Determining the effect of LEF-12 on late viral gene expression

Abstract

There are many advantages of using the baculovirus expression system, including: high expression levels, post-translational modifications, and the fact that the resulting proteins are correctly folded and biologically active. Late viral expression factors (LEFs) are required for transcription from late viral promoters, such as the polyhedrin promoter, under which the desired protein is overexpressed. For this reason, a better understanding of the LEFs is advantageous to better understand and improve upon the baculovirus expression system. The most recently identified LEF, LEF-12, was found to be necessary for transient late gene expression but its function has yet to be determined (5). We over-expressed and purified the LEF-12 protein and subjected it to DNA binding and transcription assays. Through these assays we determined that LEF-12 does not bind directly to the polyhedrin promoter and increases late viral transcription to a point. We also discovered significant sequence homology between LEF-12 and a subunit of eukaryotic RNA polymerase II. Although our next objective was to determine whether mutations in the conserved motifs abolished LEF-12 function, unforeseen difficulties have prevented this analysis up to this point. A recombinant virus in which the lef-12 gene has been interrupted by the mbol b - galactosidase gene has also been constructed. We are performing other experiments to better understand the function of LEF-12 in baculovirus late viral transcription which have not been completed at this time.