Polyoma virus assembly occurs by sequential addition of capsid proteins to the viral minichromosome to form a complete T=7d icosahedral virion.
The specific aims of this proposal address two questions concerning the assembly pathway: 1) how are the individual capsid proteins localized to the site of virus assembly, and 2) are there specific DNA binding sites for the initiation of virion encapsidation? Large amounts of the individual polyoma capsid proteins will be produced by molecular cloning in E. coli. Capsid proteins purified from E. coli will then be used to study their in vitro binding to DNA and to generate affinity purified antibodies. The intracellular localization of these proteins will be studied using immunofluorescence of individual capsid protein genes transfected into NIH 3T3 cells. Encapsidation initiation sequences will be assayed by adding polyoma DNA sequences to episomal bovine papilloma virus (BPV), introducing the modified BPV genome into C127 mouse cells, and determining whether the BPV minichromosome subsequently can be encapsidated during a lytic polyoma infection. Because the polyoma life cylce must interface with normal cellular processes, we anticipate that polyoma assembly will have significance to basic eukaryotic biological processes including the subcellular localization of proteins, chromatin structure, gene regulation, and other macromolecular assembly mechanisms. In the long term, a more complete understanding of the viral life cycle will be relevant to designing and understanding anti-viral chemotherapy, and to elucidating the role of polyoma's transforming genes.
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