Polh Promoter Research Articles

Novel Sp Family-like Transcription Factors Are Present in Adult Insect Cells and Are Involved in Transcription from the Polyhedrin Gene Initiator Promoter

We earlier documented the involvement of a cellular factor, polyhedrin (polh) promoter-binding protein, in transcription from the Autographa californica nuclear polyhedrosis virus polh gene promoter. Sequences upstream of the polh promoter were found to influence polh promoter-driven transcription. Analysis of one such region, which could partially compensate for the mutated polh promoter and also activate transcription from the wild-type promoter, revealed a sequence (AcSp) containing a CACCC motif and a loose GC box resembling the binding motifs of the transcription factor Sp1. AcSp and the consensus Sp1 sequence (cSp) specifically bound factor(s) in HeLa and Spodoptera frugiperda (Sf9) insect cell nuclear extracts to generate identical binding patterns, indicating the similar nature of the factor(s) interacting with these sequences. The AcSp and cSp oligonucleotides enhanced in vivo expression of a polh promoter-driven luciferase gene. In vivo mopping of these factor(s) significantly reduced transcription from the polh promoter. Recombinant viruses carrying deletions in the upstream AcSp sequence confirmed the requirement of these factor(s) in polh promoter-driven transcription in the viral context. We demonstrate for the first time DNA-protein interactions involving novel members of the Sp family of proteins in adult insect cells and their involvement in transcription from the polh promoter.

33] Expression of green fluorescent protein using baculovirus vectors

The expression of heterologous genes in insect cells using baculovirus vectors is commonly used to achieve a high level of recombinant protein production. The expression system exploits the promoters from a number of hyperexpressed virus genes that are activated in the very late stages of the Autographa californica nucleopolyhedrovirus (AcMNPV) replication cycle. The most widely used of these are the polyhedrin gene (polh) and p10 (p10) gene promoters. Replacement of either the polh or p10 coding region with that of a foreign gene results in the formation of a recombinant virus with the foreign gene under control of the hyperexpressed promoter. Recombinant viruses may be propagated in insect cells cultured in vitro, for example, Spodoptera frugiperda or Trichoplusia ni cells, or may be used to infect insect larvae. In the latter case, recombinant viruses are usually prepared in which the polh and p10 remain intact, so that larvae may be infected by the natural route of feeding upon a polyhedra-containing diet. In this case, the foreign gene is expressed under control of a copy of the polh or p10 promoter, located in a nonessential region of the virus genome, for example, just upstream of the polh promoter.

Expression and Mutational Analysis of the Baculovirus Very Late Factor 1 (vlf-1) Gene

We have examined the expression and function of a gene,vlf-1,ofAutographa californicanuclear polyhedrosis virus that is known to encode a regulator of very late gene transcription. Western blot analysis revealed thatvlf-1is expressed during the late phase of infection, primarily from 15 to 24 h postinfection. VLF-1 localized in the cell nucleus and was also present in the nucleocapsids of virus particles. Mapping ofvlf-1mRNA by primer extension showed that transcription initiates at a TAAG motif 71 bp upstream of thevlf-1open reading frame. Disruption of this TAAG motif abolished the ability ofvlf-1to stimulate transcription from the very late polyhedrin gene (polh) promoter in transient expression assays, suggesting thatvlf-1expression is controlled by the TAAG motif. Using a highly efficient system to construct recombinant viruses with modifications invlf-1,we confirmed that the TAAG motif was essential. Furthermore, efforts to construct null mutants ofvlf-1failed, suggesting thatvlf-1is an essential gene for virus replication. Computer-assisted sequence homology searches placevlf-1in the λ phage integrase family (McLachlin and Miller, 1994). None of the strictly conserved residues of this family which are found invlf-1could be changed in the viral genome, implying that the putative integrase activity of VLF-1 is associated with the essential function ofvlf-1.However, mutation of a crucial active-site tyrosine did not affect the ability ofvlf-1to transactivate thepolhpromoter in transient expression assays, indicating that the very late transcriptional activity of VLF-1 does not require the integrase activity.

