Temporal And Spatial Expression Of AcMNPV Lef-10in Sf9Cells And Identification Of Its Functional Domain

Baculovirus mainly infect a large number of invertebrates, with most hosts fromLepidoptera. The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is themost intensively studied type species, its genome is about134Kb, and encodes154ORFs.Viral DNA replication is essential for late gene transcription. Late genes expression dependson early genes and their products, and transcript by viral RNA polymerase. In addition, someprotein factors such as late expression factor (lef) are needed for regulating expression. Thereare19AcMNPV genes have been identified as late expression factor. The function of lef-10has not been identified. This research provides a fundermental basis for further studies oflef-10function.The expression vector of HaNPV lef-10was constructed. The HaNPV LEF-10complexwas detected by western-blot, indicating that the complex may be required for itsphysiological function. Due to the low titer antibody, we constructedpTriEx-innateP-lef10-GFP plasmid and cotransfected Sf9cells with bacmid to generaterecombinant baculovirus to infect Sf9cells at various time intervals. Then the cells wereobserved through laser scanning confocal fluorescence microscope to identify expression andcellular localization of LEF-10under the control of natural promoter in Sf9cells by detectingGFP. We identified that lef-10drives expression in the early phase after infection;Subsequently, LEF-10moved to nucleus gradually in the late and very late phase. Theseresults also indicate that LEF-10play a role as a late expression factor in infected cells, andregulate other late genes transcription. To identify the nuclear localization sequence of lef-10,we constructed ten plasmids containing various truncated lef-10gene respectively to analyseexpression and localization of LEF-10in Sf9cells. It is amazing that no LEF-10localized ininfected cells nuclear. In addition，previous studies showed that LEF-10forms complex in Sf9cells, so we infer that LEF-10may transfer into nuclear by interacting with other proteins. Todefine its positioning mechanism, further experiments are needed to reveal and verify ourspeculation.In summary, this experiment studies the universality and physiological function offorming LEF-10complex, the expression and cellular localization of LEF-10under the control of natural promoter and the potential nuclear localization sequence of lef-10ininfected Sf9cells. This has laid the foundation for further studies on the biological function oflef-10.