Transcriptional regulation of cell line-dependent, baculovirus-mediated expression of foreign genes

Abstract

Cell line-dependent expression of foreign genes in the baculovirus system was investigated using a recombinant vAcβhCG-luc virus carrying two reporter genes-β subunit of human chorionic gonadotropin (βhCG) and luciferase (luc)-placed under the transcriptional control of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin gene promoters. Five different lepidopteran cell lines derived from Spodoptera frugiperda (Sf21 and Sf9), Bombyx mori (BmN and Bm5), and Trichoplusia ni (TN368) were used as host cells. TN368 expressed both βhCG and LUC to maximum levels, followed by BmN, Sf21, and Sf9 in descending order. Bm5 did not show any evidence of synthesis of the two proteins. Dot blot analysis of DNA from the vAcβhCG-luc-infected cells revealed that the level of entry of viral DNA was the same for all the five cell lines. After the completion of viral DNA replication (18 hr post infection), the level of viral DNA was the same for all the cell lines except for Bm5 where viral DNA replication did not take place and the residual virus was cleared from the cells. Analysis of RNA from the four expressing cell lines revealed a direct correlation between protein levels and levels of mRNA, suggesting transcriptional control. Differences in mRNA stability between cell lines was also evident. Gel retardation analysis of a host factor binding to transcriptionally important sequence motifs within the AcNPV polyhedrin gene promoter revealed an inverse correlation between the levels of this polyhedrin promoter-binding protein (PPBP) and reporter gene expression. Cold competition and mutation analyses of the DNA-protein complexes indicated that PPBP present in different cell lines recognized the same DNA sequence motifs present within the polyhedrin promoter.