9506233 Guarino Baculovirus late and very late genes are expressed at different times post infection. The late genes encode viral structural proteins and their expression is maximal between 24 - 36 hr post infection. Polyhedrin is an example of the very late genes which are involved in the formation of occlusion bodies in the nuclei of infected cells. Transcription of the very late genes is relatively low at 24 hr and reaches maximal levels at 48 hr, after the synthesis of late proteins and assembly of virions. This suggests that the late and very late promoters are controlled by different factors, although the mechanisms that regulate differential transcription of the two classes of genes are unknown. The goals of this project are to purify and identify the viral proteins that are directly involved in transcription of the late and very late genes. For this research, hybrid templates have been constructed that contain late or very late promoters linked to a synthetic DNA fragment that lacks cytidine residues on the transcript strand. In vitro transcription reactions performed in the absence of CIP yield strong signals resulting from accurate initiation at the correct sites. These constructs were used to monitor the purification of active transcription complexes from infected cell extracts prepared at 36 hr post infection. Phosphocellulose chromatography yielded two fractions that differentially transcribed the late and very late templates. Addition of the follow-through fraction to either of the active fractions resulted in an increase in transcription from both late and very late promoters, although the flow-through fraction had no transcription activity when tested separately. The very late transcription complex was purified on three additional columns. The purified complex contains only five proteins and transcribed the polyhedrin and 39k C-free constructs in the absence of additional factors, indicating that the complex has both promoter recognition and RNA polymerase activities. Next, the late complex will be purified in order to compare the protein compositions of the two complexes. Proteins in the two complexes will be identified using a combination of methods including V8 protease mapping, immunochemical analyses, and direct protein sequencing. The DNA binding activities of the two complexes will be compared and proteins that differ between the two complexes will be targeted for further study, including an analysis of their promoter recognition. The factors that increase expression of late and very late genes will be purified and the corresponding genes identified. %%% This research should aid in understanding the mechanisms of transcriptional regulation of baculovirus late and very late gene expression. The baculoviruses late transcription system is unique among eukaryotic viruses. In some respects, it seems very primitive. The fact that late and very late promoters are preferentially transcribed by different RNA polymerase complexes is reminiscent of bacterial RNA polymerase and sigma factors. However, transcription is increased by the addition of separable factors, a strategy of eukaryotic transcription systems. In addition, results from this research may have a practical impacts on biotechnology. The baculovirus expression system is widely used for the production of large amounts of medically important proteins for use as vaccines, diagnostic tools, or therapeutic agents. However, the baculovirus system has limitations: it is transient because the viral vectors kill the cells; and recombinant proteins are expressed late when cellular processing pathways are compromised. A knowledge of the mechanisms that control baculovirus polyhedrin expression may aid in the design of vectors that work in the absence of virus infection and thus do not kill the host or impair protein processing. ***
