Functional Genomics of Insect Baculoviruses

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Insect baculoviruses infect many insect pests of agriculture and forests and nuisance pests like mosquitoes and blackflies giving them a major role as biological control agents. Baculoviruses are also used as efficient expression vectors (e.g. Cervarix vaccine). Its large dsDNA genome encodes some 150 genes important in the virus replication cycle. The functions of some of these baculovirus genes are known, some for initial infection, production of virion proteins, egress from the cell, cell and host regulation and virus transmission. Much less is known about other genes. Among these is the me53 gene, a major early gene encoding a 53 kDa protein. Me53 is transcribed shortly after infection and later. We showed it was an important viral protein because in its absence virus replication is severely impacted. Since it has protein domains consistent with transcription factors, we felt it must work in the nucleus to regulate transcription. We found that not only is ME53 located in the nucleus (as expected of a transcription factor) but surprisingly also in foci at the cell periphery where it colocalizes with a viral envelope protein GP64. From this we surmised that ME53 might help virions bud through the membrane at these ME53/GP64 foci. But we are still stuck with the question, what is the role(s) of ME53? We posited two hypotheses, one on its role in transcriptional regulation and one on virus egress. One of our objectives is to follow differences in the virus replication cycle of ME53 deleted viruses (deltaME53) compared to wild type virus. We will start our search by electron microscopy for an ultrastructural comparison (e.g. looking at virogenic stroma and assembly/transit of virions in the nucleus and at the cell membrane). We will also compare virus (and cellular) transcriptional profiles. To differentiate the nuclear-specific function from the cell membrane one of ME53, a second objective is to map domains of ME53 needed for nuclear translocation (NTD) and those for membrane translocation (MTD). By mutating those translocation domains we hope to assess the roles of ME53 at different intracellular sites. For example mutating NTD will prevent nuclear translocation and any differences observed (e.g. lack of regulation of certain viral, or even host, genes for the NTD mutant), must be due to its nuclear function, and vice versa for the MTD mutants. While ME53 associates with GP64 and the capsid protein VP39, we suspect it also interacts with other viral (or host) proteins, for example to help ME53 translocate to the nucleus or plasma membrane. For this we will be looking for ME53 protein partners (the ME53 interactome) using a genetic, yeast two hybrid screen, and biochemical “pull down” experiments to remove ME53 from a cell preparation but with associated proteins still attached. Binding proteins from those “pull downs” will be identified by very precise spectroscophotometric measures. This would be followed by further mutagenesis to try to identify domains in ME53 important for binding to the different partners (and mutate the partners themselves). ME53 mutations unable to bind to certain partners will then be assessed for their affect on virus replication. Any differences noticed between the binding domain mutant ME53 and normal ME53 must relate to the importance of that particular protein. From this work we should better understand the function of ME53 in the baculovirus life cycle. While such discoveries advance fundamental science we cannot predict if there will be any practical or commercial value to this knowledge. As these viruses are used as biological control agents, perhaps this knowledge might be used to improve their efficacy and better protect against Canadian pest insects, or improve vaccine production.