AcMNPV Lef-10Knockout And Precisely Identify Functional Domain Of Acmnpv LEF-10Carboxyl-terminal

Baculoviruses mainly infect insects and crustaceans invertebrates. The Autographacalifornica multicapsid nucleopolyhedrovirus (AcMNPV) is the most intensively studied typespecies, its genome is134kbp in size, and encodes154ORFs. According to the time of geneexpression, The ORFs can be divided into early gene and late genes and late gene expression isregulated by late expression factor (lef), lef-10is one of them in our study. Previous studies haveconfirmed that BmNPV lef-10is an essential gene, and lef-10deletion would affect replication ofthe viral DNA, late gene transcription and expression levels of the early genes. In order to studywhether AcMNPV lef-10is an essential gene, we constructed AcMNPV lef-10-KO-bacmid, andanalyzed the physiological function of the gene in insect cells which infected by the recombinantvirus. We also precisely identified functional domain of AcMNPV LEF-10carboxyl-terminal.These results provide theoretical basis for further studies of lef-10.Early confirmed the10bases deletion downstream the initiation codon ATG of AcMNPVlef-10gene was not affect the vp1054gene following lef-10gene expression. We used Red/ETrecombination technology, through the introduction of AMP resistance screening marker,successfully constructed AcMNPV lef-10gene deletion recombinant bacmid. We alsoconstructed a recombinant plasmid pTriEx-innateP-lef10driven by innate lef-10promoter. Then,Sf9cells were co-transfected with lef-10-KO-Bacmid DNA and pTriEx1.1, or lef10-KO-BacmidDNA and pTriEx-innateP-lef10or wtBacmid DNA and pTriEx1.1respectively, vAclef-10-KO(lef-10knockout vAc) could not produce cytopathic effect observing by a microscope, While theopposite the vAcwtand vAclef-10-KO-REP(lef-10revertant vAc) could produce similar cytopathiceffect. These results indicated that insect cells infected by vAclef-10-KOcould not produceinfectious viral particles, and AcMNPV lef-10is an essential gene.Considering the overlap between lef-10and vp1054, and avoiding the interference ofvp1054gene expression, When lef-10gene knockout, we introduced a single point mutation intothe AcMNPV lef-10by Red/ET recombinant system in combination with rpsL counter-selection.The method of introducting point mutations is by two Red/ET recombination: the first recombination is introducting rpsL-AMP resistance selection marker, the second recombinationis using streptomycin to remove rpsL-AMP resistance selection marker, while a point mutationwas been introducted. Subsequently, we constructed vAcwt, vAclef-10-KOand vAclef-10-KO-REP. Sf9cells were infected by these viruses, cytopathic effect was observed under a microscope. Wefound vAclef-10-KOcan affect viral replication and could not produce infectious BV, which weresimilar to the lef-10of BmNPV.To study the function domain of LEF-10, we constructed a series of truncated LEF-10plasmid, Sf9cells were co-transfected by the plasmid with the truncated LEF-10andlef-10-KO-bacmid, we found the function domain of LEF-10is the48amino acid of N-terminalby flow cytometry analysis and microscopic observation.