Trans-Activation of a Cell Housekeeping Gene Promoter by the IE1 Gene Product of Baculoviruses

Abstract

Protein IE1 is the product of a baculovirus gene, ie1, that is activated immediately upon entrance of the viral genome into the cell nucleus. This protein was previously shown to be a trans-regulator of viral genes whose products are required for initiation of the infectious cycle including viral DNA replication. To test whether the IE1 protein is also capable of trans-regulating nuclear genes of the host in vitro and in vivo, we transfected the ie1 gene of Bombyx mori nuclear polyhedrosis virus (BmNPV) into silkworm Bm5 tissue culture cells together with expression cassettes directing expression of chloramphenicol acetyl transferase or juvenile hormone esterase under the control of the cytoplasmic actin A3 gene promoter of B. mori. Cotransfection with the ie1 gene resulted in a dramatic increase in the amount of the two enzymes expressed in the transfected cells. The increased enzyme activities correlate with an increased accumulation of the corresponding mRNAs, and the latter is caused by an increase in the rate of transcription directed by the cytoplasmic actin gene promoter. The chromosomal cytoplasmic actin gene of Bm5 cells is also upregulated upon transfection of the cells with the ie1 gene. However, infection of cells with BmNPV does not cause an increase in the level of expression of the endogenous cytoplasmic actin gene. Thus, the effect of IE1 on the transcriptional properties of the cytoplasmic actin gene vary depending on whether IE1 is expressed in isolation or in the context of a viral infection. The trans-activating effects of BmNPV ie1 gene expression on the silkmoth actin promoter are also evident in Spodoptera frugiperda Sf21 and Choristoneura fumiferana Cf1 tissue culture cells. Finally, the ie1 gene of Autographa californica nuclear polyhedrosis virus can substitute for its BmNPV counterpart in all cell lines tested.

A Baculovirus (Bombyx mori Nuclear Polyhedrosis Virus) Repeat Element Functions as a Powerful Constitutive Enhancer in Transfected Insect Cells

“…Furthermore, we also reported previously that a baculovirus transcriptional activator, BmIE1, that is encoded by an immediate early gene of BmNPV, functions as co-activator of the cytoplasmic actin gene promoter in transfected cells and increases the level of transcription from this promoter by two orders of magnitude (20).…”

“…2A). Plasmids pIE1/133 and pIE1/153, which contain the HR3 enhancer and the ie1 gene of BmNPV (20), were made by inserting a 3.8-kb ClaI fragment containing the ie1 gene into the ClaI site of plasmids p133 and p153, respectively, removing unwanted restriction sites in the polylinker of these plasmids by double digestion with SacII and BamHI, blunt-ending with T4 DNA polymerase and self-ligating the resultant plasmids. The double-enhanced expression cassettes pIE1/133A and pIE1/153A were generated by inserting a 2.2-kb SacI fragment containing the basic actin expression cassette from plasmid pBmA (17) into the unique SacI sites of plasmids pIE1/133 and pIE1/153, respectively.…”

“…The recombinant expression vectors pIE1/133A.cat and pIE1/153A.cat were generated by inserting a 0.9-kb BamHI fragment, obtained from plasmid pBmA.cat and containing the ORF for CAT, into the unique BamHI sites of the expression cassettes pIE1/133A and pIE1/133A, respectively. The construction of plasmid pBmA.jhe(kk) containing the ORF of a modified version of the juvenile hormone esterase (JHE) cDNA of Heliothis virescens (23) under the control of the actin gene promoter was previously reported (20). Finally, the double-enhanced expression vector pIE1/153A.jhe(kk) was generated by first isolating a 1.8-kb EcoRI fragment containing the ORF for JHE (kk) from plasmid pAcUW21-KK (23), ligating NotI linker to its ends, digesting with NotI, and inserting it into the unique NotI site of the double-enhanced expression cassette pIE1/153A.…”

The genes encoding the P39 and CG30 proteins of Bombyx mori nuclear polyhedrosis virus

“…At the end of the incubations, the DNA templates were hydrolysed with RNasefree DNaseI (Boehringer Mannheim), and the riboprobes were purified by Sephadex G50-50 spin column filtration. Total nucleic acid was isolated from transfected Bin5 cells, resolved on denaturing agarose gels and hybridized to the labelled riboprobes as previously described (Fotaki & Iatrou, 1988;Skeiky & Iatrou, 1991;Lu et al, 1996).…”

“…Because cg30 is a delayed early gene whose expression may be dependent on the presence of one or more immediate early gene products of the virus, we tested the effect of expression of the immediate early gene iel of BmNPV on the activity of the putative cg30 gene promoter sequences by co-transfecting Bin5 cells with pCG30.cat and plasmid pBmIE1 that contained the iel gene of BmNPV (the latter can be expressed in transfected Bm5 cells in the absence of other viral products ;Lu et al, 1996). Strong CAT activity was observed in the cotransfected cells (Fig.…”