Study On The Functional Domain Of Baculovirus VLF-1 And Screening Of Its Interactive Proteins

Vlf-1 is one of the core genes of baculovirus.It is the essential gene of virus infection and exists in all the sequenced baculovirus.The first study found that the gene-encoded protein affected the over-expression of very late genes,so it is named very late expression factor 1(VLF-1).Then it was found that VLF-1 was necessary for viral nucleocapsid formation,and the empty tube shell structure could be observed in the cells transfected with the vlf-1-deleted mutated virus,indicating that VLF-1 was involved in the assembly of viral nucleocapsid.The genomic DNA of mutated virus can be completely degraded by DNA enzyme.The deletion of some other genes of virus also produces empty-tube chitin viruses,and about 35%of the viral genome DNA is protected from degradation by DNA enzymes,which suggests that VLF-1 may be involved in regulating the DNA envelope of the virus genome.In addition,VLF-1 is a nucleocapsid structural protein located at the end of the nucleocapsid.Therefore,VLF-1 plays a key role in the final stages of DNA processing,packaging and nucleocapsid assembly.So the function of VLF-1 was further studied from two aspects.First,by means of the prediction of VLF-1 protein structure,the vlf-1 gene sequence was truncated according to the conservative domain,integrase family domain,protein binding region,DNA binding region and RNA binding region.Twelve truncated and rescue-type recombinant baculovirus plasmids were constructed in the Bac-to-Bac system and transformed into Sf9 cells.The replication of recombinant virus in Sf9 cells was observed by light microscope and fluorescence microscope,and the effect of truncated VLF-1 on virus replication was analyzed by drawing one-step growth curve of virus.Second,construction of total cDNA library of AcMNPV infected Sf9 cells by yeast two-hybrid method.The protein interacting with VLF-1 was gradually verified by binding method,and then the defective medium was used to verify the protein interacting with VLF-1 step by step.After the preliminary results were obtained,the interaction proteins were further verified by Co-IP,bimolecular fluorescence complementation techniques.Finally,through subcellular localization and other ways to analyze the location of interaction in the cell.The regulation of viral nucleocapsid assembly and very late transcription mechanism are explored.The results showed that in a series of truncated recombinant viruses,the replication and infection of truncated type 1-1044 in Sf9 cells was similar to that of wild type and rescue type,so its key functional domain might be located in this region.In the screening of interacted protein with VLF-1,19 proteins that may interact with VLF-1 were screened out.Then,the following AcMNPV encoded proteins39K(Ac36),VLF-1(Ac77),CG30(Ac88),VP39(Ac89),LEF-5(Ac99),P6.9(Ac100)and ME53(Ac139)were selected for further verification.The verification results showed that 39K(Ac36),CG30(Ac88),LEF-5(Ac99)interacted with VLF-1.The subcellular co-localization test showed that 39K and VLF-1 were co-localized in the nucleus.The subcellular localization test showed that 39K was localized in the nucleus,CG30 was localized in the nuclear membrane and LEF-5 was localized in the nucleus.Among them,39K is a protein that can bind to single-and double-stranded DNA;CG30 contains the structure of a zinc finger and leucine finger,and the titer of the virus drops sharply after the deletion of the protein;LEF-5 is a late expression factor,which contains a domain similar to RNA polymerase ? extension factor(TFIIS),its main function is to regulate late gene transcription.We speculate that VLF-1 may be involved in the processing of viral DNA by interacting with 39K,involved in the assembly of nucleocapsid through interacting with CG30,and regulate the over-expression of very late genes through interacting with LEF-5.Its specific mechanism remains to be clarified.