Functional Studies On Bombyx Mori Nucleopolyhedrovirus Late Expression Factor LEF-9

The BmNPV lef-9and lef-4, lef-8and p47encoded protein subunits of a complexes canstart polh promoter transcription in vitro. But the role of lef-9in viral replication, assemble andgene transcription are still not clear. In order to study the biological function of lef-9, weconstructed lef-9deficient virus（lef-9-KO-Bacmid）using the Red recombination system, andlef-9repaired viru（slef-9-Re-Bacmid）using the Bac-to-Bac system. After transfecting BmN cellswith wtBacmid, lef-9-KO-Bacmid and lef-9-Re-Bacmid DNA, we found that the lef-9deletionvirus does not generate infectious virus particles, while repaired virus can restore infectivity,indicating that lef-9is critical for the formation of infectious budded virus. In order to furtherstudy the effect of lef-9deletion on BmNPV gene replication and transcription,4genes wereselected to investigate the role of lef-9in viral replication and transcription. Deletion of lef-9didnot reduced the replication level of the viral genome（P>0.05） and lef-9deletion significantlyreduced the transcriptional level of early genes ie-1and lef-3, detected by qPCR（P<0.05）,moreover, the transcription level of late gene vp39and very late gene p10were reduced also（P<0.05）. Furthermore, the expression level of early gene, late gene and very late gene in theabsence of lef-9were also examined. Western blotting results also showed that the expressionlevel of LEF-3, VP39and P10were significantly lower than the wild type and repairedrecombinant viru（sP<0.05）. Electron microscopy was used to observe the BmN cells infected bylef-9-KO-Bacmid, lef-9-Re-Bacmid and wtBacmid. The results showed that there were no BVparticles in lef-9-KO-Bacmid infected cells; however, there were a large number of mature virusparticles in lef-9-Re-Bacmid and wtBacmid infected cells, indicating. These results indicated thatBmNPV lef-9is not essential for virus DNA replication, but it is very important to the virus genetranscription and expression. Electron microscopy results also confirmed the above results.