Baculovirus-mediated expression of cytochrome P4502C8 and human NADPH-cytochrome P450 reductase : optimization of protein expression

1. High expression levels of cytochrome P450 (CYP) 2C8 and NADPH-cytochrome P450 oxidoreductase (OxR) in Spodoptera frugiperda (Sf21) cells have been achieved using the baculovirus expression system. 2. The baculovirus dual expression plasmid, pAcUW31, was used to insert CYP2C8 and OxR cDNAs downstream of the polyhedrin (polh) or p10 promoters, either separately or together, generating four recombinant baculoviruses; two expressing single proteins (CYP2C8 driven by the p10 promoter, bVp10.2C8 or OxR driven by the polh promoter, bVpolh.OxR) with another two coexpressing both CYP2C8 and OxR under reciprocal control of the polhand p10 promoters (bVpolh.OxR-p10.2C8 and bVpolh.2C8-p10.OxR). 3. High levels of singly expressed CYP2C8 and OxR were achieved from bVp10.2C8 and bVpolh.OxR, with levels of 0·7-1·2 nmol CYP/mg protein and 400-500 nmol cytochrome c reduced/min/mg protein respectively. 4. The two dual gene clones (bVpolh.OxR-p10.2C8 and bVpolh.2C8-p10.OxR) showed, in general, greater variation in CYP content and OxR activity than single gene clones. Screening was therefore necessary for the selection of dual gene clones expressing both proteins optimally. 5. Sf21 microsomes infected by selected dual gene clones were, on average, 14 times more active intolbutamide hydroxylase activity than those expressing CYP2C8 alone, with a mean spectral CYP content of 79 pmol/mg cell lysate protein and a mean OxR level of 600 nmol/min/mg cell lysate protein.

Expression of lacZ reporter gene under the control of the polyhedrin promoter of Spodoptera litura nuclear polyhedrosis virus

Promoter function of the putative polyhedrin-encoding gene ( polh) of Spodoptera litura nuclear polyhedrosis virus (S1MNPV) was determined by transferring it to the Autographa californica nuclear polyhedrosis virus (AcMNPV) through the AcNPV polh based vector, pVL1393. Three transfer vectors pCBT2, pCBT3 and pCBT4 were constructed by substituting the promoter and the neighbouring sequences of AcNPV in pVL1393 by that of SINPV. The Escherichia coli lacZ gene was placed downstream from the S1NPV polh promoter in the hybrid transfer vector (pCBT) constructs. Co-transfection of Spodoptera frugiperda cells (Sf9) with each of the pCBTlacZ vector and wild-type AcNPV DNAs led to synthesis of β-galactosidase (βGal). The plaque-purified recombinant viruses (S1AcNPV.lacZ) expressing lacZ under the polh promoter of S1NPV are stable. The highest βGal activity was obtained with S1AcNPV4.lacZ. Production of βGal with recombinant virus, S1AcNPV3.lacZ in which S1NPV polh promoter is in the reverse orientation in the AcNPV genome, is 83% of that produced by S1AcNPV4.lacZ. These results indicate that the S1NPV polh promoter is active in the genetic environment of AcNPV; the polh of S1NPV is phylogenetically related to AcNPV like other baculoviruses.

Heterologous promoter recognition leading to high-level expression of cloned foreign genes in Bombyx mori cell lines and larvae

The baculovirus expression system using the Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for high-level expression of cloned foreign genes, driven by the strong viral promoters of polyhedrin (polh) and p10 encoding genes. A parallel system using Bombyx mori nuclear polyhedrosis virus (BmNPV) is much less exploited because the choice and variety of BmNPV-based transfer vectors are limited. Using a transient expression assay, we have demonstrated here that the heterologous promoters of the very late genes polh and p10 from AcNPV function as efficiently in BmN cells as the BmNPV promoters. The location of the cloned foreign gene with respect to the promoter sequences was critical for achieving the highest levels of expression, following the order + 35 > + 1 > − 3 > − 8 nucleotides (nt) with respect to the polh or p10 start codons. We have successfully generated recombinant BmNPV harboring AcNPV promoters by homeologous recombination between AcNPV-based transfer vectors and BmNPV genomic DNA. Infection of BmN cell lines with recombinant BmNPV showed a temporal expression pattern, reaching very high levels in 60–72 h post infection. The recombinant BmNPV harboring the firefly luciferase-encoding gene under the control of AcNPV polh or p10 promoters, on infection of the silkworm larvae led to the synthesis of large quantities of luciferase. Such larvae emanated significant luminiscence instantaneously on administration of the substrate luciferin resulting in ‘glowing silkworms’. The virus-infected larvae continued to glow for several hours and revealed the most abundant distribution of virus in the fat bodies. In larval expression also, the highest levels were achieved when the reporter gene was located at +35 nt of the polh.

