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Global analysis of the vaccinia genome.


Vaccinia virus, a close relative of variola virus the causative agent of smallpox, is a large double-stranded DNA virus encoding more than 260 genes that replicates in the cytoplasm of the host cell. To date the function or location of only ~100 viral proteins has been determined although the genome has been available for 11 years. The project aims to take a global genomic approach to identify new vaccinia proteins involved in viral morphogenesis and those responsible for the dramatic cellular effects observed during vaccinia infection. These include stimulation of cell motility, changes in cell adhesion, re-organization of the actin and microtubule cytoskeletons as well as destruction of the centrosome to name but a few. I will initially PCR all vaccinia genes and the resulting open reading frames will be cloned into a series of viral and CMV expression vectors containing epitope and GFP tags. These clones will be used to localize each gene product in fixed and live cells in both infected and non-infected cells. This first screen will immediately provide large amount of information as localization will often implicate function and will identify viral proteins that induce profound cellular changes. I will subsequently further characterize proteins that are associated with the cytoskeleton or induce changes in cell morphology, adhesion or motility when expressed although other interesting proteins will not be neglected. Selected proteins will be studied using a combination of approaches including GFP imaging, biochemical isolation coupled with mass spectrometry analysis, anti-sense expression and RNA interference and the ability to knockout viral genes by homologous recombination.


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