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.
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.
Windl
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Institut für Virologie und Immunbiologie, Universität Würzburg, Germany.
Dörries
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D000256:Adenoviridae / Q000235:genetics*; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D007577:JC Virus / Q000276:immunology* / Q000502:physiology; D007668:Kidney / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014779:Virus Replication
D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D011994:Recombinant Proteins