Expression of human polyomavirus JC T antigen by an adenovirus hybrid vector and its binding to DNA sequences encompassing the JC virus origin of DNA replication

Institut für Virologie und Immunbiologie der Universität Würzburg, Versbacherstrasse 7, D-97078 Würzburg, Germany In the search for factors that influence the outcome of human polyomavirus JC (JCV) infection, the roles not only of host-related immunological control but also of virus-dependent regulatory steps have to be taken into account. Besides cell-specific control of early expression of the multifunctional virus protein large tumour antigen (T Ag), control mechanisms involve individual steps of the DNA replication process. For the analysis of T Ag DNA binding, the protein was expressed by an adenovirus hybrid vector in the 293 cell line to provide saturating amounts of JCV T Ag. After determination of the size and immunoreactivity, functional activity was analysed by specific DNA binding. To avoid the interference of cellular proteins, T Ag was immunoprecipitated prior to the reaction. Binding to T Ag-binding sites I and II within a 141 bp DNA segment in the control region was analysed using deletion mutants of a JCV subtype from brain tissue of a patient with fatal central nervous system disease. The specificity of the binding was confirmed by recombinant T Ag binding to origin of DNA replication (ori) sequences of wild-type JCV genomes. These data document that recombinant T Ag overexpressed by the adenovirus vector in eukaryotic cells was JCV-specific, had the expected length and exhibited specific ori-binding activity, thus providing the essential tool for future analysis of virus-host interactions at the level of viral DNA replication. * Author for correspondence. Fax +49 931 201 39 34. Present address: AFRC Centre for Genome Research, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3JQ, UK. Received 18 August 1994; accepted 25 August 1994.