xwith in situ mutations of LEF-10 protein was constructed.It was also observed that only recombinant viruses wild type,L21I,L21V,L21F,L21C,L21M,L21A can produce infectious progeny virus particles.The results of ectopic insertion of are consistent with the results of in situ mutations,suggesting that the 21st position of LEF-10 is important for the structure and function of LEF-10,and only six amino acid residues?isoleucine,valine,phenylalanine,cysteine,methionine,alanine?can functionally replace leucine residues.Analysis of the effects of the above seven in situ mutant recombinant viruses on late gene expression by flow cytometry found that when Sf9 cells were infected with high MOI,only four recombinant viruses?L21A,L21F,L21C,L21I?maintained higher levels of late gene expression.Subsequently,in this study,a recombinant virus Bacmid p?Chi A?lef-10^WT/L21A overexpressing LEF-10 on bacmid was constructed.Detection of late gene expression revealed that Bacmid p?Chi A?lef-10^L21A recombinant virus can better express late genes under high MOI infection,which is consistent with the previous report of this research group.In the second part,this study constructed the recombinant virus Bacmid lef-10-His.Although the specific DNA sequence bound by LEF-10 could not be accurately located through the AC-Seq experiment.The sequencing results in the two repeated experiments were consistent and suggested that there may be certain Biological significance.Subsequently,in this study,LEF-6,LEF-12 and P47 were fused to express GST as bait protein for Pulldown experiment.The results showed that the above three GST fusion proteins did not interact with LEF-10-His.However,Bi FC results show that there is an interaction between LEF-10 and LEF-6,LEF-12 and P47.The results of the Bi FC experiment can be confirmed with the results of the yeast two-hybrid experiment,suggesting that there may be potential interactions between LEF-10 and LEF-6,LEF-12 and P47.In summary,this study proves that the highly conserved amino acid residue 21 of LEF-10is important for LEF-10 function,and the replacement of hydrophobic amino acid residues at this site can better maintain the function of LEF-10.The LEF-10^L21A mutant can relieve the prion state of LEF-10 to a certain extent under high MOI infection and up-regulate the expression of late baculovirus genes.This study laid the foundation for the functional study of LEF-10 and provided new ideas for further optimization of the Baculovirus Expression Vector.