Molecular virology of insecr baculoviruses

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While much is known (and still unknown) about the interactions between mammalian viruses and their host cells that between insect viruses and insect cells is poorly understood. Baculoviruses are unique to insects and as such their study should reveal novel aspects of their replication strategies. This proposal involves two baculoviruses. CfMNPV infects the spruce budworm (a major defoliator in Canadian forests) and AcMNPV infects many lepidopteran insects, particularly pests of agricultural crops. With about 150 baculovirus genes, there is a need to regulate when and at what levels those genes are expressed. It would be wasteful if all genes were expressed simultaneously and at the same level. Some virus products are needed at only low amounts and early in infection (e.g. enzymes for DNA replication) whereas others are needed in higher amounts and later in infection (e.g. virion proteins). Similarly viral DNA replication, which occurs before viral assembly, requires prior production of viral DNA polymerase. This study focuses on genome transcriptional regulation and viral DNA replication. In particular we will characterize two baculovirus proteins, ME53 and Cf103 both of which may be involved in regulation of gene expression. We will determine when these proteins and their transcripts are made, where in the cells these proteins are localized, what viral and cellular proteins or DNA sequences they interact with (and how) and evaluate how they influence the expression of other baculovirus genes (we already know that Cf103 delays expression of late genes). We noted that CfMNPV generates several antisense, non-coding RNAs. While this may be a natural consequence of transcription of upstream sense mRNAs we feel some of the antisense transcripts have a regulatory role in the virus replication cycle, perhaps using RNA silencing, and we wish to address that hypothesis. In addition, since viral DNA replication is such a central feature of the virus replication cycle we wish to determine the mechanism of DNA replication. To that end we will try to identify where on the DNA genome replication actually starts (an ori of DNA replication) and what viral and cellular components exist at the origin of replication complex (ORC).