Studies On The Regulation Mechanism Of LEF3 Acetylation In The Process Of Bombyx Mori Nucleopolyhedrovirus Infection

Bombyx mori Nucleopolyhedrosis virus(BmNPV),an enveloped virus with a circular double-stranded DNA genome,is one of the most global diseases in the breeding industry of silkworm and its infection mechanism has always been a hot topic in the field of insect virology.Late expression factor 3(LEF-3),a single-stranded DNA binding protein of BmNPV,plays an important regulatory role during virus infection.However,the possible regulatary role of LEF3 acetylation modification has not been reported.In our previous acetylated profiling upon BmNPV infection,we found that not only a large number of host proteins were undergone acetylation changes,but also many viral proteins were also changed during the interaction between BmNPV and BmN cells.Particularly,the acetylation changes of LEF3 protein is the most significant one among viral proteins.The analysis results showed that during BmNPV infection,the acetylation level of total 4 Lysine sites(K18、K27、K325 and K326)within LEF3 protein undergoes changes at 36 h.p.i.Among the modification sites,two of them(K18 and K27)are located in the N-terminus conserved nuclear localization sequence region,and the acetylation level of K18 is up-regulated approximately 7.4 times.In order to further understand the effect of LEF3 acetylation on its function and potential regulatory role during virus-host interaction,LEF3 protein was selected as the research object in this study and lef3 knockout,repaired and lef3 Lysine site-specific mutant virus were constructed,respectively by site-directed mutation,Red recombination system and Bac-to-Bac recombination technologies.The fluorescence analysis results showed that the proliferation capability of the virus was significantly reduced after LEF3 acetylation.Furthermore,the quantitative q PCR analysis results demonstrated that the replication level of the viral DNA was significantly reduced by LEF3 acetylation.The K18 is the crucial acetylation site of LEF3,which might affect the function of acetylation on other three sites.The results of laser confocal microscopy analysis indicated that the acetylation at K18 caused LEF-3 to lose its nuclear targeting ability and affected the interaction between LEF3 and P143.In addition,the analysis results of the effect of LEF3 acetylation on the defense system of BmN cells showed that the transcriptional level of toll-like receptor 4(TLR-4)and Spatzle-1 were significantly decreased when the K18 of LEF-3 was acetylated,indicating that the acetylation might reduce the pathogenicity of BmNPV,thereby lowering its capacity to activate the Toll-like receptor signaling pathway of the host cells.In conclusion,the acetylation of BmNPV LEF3,especially the acetylation at K18 site,affects the nuclear localization of LEF3 and the interaction between LEF3 and P143,which results in the reduction of viral DNA replication level.The acetylation of LEF3 might be one of the strategies of silkworm to against virus infection.This study would lay the foundation for further research on the interaction mechanism between BmNPV and silkworm host,and may also provide new ideas for antiviral breeding of silkworm.