Temporal Regulation of Baculovirus Gene Expression

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Baculoviruses are unique among eukaryotic DNA viruses because the early genes are transcribed by the host RNA polymerase II, while late and very late genes are transcribed by a viral-encoded RNA polymerase. The viral RNA polymerase is a four subunit complex with intrinsic promoter recognition, mRNA capping, termination, and polyadenylation activities. The first goal of this project is to identity the cap methyltransferase that participates in the formation of 5' caps on viral RNAs. Two complementary approaches will be used; one is to purify cap methyltransferase from infected cells; the other is to test orf69 protein for cap methyltransferase activity. This protein, which is a homolog of the rRNA methyltransferase FtzJ, is the only viral protein with an S-adenosyl-methionine binding motif. The second aim of this project is to define the mechanisms that allow RNA polymerase to discriminate between late and very late templates. Again, two approaches to this problem will be used. First, a targeted analysis of the in vitro activity of VLF-1, which stimulates very late gene expression in vivo, will be conducted. Then, factors that alter the promoter specificity of RNA polymerase will be purified from infected cells. The third aim of this project is to characterize the baculovirus protein LEF-5, which has homology with transcription elongation factor TFIIS. To test whether LEF-5 functions as an elongation factor, experiments will be performed to determine whether LEF-5 binds RNApol and whether it stimulates the RNA cleavage activity of RNApol; these two activities are characteristic of TFIIS and are required for its function.This project will elucidate the mechanisms that regulate temporal expression of baculovirus genes, and should also have practical impacts on biotechnology. The baculovirus expression system has been used to produce thousands of different proteins used for basic research and commercial projects. The baculovirus RNA polymerase is central to this expression system, and an understanding of its unique properties will lead to improvements to this technology.