Study On The Regulation Mechanism Of Late Expression Factor 6 Acetylation Modification In The Process Of Bombyx Mori Nucleopolyhedrovirus Infection

The silkworm has been an important economic insect since ancient times,and its economic value lies in the silk.Up to now,our country’s production of silkworm silk and cocoon have ranked first in the world.Because of its huge economic benefits,the silkworm industry has naturally become an important part of China Agriculture.Bombyx morinucleopolyhedrovirus(BmNPV)is a type of double-stranded circular DNA virus.It is a key viral pathogen that causes disease in the silkworm and can cause nearly 80% of the economic loss of the silkworm industry.Therefore,the research on the interaction between silkworm and BmNPV has always been a hot issue in the field of insect virology,but its infection mechanism is not yet fully understood.Late expression factor 6(LEF-6)is a non-essential factor for virus replication.Knock-out of it can delay the transcription of late viral genes and reduce the production of virus particles.However,its specific mechanism of action on the regulation of virus proliferation is unclear.Through the differential omics analysis of protein modification in the early stage of our research group,we found that after BmNPV infects silkworm cells,not only a large number of host protein-related lysine sites have undergone acetylation modification,but also a variety of viral protein-related lysine sites have also occurred.LEF-6 protein is one of the most significant viral proteins undergoing acetylation modification.The results show that LEF-6 protein has two significant acetylation modification sites: K85 and K94.In order to understand the effect of LEF-6 protein acetylation modification on its function and its regulatory mechanism during virus infection.First of all,we used the prokaryotic expression and purification system to get the LEF-6 polyclonal antibody;secondly,we used the Red recombination technology and the Bac-to-Bac system to construct lef-6 knock-out Bacmid(lef-6-KO-Bacmid)and the repaired Bacmid(lef-6-RE-Bacmid),and then we constructed the mutants Bacmid by substitute lysine(K)to glutamine(Q)to simulate acetylation modification and substitute lysine(K)to arginine(R)to simulate deacetylationmodification by using site-directed mutagenesis,we got each mutant Bacmid(lef-6-85Q-Bacmid,lef-6-85R-Bacmid,lef-6-94Q-Bacmid,lef-6-94R-Bacmid),Transfect the successfully constructed virus DNA into silkworm cells,the viral genome was extracted after 72 h and quantitatively analyzed by q PCR.The results show that the copy number of lef-6-KO-Bacmid is significantly reduced,but the virus still has the ability to replicate.The acetylation modification of the two lysine sites can significantly inhibit the replication of the viral genome,while the deacetylation modification has no significant effect;then the virus proliferation viability test and virus titer are further carried out.Analysis results show that acetylation modification can inhibit virus proliferation and reduce the production of progeny viruses;in addition,LEF-6 acetylation modification significantly inhibits the transcription of late and very late genes(vp39,p6.9,p10);finally,we found that the acetylation modification can affect the entry of LEF-6 protein into the nucleus and make it mainly locate in the cytoplasm by laser confocal microscopy.This study can provide new ideas for further exploring the mechanism of action between BmNPV and silkworm host cells.