The Effects Of Knocking Out AcMNPV RNA Polymerase Genes On Viral Replication And Late Gene Expression

Baculoviruses are a family of enveloped viruses with double-stranded DNA genome. The gene expression of baculoviruse is regulated in time course. It could be divided into early, late and very late stages. Transient assays show that the expression of early genes relys on host RNA polymerase; while, the late genes expression relys on virus-induced RNA polymerase. The viral RNA polymerase consists of four subunits, LEF4, LEF8, LEF9and P47. In this paper, we study the effects of knockout of the AcMNPV RNA polymerase subunit genes on viral replication and late gene expression.I. The Ief4, lef8, lef9and p47genes were separately knocked out from the AcMNPV genome, using λ-Red homologous recombination system. By using a Bac-to-Bac system, a copy of the enhanced green fluorescent protein gene was inserted into the individual gene knockout mutants and the wild type bacmid, constructing reporter bacmids.Ⅱ. The Ief4、Ief8、lef9、p47gene deletion mutants and wild type bacmid which contained early expression reporter gene were separately transfected into Sf9cells.Then, we used Fluorescence microscope to observe the expression of fluorescent protein at different time points. The result showed that fluorescence could be seen in the Sf9cells transfected with the individual recombinant or wild type bacmids. Flurescence-containing cells were also observed in the culture infected with the supernatant from the transfection with the wild type bacmid but not observed in the cultures inoculated with the supernatants from the transfections with the individual gene knockout mutants. These phenomena showed that lef4、lef8、lef9and p47genes are essential for virus replication. The absence of lef4、lef8、lef9or p47gene resulted in that BV could not propagate.Ⅲ. The lef4、lef8、lef9、p47deletion mutants and wild type bacmid containing egfp under control of a late promoter were separately transfected to Sf9cells to test expression of EGFP in the transfected cells. It was found that a large number of fluorescent cells appeared in the culture of the Sf9cells transfected with the wild type bacmid, and a few fluorescent cells were also seen in the cultures transfected with the individual deletion mutants.In addition, we used lef4、lef8、lef9or p47deletion mutants containing the reporter gene luc under control of a late promoter to transfect Sf9cells, respectively. Then we quantitatively detected the luciferase activity, and found that the luciferase expression levels of lef4、lef8、lef9and p47deletion mutants were fell by99.79%.99.89%、99.87% and99.97%, in comparison with wild type bacmid. These results showed that the absence of lef4、lef8、left and p47genes resulted in dramatical reduction in late gene expression, but low levels of late expression still remained.Ⅳ. We used the DNA polymerase inhibitor aphidicolin and the RNA polymerase inhibitor a-amanitin to treat the Sf9cells which were transfected with lef4、Ief8、lef9and p47deletion mutants, then we quantitatively detected the luciferase activity. The result showed that after treating with the aphidicolin, the luciferase expression level of lef4、 lef8、lef9and p47deletion mutants decreased from98.64%to99.96%, comparing with wild type bacmid. However, there was no significant effect when cells were treated with a-amanitin.