Three baculovirus genes involved in late and very late gene expression: ie-1, ie-n, and lef-2.

We developed a method to identify baculovirus genes required for late and very late gene expression that is based on subtraction of clones from an Autographa californica nuclear polyhedrosis virus genomic library which is able to trans activate promoters of reporter plasmids in transient expression assays. Using this assay, we found that three genes located between 83.7 and 7.5 map units of the Autographa californica nuclear polyhedrosis virus genome were involved in expression from the late capsid protein (vp39) and very late polyhedrin (polh) gene promoters. Two of these genes, ie-1 and ie-n, trans regulate early genes in transient expression assays. Although ie-1 was necessary and sufficient for expression from the early promoter in our assay, it was necessary but not sufficient for expression from the vp39 and polh promoters. The presence of ie-n increased expression from the early, late, and very late classes of promoters tested. However, a third gene identified in this region was specifically required for expression from the vp39 and polh promoters. This gene, a previously sequenced 630-nucleotide open reading frame, was renamed lef-2 for late expression factor 2. We also found that other genes in the region between 83.7 and 7.5 map units were not required for expression from the promoters used in this assay, although we did not eliminate the possibility that they subtly modify expression. These genes include pe-38 and me53, early genes with zinc finger-like motifs, and the upstream exon of ie-0, which specifies an alternate form of IE-1.

Transient expression assay in a baculovirus system using firefly luciferase gene as a reporter.

Transient gene expression assays were developed to assess the function of the regulatory sequences of baculoviruses Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa californica nuclear polyhedrosis virus (AcNPV) in insect cells of Bombyx mori and Spodoptera frugiperda, respectively. DNA sequences encoding luciferase (luc) of the firefly Photinus pyralis was successfully employed in the expression assay as a reporter gene. Recombinant plasmids were constructed containing the luc gene under control of baculovirus-specific or heterologous promoters. Cotransfection of Bombyx mori and Spodoptera frugiperda cells with recombinant plasmids carrying virus-specific promoter sequences and BmNPV and AcNPV DNA, respectively, gave rise to efficient synthesis of luciferase (Luc), while heterologous promoters induced a low level of luc expression. We found that flanking sequences of the AcNPV DNA in the transfer plasmid contained an unknown promoter conferring an efficient luc expression. The activity of this promoter was modulated by the polh promoter sequences. The assay allows one to conduct highly sensitive monitoring of the transient expression of foreign genes from the transfecting plasmids prior to construction of recombinant viruses.

Differential gene expression mediated by late, very late and hybrid baculovirus promoters

A series of recombinant viruses was constructed to study the regulation of expression from the promoter (P cap ) for the major capsid-protein-encoding gene ( vp39) of the baculovirus, Autographa californica nuclear polyhedrosis virus. Each virus of this series contains the cat reporter gene under the control of P cap or portions thereof. P cap regulation of cat was determined at the RNA and enzyme activity levels, and was compared to cat gene expression driven by P polh , the promoter of the polyhedrin gene ( polh). Although optimum expression was achieved with a 464-bp region, which included all three P cap transcription start points ( tsp), a 99-bp segment of P cap containing a single tsp was sufficient to direct late cat expression. P polh and P cap - mediated regulation differed temporally; P cap -cat constructs were expressed at 12 h post-infection (p.i.) and continued through 48 h p.i., whereas P polh - cat were not expressed at 12 h, but were initiated around 18 h p.i. and underwent a burst of expression between 24 and 48 h p.i. A recombinant virus carrying a hybrid vp39 and polh promoter (P cappolh ) was also constructed and studied; P cappolh contained the two distal tsp of P cap and a proximal tsp of P polh P cappolh exhibited both ‘late’ and ‘very late’ regulation; the distal tsp were regulated as late sites and the proximal tsp was regulated primarily as a very late site although it also showed a weak late response. Hybrid late/very late promoters should prove useful in optimizing foreign gene expression using baculovirus vectors.