FIG. 3.

Agno protein inhibits T-antigen-mediated JCV DNA replication. A replication-competent plasmid, pBLCAT₃-Mad-1L (5 μg), containing the regulatory region of the Mad-1 strain of JCV was transfected alone or in combination with the expression plasmids CMV-T-antigen and CMV-Agno into U-87MG cells as described in Materials and Methods. Plasmid concentrations used in transfections are indicated on top (in micrograms). The total amount of DNA transfected into the cells was normalized with appropriate empty vectors. At 72 h posttransfection, low-molecular-weight DNA was isolated by the Hirt method (23), digested with SacI and DpnI enzymes, resolved on a 0.8% agarose gel, and analyzed by Southern blotting. The bands corresponding to the replicated DNA bands were quantitated by densitometry (see Materials and Methods) and expressed as percent inhibition with respect to the viral DNA replication in the presence of T antigen alone. The results for percent inhibition are shown at the bottom.