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

Abstract

It has been previously reported that baculovirus homologous regions, the regions of baculovirus genomes that contain the origins of DNA replication, can augment the expression of a small number of baculovirus genes in vitro. We are now reporting that a region of the genome of Bombyx mori nuclear polyhedrosis virus (Bm-NPV) containing the homologous region 3 (HR3) acts as an enhancer for the promoter of a nonviral gene, the cytoplasmic actin gene of the silkmoth B. mori. Incorporation of the HR3 sequences of BmNPV into an actin promoter-based expression cassette results in an augmentation of transgene expression in transfected cells by two orders of magnitude relative to the control recombinant expression cassette. This increase is due to a corresponding increase in the rate of transcription from the actin promoter and not to replication of the expression cassette and occurs only when the HR3 element is linked to the expression cassette in cis. A comparable degree of enhancement in the activity of the silkworm actin promoter occurs also in heterologous lepidopteran cells. Concomitant supplementation of transfected cells with the BmIE1 trans-activator, which was previously shown to be capable of functioning in vitro as a transcriptional co-activator of the cytoplasmic actin gene promoter, results in more than a 1,000-fold increase in the level of expression of recombinant proteins placed under the control of the actin gene promoter. These findings provide the foundation for the development of a nonlytic insect cell expression system for continuous high-level expression of recombinant proteins. Such a system should provide levels of expression of recombinant proteins comparable to those obtained from baculovirus expression systems and should also have the additional advantage of continuous production in a cellular environment that, in contrast to that generated by a baculovirus infection, supports continuously proper posttranslational modifications of recombinant proteins and the capability of expression of proteins from genomic as well as cDNA sequences.

An inducible system for highly efficient production of recombinant adeno-associated virus (rAAV) vectors in insect Sf9 cells

“…Earlier Habib et al (18) reported that AcMNPV hr1 enhances transcription from the polyhedrin promoter in a classic enhancer-like manner. Other BEV hrs have also been shown to display a potent enhancer function on exogenous and endogenous promoters in the absence of any viral trans-activator, suggesting that the binding of host factors might be involved in the enhancer mechanism (19). Considering the enhancing propensity of hrs to mediate transcription even without the BEV-encoded IE-1 factor, one would predict some basal level of transcription from the integrated rep genes.…”

A Virus Essential for Insect Host-Parasite Interactions Encodes Cystatins

“…The expression cassette pIE1/153A (23,45) was employed for expression of recombinant cystatin 1 in insect cells. Plasmid pIE1/153A.cyst was generated as follows.…”

Production of Japanese encephalitis virus-like particles in insect cells

“…[16][17][18] Use of the IE-1 transactivator and the HR3 enhancer with the actin promoter has resulted in a more than 1000-fold increase in the stimulation of foreign gene expression through the actin promoter. 19,27 We used plasmid vectors that contained the B. mori actin promoter downstream from the BmNPV IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression, together with either a blasticidin or a neomycin resistance gene for use as a selectable marker (Fig. 1).…”