| Nuclear polyhedrovirus（Nucleopolyhedrovirus）is a type of double-stranded circular virus that specifically infects arthropods,most of its hosts are Lepidoptera and Hymenoptera insects.Bombyx mori nucleopolyhedrovirus（BmNPV）is a baculovirus that specifically infects silkworms.On the one hand,BmNPV is very destructive to the sericulture production,on the other hand,BmNPV-silkworm is a high-efficient bioreactor.Therefore,no matter whether it is for virus prevention or efficient protein production,it has a positive effect for in-depth study of BmNPV infection interaction model of silkworm.In recent years,more and more research results have shown that protein post-translational modification plays an extremely important regulatory role in the life activities of organisms.Recent studies on differential proteomics of proteins post-translational modification show acetylation is important in BmNPV infected host Bm N cells after 36 h,the level of various viral proteins acetylation modification has changed,among of them,the late expression factor-11（LEF-11）is one of the most significantly down-regulated proteins(the 83rdLys site is down-regulated by 0.768 times).Existing research shows that lef-11 is one of the late expression factors encoded by the BmNPV genome witch is an essential gene for viral DNA replication,and its size is 336 bp,encoding 112 amino acids.The molecular weight is about 13.1 KDa,which exists in most known baculovirus genome,and it is important functional genes regulating the virus life activities,however,the role of acetylation modification in the process of BmNPV infection has not been reported.To further study the possible regulatory mechanism in the process of virus infection on LEF-11 K83 acetylation modification（Kac）,firstly,we knock out the original lef-11 gene in the BmNPV by Red recombinant technology,then,we designed the primers containing simulated（de）acetylation mutation sites with site-directed mutation technique to obtain lef-11 with specific lysine site mutants by overlapping PCR,inserted into the transfer vector p Fastbac1;finally,we respectively repaired the missing gene（or mutant gene）constructed the recombinant virus with ectopic compensation lef-11 gene and its mutants by Bac-to-Bac technology.Transfected Bm N cells with lef-11-knockout virus,lef-11-repair type and mutants DNA to obtain progeny virus,then reinfection of silkworm cells for virus titer detection.The results showed that after lef-11 knockout,the cells could not be infected,indicating it is an essential gene for BmNPV replication;however,it has a promotion effect on the generation of progeny viruses with the acetylation modification of LEF-11 K83.Furthermore,with fluorescence quantitative PCR technology,we detected the replication of BmNPV genomic DNA,and the results further confirmed that the acetylation modification of LEF-11 K83 could promote the replication of the viral genome.In addition,the LEF-11 protein fusion with EGFP was constructed,in order to investigate the effect of acetylation modification on LEF-11 nuclear localization,the results showed that acetylation of LEF-11 K83 would affect the function of LEF-11 nuclear localization.This study will lay a foundation for further understanding the interaction mechanism of BmNPV infection host silkworm cells,and may also provide new ideas for the antivirial research of silkworm. |
