Abstract
The Autographa californica multinucleocapsid polyhedrosis virus homologous region sequence hr1 enhances transcription from the viral polyhedrin promoter in Spodoptera frugiperda insect cells and independently functions as an origin of replication (ori) sequence. The binding of the host nuclear protein, hr1-binding protein (hr1-BP), is crucial for the enhancer activity (Habib, S., Pandey, S., Chatterji, U., Burma, S., Ahmad, R., Jain, A., and Hasnain, S. E. (1996) DNA Cell Biol. 15, 737-747 and Habib, S., and Hasnain, S. E. (1996) J. Biol. Chem. 271, 28250-28258). We demonstrate that hr1 can also enhance transcription from non-baculoviral promoters like cytomegalovirus and hsp70 in mammalian cells but does not support ori activity in these cells. Unlike insect cells, hr1 can also function in mammalian cells as an enhancer when present in trans. hr1 DNA sequence binds with high affinity and specificity to nuclear factors in the mammalian cells. The insect hr1-BP- and the hr1-BP-like proteins from mammalian cells (mhr1-BP) have different properties with respect to ion requirements, DNA groove binding, and molecular size. When mammalian cells are infected with a recombinant baculovirus containing two promoters, the baculovirus polyhedrin and Drosophila hsp70 gene promoter, the hsp70 gene promoter alone is active in these cells, and this activity is further enhanced by the presence of an additional hr1 in the recombinant virus. hr1 may thus also have a role in baculovirus-mediated gene delivery in mammalian cells.
Highlights
Introduction of anAdditional hr1 in the Recombinant Virus Increases the Expression from the hsp70 Promoter in Mammalian Cells—Having shown that the hr1 sequence element can enhance transcription from hsp70 promoter in transient transfections in mammalian cells, we checked for hr1-mediated enhancement when it is present as an additional copy in the virus
We describe the ability of the baculovirus hr1 to function as an enhancer, both when placed in cis as well as trans, but not as an ori sequence in heterologous mammalian cells
Spodoptera frugiperda insect cells (Sf9) are the natural hosts for Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV), which follows a temporal profile of gene expression and has been used to express heterologous genes. hr1 can act to enhance the expression of genes during all the different phases of infection; the immediate early gene ie-N, the delayed early 39k gene [7, 15, 16], the late gene cor and very late polh [8]
Summary
Cells and Virus—Spodoptera frugiperda cells (Sf9) were grown in TNMFH medium (Invitrogen) containing 10% fetal bovine serum as described previously [11]. To ascertain that equal amounts of viral DNA from the different recombinants had entered the insect cells, equal amounts of the reaction mixtures were dot-blotted onto a nylon membrane after the luciferase assay, followed by probing with the luc cDNA and densitometric scanning [8]. After 6 h, 1 ml of 20% fetal bovine serum-containing media was added to the cells followed by infection with either wild type or recombinant viruses or transfected with transfer vector plasmid DNAs. Aliquots of infected and/or transfected cells were harvested at different time points, and luciferase or -galactosidase enzyme expression levels were monitored as described [8, 11]. DpnI Sensitivity Assay—Plasmid DNA (2 g) was transfected into insect and mammalian cells, followed by infection with wild type AcMNPV virus after 24 h or no infection at all as mentioned in the respective figure legends. The digested DNA was Southern-transferred and hybridized to 32P-labeled pUC18 probe
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