Model systems based on viral genes are among the most useful and important systems available for studies of gene regulation in animal cells. Baculoviruses are large DNA viruses that are transcribed and replicate in the nuclei of insect cells and as such, provide an excellent model for studies of gene regulation in insect cells. After entry into the cell, some baculovirus early promoters are immediately recognized and transcribed by the host cell machinery. Studies of baculovirus early promoters will be important in our basic understanding of promoter recognition and regulation in insect cells. The baculovirus major envelope glycoprotein (gp64)gene is recognized and transcribed immediately upon entry into the cell and gp64 is believed to play an important role in the viral infection cycle. Additionally, the transcriptional and translational regulation of gp64 appear to be complex. Gp64 is transcribed from different promoters at early and late times in the infection cycle, resulting in mRNAs which may be translated with different efficiencies. As such, the baculovirus gp64 gene provides a convenient multipurpose model system for the study of several aspects of the insect cell and the viral-cell interactions. In these studies, we will examine transcriptional regulation in the insect cell and recognition of early viral promoters, regulation and selective utilization of viral late promoters, and translational regulation in insect cells. In addition, elucidation of the mechanisms regulating expression of this important envelope glycoprotein will provide insight into the complex replication strategy utilized by this group of viruses and may have implications for other large DNA viruses. The regulation of gp64 expression will be examined by multiple complementary methods. Recognition of the immediate early gp64 promoter by the insect cell will be examined in the following manner: Deletion, linker scanning, and saturating point mutations will be generated and used for functional analyses (CAT and primer extension assays) to identify cis acting elements which determine the rate and accuracy of transcription initiation. DNA binding sites for cellular proteins will be located by gel mobility shift and DNAase footprinting. Functionally important cellular DNA binding proteins will be isolated, cloned and sequenced from Spodoptera frugiperda and/or Lymantria dispar cells. Transcriptional regulation of the gp64 late promoter will be examined by similar methods and viral genes encoding proteins which directly interact with late promoters will be cloned and sequenced. Regulation of gp64 expression from the early and late gp64 mRNAs will be examined by determining transcription initiation rates of early and late promoters, stabilities of the early and late mRNAs, and translational efficiencies of earl and late mRNAs.
