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Fig. 1. Characterization of cells harboring the recombinant EBV expressing HA-tagged EBNA1. (A) Expression of HA-tagged EBNA1 protein in the cells that were infected with the recombinant EBV. Whole cell extracts of Akata cells harboring naturally infected EBV (lane 1, referred to as wild-type EBV hereafter) and four independent cell clones harboring the recombinant EBV (lanes 2-5) were analyzed by western blot analyses. EBV-immune human serum (left panel) and anti-HA antibody (right panel) were used as primary antibodies. (B) Detection of EBV genomes by FISH analyses. Akata cells harboring wild-type EBV (top panels) or cells harboring the recombinant EBV (bottom panels) were processed for FISH analyses before (left panels) and after 5 weeks of hydroxyurea (HU) treatment (right panels). An EBV genome-derived FISH probe was used for the analyses. Note that the majority of HU-treated cells are free of FISH signals (right panels). (C) EBV copy number estimation by Southern blotting. Akata cells harboring wild-type EBV or the recombinant virus were treated with HU for 5 weeks. Genomic DNAs were prepared before (lanes 1 and 4) and after HU treatment [either after 3 weeks (3 wk) or 5 weeks (5 wk) of treatment]. The genomic DNAs (10 µg) were digested by NcoI and analyzed by Southern blotting using a part of BamHI-C fragment (EcoRI-BamHI fragment) as a probe. The NcoI fragments of 21-kb size are shown. The copy number controls, which were prepared from a BAC clone DNA of the EBV genome, are also indicated.

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