JCV Small t Protein Functions Figure 7. JCV tAg fails to complement replication of a tAg- deficient JCV genome in trans. PHFG cells in 60 mm plates were co- transfected in duplicate with 400 ng of a tAg-deficient (T+/t2/T’+) JCV genome and 400 ng of a JCV DNA construct expressing either wild type tAg (T2/t+/T92) or a J domain mutant tAg (T2/H42Qt+/T92) under the control of the JCV promoter-enhancer. Cells were also transfected with 400 ng of either a T9-deficient (T+/t+/T’2) JCV genome or a positive replication control (T+/t+/T’+). Duplicate, independent samples repre- senting each DNA construct were extracted by the method of Hirt [39] on days 0, 7, and 10 p.t. and analyzed using the Dpn1 assay as described in the legend to Figure 5. The marker shown in the first lane of each blot is 1 ng of linear JCV DNA (5130 bp), and the position of Dpn1-resistent replicating genomes is denoted by an arrow at days 7 and 10 p.t. Dpn1- and EcoRI-sensitive input DNAs are noted at the day 0 time point. doi:10.1371/journal.pone.0010606.g007 We created the C157A tAg mutant to determine if the conserved cysteine in the newly recognized LxCxE domain of JCV tAg was required for Rb binding. However, once made, we also tested whether the mutant displayed altered PP2A binding relative to wild type tAg. In one cell line, we found that the C157A tAg bound PP2A with wild type efficiency. We consistently observed; however, that the same mutant tAg expressed in a second, independently derived cell line bound PP2A even better than wild type tAg. A preliminary comparison of the two cell lines expressing the C157A tAg indicates that the line in which more efficient PP2A binding occurred had a shorter doubling time and more transformed morphology. This observation will require additional investigation to determine if enhanced PP2A binding is associated with a transformed phenotype. It should be noted that to detect PP2A binding in this study, we employed antibodies that recognized the catalytic subunit of PP2A. PP2A is found abundantly as either a holoenzyme consisting of the AC core plus a regulatory B subunit, or as the AC core alone. High levels of the A and C subunits are not found as free proteins in cells [56], and SV40 tAg does not bind Figure 8. JCV early genes expressed from a CMV promoter induce replication of a JCV origin plasmid. PHFG cells in 60 mm plates were co-transfected in duplicate with 400 ng of the JCV replication origin plasmid, pM1o, and 400 ng of one of the following expression vectors that encode JCV early genes under the control of the CMV promoter: pCMV:T+/t+/T9+ (lanes 2, 3; all five tumor proteins expressed), pCMV:T+/t2/T9+ (lanes 4, 5; TAg and 3 T9 proteins expressed) or pCMV:T+/t2/T92 (lanes 6, 7; TAg only expressed). The ability of the proteins produced by the second plasmid to drive replication of the origin plasmid in trans was tested. Duplicate, independent samples representing each DNA construct were extracted by the method of Hirt [39] on days 0, 3, 4 and 5 p.t. and analyzed using the Dpn1 assay as described in the legend to Figure 5. The marker (M) shown in lane 1 of each blot is 1 ng of linear pM1o (,2400 bp), and the position of Dpn1- resistent replicating genomes is denoted by an arrow at days 3–5 p.t. Dpn1- and EcoRI-sensitive input DNAs are noted at the day 0 time point. doi:10.1371/journal.pone.0010606.g008 detectably to purified free C subunit [57]. Given this information, our data suggest JCV tAg binds the scaffold subunit (A) of the AC core of PP2A in rat and mouse fibroblasts and not directly to the C subunit. As noted above, the C157A tAg mutant was created to see if alteration of the second LxCxE motif would affect Rb binding. In addition, the CxxxPxC motif containing the P99A mutation overlaps the first LxCxE domain in the JCV protein. Neither mutation had a detectable affect on the ability of tAg to bind p107 (Fig. 4) or p130 (data not shown). It is likely that both LxCxE motifs contribute to Rb binding, perhaps redundantly, and that to abolish binding activity, double mutants will have to be created PLoS ONE | www.plosone.org 10 May 2010 | Volume 5 | Issue 5 | e10606