Baculoviruses are unique among eukaryotic DNA viruses in that 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 contains four equimolar subunits, LEF-4, -8, -9, and p47. LEF-8, the largest of the four subunits, contains a 13 amino acid motif conserved among beta subunits of RNA polymerases. LEF-9 contains a 7 amino acid sequence with homology to a highly conserved beta-prime sequence. These similarities suggest that LEF-8 and LEF-9 are the catalytic subunits of RNA polymerase. However, these regions of homology are considerably more restricted than is common among RNA polymerases. To examine the functional relevance of these sequences, alanine mutagenesis will be conducted on the conserved motifs, and the effects of the mutations on transcription activity will be determined. In addition, the individual subunits will be assayed for RNA polymerase, promoter binding, and NTP binding activities. Additional late transcription factors will be assayed for the ability to stimulate viral RNA polymerase and for DNA and RNA binding activities.
The viral protein VLF-1 is the best candidate for the factor that differentially regulates expression of late and very late genes. VLF-1 shows strong homology to a family of proteins that function as integrases, yet there is no obvious role for an integrase in the life cycle of baculoviruses. Thus, VLF-1 probably acts on DNA in a manner analogous to integrases and binds to DNA in a sequence-specific manner and modifies it in some way. Purified VLF-1 will be characterized with respect to its activity in assays for DNA/RNA binding, topoisomerase activity, and stimulation of in vitro transcription.
This research should aid in understanding the mechanisms that regulate temporal expression of baculovirus genes, and have practical impacts on biotechnology. The baculovirus expression system is widely used for the production of 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.
