STUDIES ON THE PENTAMERIZATION OF SV40 VP1 IN VITRO

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Simian Virus 40 (SV40) belongs to the papovavirus family of oncogenic DNA animal viruses. The nuclear site of assembly of the virus as well as the nuclear activities of its large-Tumor-Antigen, have rendered it an ideal model system for the study of nuclear localization and nuclear localization signals. Regulated nuclear protein import of different cellular proteins, including the rel oncoprotein, the glucocorticoid receptor, and the dorsal early developmental protein, is essential to normal cell growth and development. Understanding the nuclear transport machinery is a first step towards studying disease states caused by mislocalization of nuclear proteins. The SV40 capsid is comprised of three structural proteins --Vp1, Vp2, and Vp3. Vp1 is the major capsid protein of SV40 accounting for at least 70% of the total virion protein by weight. VP1 forms pentamers in the cytoplasm of the infected cells and is then transported to the nucleus. The chemical nature of the bonds that stabilize the VP1 pentamers has not been established. We have been interested in identifying the nature of the interactions that stabilize the VP1 pentamers, as well as studying the role of pentamerization in the nuclear transport of VP1. In the past year, results of VP1 reduction/alkylation experiments obtained in our laboratory strongly implicate the involvement of disulfide bridges in the formation of Vp1 pentameters in vitro. SV40 Vp1 contains seven cysteine residues, any or several of which could be involved in disulfide linkage(s). We have initiated studies to identify Vp1 cysteine residues involved in those interactions. Based on a model of how two internal domains of single Vp1 peptide chain interact to form an intramolecularly folded Vp1 protein and may be available for intermolecular interactions with other Vp1's. Oligo-directed mutagenesis of Cys10 and Cys50 is presently in progress. In vitro protein-protein interaction assays will enable a rapid screening of generated mutants. Eventually, all seven cysteines of Vp1 will be mutagenized and assayed. Mutants defective in multimerization in vitro will be further studies for the extent and efficiency of their nuclear transport in vivo. The project provides an excellent opportunity for student involvement in both basic and advanced molecular biological technique, as well as for development of skills and interests in biomedical research. Currently, 3 MBRS students are involved in these studies in our laboratory. They will be presenting their results shortly at National meetings including the MBRS annual symposium and the Annual Meeting of the Society for Cell Biology.