Molecular Characterization Of BmNPV Bm11 Gene

Baculoviruses are a unique group of insect-specific viruses with double-stranded,circular,supercoiled genomes packaged into rod-shaped nucleocapsids.During the biphasic life cycle,baculoviruses produce two virion phenotypes,the budded virus(BY)and the occlusion-derived virus(ODV).BVs mediate cell-to-cell infection inside the individual host,whereas ODVs facilitate oral infection(primary infection)by binding specifically to host epithelial cells.Bombyx mori nucleopolyhedrovirus(BmNPV)belongs to the alphabaculovirus genus of the baculovirus family and is the main pathogen of Bombyx mori,causing serious economic losses to the silk industry.At present,the role of most BmNPV genes in their life cycle has been elucidated.However,the biological functions of some genes are still unknown.In this study,the function of bmll,a highly conserved BmNPV gene,was investigated.The recombinant virus were constructed by ?-red recombinant system and Bac-to-Bac system.The transcription,expression,localization,function and mechanisms were systematically studied.The main results are as followings:1.Bioinformatics analysis of BmNPV bmll gene and its encoded protein,bmll is located at 10,459-10,791 nt in the genome of BmNPV and the C-terminal region is more conserved.Bmll homologues have been shown to constitute DUF1477 baculovirus protein family of unknown function and have multiple transcriptional activator sites.The results of RT-PCT,qPCR and Western Blotting analysis demonstrated that bmll was a late gene.Confocal microscopy observations suggested that Bmll predominantly exhibited a ring zone distribution at the very late phase of infection.However,fractionation showed that Bmll was neither associated with BY or ODV,a phase separation assay suggested that Bmll was nor an integral membrane protein.2.The effect of bmll deletion on BmNPV infection was explored by constructing recombinant bacmid.Fluorescence microscopy,qPCR and TCID50 analysis all demonstrated that bmll was not essential for infectious BV production or viral DNA replication.However,bmll was involved in efficient OB production in vitro and in vivo.Electron microscopic analysis showed that the deletion of bmll did not affect the nucleocapsid assembly,virus-induced intranuclear microvesicle formation or the BV and ODV production,but resulted in a remarkable effect on OBs with fewer ODVs,which might thus reduce the BmNPV oral infection efficiency.3.The key sequence and amino acid site of Bmll were determined by truncation knockout and site-directed mutation.The results showed that 80-110 aa at the end of C terminal was required for OB production and ODV embedding.Furthermore,point mutation results showed that Q94 was the key amino acid to mediate the above process.4.The viral interaction protein of Bmll was identified and its nuclear import mechanism was therefore elucidated.Bmll was expressed transiently alone and distributed in the cytoplasm.When transfected with vBmWT,Bmll was distributed throughout the cell.These results suggested that the nuclear entry of Bm11 required the assistance of viral proteins.Twelve integral proteins were screened and hybridized with Bm11.The interaction between PEF4 and ODV-E66 with Bm11 was found by the yeast two hybrid system.Furthermore,the strong interaction between PIF4 and Bm11 was confirmed by co-immunoprecipitation validation and the subcellular localization of the interaction was determined by confocal microscopy.It was found that the interaction mainly occurred in the cytoplasm,nuclear membrane and ring zone of nucleus during virus infection.These results indicate that Bm11 bind indirectly to the nuclear membrane by interacting with PIF4,thus clarifying the nuclear entry mechanism5.The Bmll was determined as a transcription factor and the deep mechanism of decreased OB production was thus elucidated.In the late stage of BmNPV infection,the deletion of bmll caused down-regulation of transcription levels of the late and very late genes and polyhedrin-related genes,but had no significant effect on ODV envelope protein-coding genes.The dual luciferase reporter system was constructed and it was found that Bm11 had a positive regulatory effect on the promoters of v-cath,fp and vlf-1.Based on the results above,it can be concluded that the deletion of bmll down-regulated the activity of vlf-1 and fp promoter,affecting the transcriptional levels of polh and p10,which in turn lead to the decrease of OB production.