The mechanism(s) by which proteins are transported to the nucleus in eucaryotic cells will be examined. The simian virus 40 (SV40) large tumor antigen (T-ag) offers an exceptional model system for the analysis of nuclear transport; the T-ag polypeptide is well characterized and the nucleic acid sequence encoding T-ag can be easily manipulated. A mutant of SV40 has been constructed which is defective in the transport of T-ag to the nucleus. Infection of cells with the mutant virus results in the accumulation of T-ag in the cytoplasm. The mutation has been localized on the SV40 genome by the marker rescue technique. The first specific aim will be to determine by nucleotide sequencing the exact nucleotide alteration resulting in the mutation. The possible effects of the nucleotide alteration on amino acid sequence, protein conformation, and protein structure will be deduced. Secondly, a domain on the T-ag polypeptide governing nuclear transport will be defined at the nucleotide level by using site-specific mutagenesis to create additional transport-defective mutants. Transport to the nucleus will be investigated at the polypeptide level by microinjection. The third specific aim will be to determine by microinjection the capacities of several different forms of T-ag (monomeric, multimeric, cell surface-associated) for nuclear transport. Transport-defective T-ag will be microinjected into the cytoplasm and the nucleus of recipient cells to distinguish whether the mutation affects transport across the nuclear membrane or retention within the nucleus. Previous experiments have revealed that the cytoplasmic T-ag phenotype is dominant over the nuclear T-ag phenotype since co-infected cells synthesizing both wild-type and transport-defective T-ag polypeptide do not transport wild-type T-ag to the nucleus. The fourth specific aim will be to determine the mechanism responsible for the dominance of the cytoplasmic T-ag phenotype by microinjection of various combinations of mutant and wild-type T-ags. Fifth, a domain on the T-ag polypeptide governing nuclear transport will be determined at the polypeptide level by microinjection of proteolytic fragments of T-ag. Finally, partial phenotypic revertants of the cT mutation will be examined. This analysis of the structural properties of the SV40 T-ag which specify its transport to the nucleus will provide insights into the general process of nuclear transport in eucaryotic cells. Elucidation of the mechanism(s) governing nuclear transport and nucleocytoplasmic interactions has broad implications to understanding the regulation of gene expression and the control of early embryonic development.
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