Effect of LEF-10 21 mutation on AcMNPV activity.
Objective: A point saturation mutation of the 21st conserved amino acid residues of the late expression factor LEF-10 of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was performed and its effect on AcMNPV activity was studied to provide foundation for further research on characteristics of LEF 10 prion and role of prion in viral replication. Methods: Based on homology sequence alignment, it was found that the leucine residue at position 21 of LEF-10 was highly conserved. The leucine residue at position 21 of LEF-10 on pTriEx plasmid vector was subjected to saturation mutation by ectopic backfill. The constructed recombinant plasmid was co-transfected with linearized BacmidΔlef-10 into Sf9 cells of Spodoptera frugiperda to construct recombinant virus, and the effect of different amino acid residues at the 21 position of LEF-10 on virus activity was studied. The LEF-10 gene of AcMNPV Bacmid was in situ mutated using the Red/ET gene knockout system and the rpsl reverse sieving system. The mutant linearized Bacmid and pTriEx-p(p10) dsRed plasmid were co-transfected into Sf9 cells of Spodoptera frugiperda to construct an in situ mutant virus, and the effect of point mutation on activity of the recombinant virus was clarified. The effect of mutant viruses on late gene expression was also detected by flow cytometry. Results: The results of transfection and infection of Sf9 cells with ectopic backfill point mutation recombinant virus and in situ mutation recombinant virus showed that the mutation of leucine residue at position 21 of LEF-10 to an isoleucine residue, alanine residues, valine residues, proline residues, cysteine residues and methionine residues would rescue LEF-10-deficient viruses and produce infectious recombinant viruses, while other mutations recombinant viruses were lethal. Seven in situ mutant recombinant viruses were used to infect Sf9 cells with high and low MOI, respectively. The results showed that expression levels of foreign proteins were equivalent with low MOI, while they were significantly different with high MOI. The recombinant virus mutated to an alanine residue maintained the highest expression level of foreign proteins. Conclusion: The amino acid residue at position 21 of LEF-10 plays a vital role in the function of LEF-10, and its properties affect AcMNPV activity.