Abstract
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.