JCV Small t Protein Functions Figure 5. Replication efficiencies of DNA constructs expressing combinations of JCV early proteins. PHFG cells in 60 mm plates were transfected in duplicate with 400 ng of wild type (T+/t+/T’+) or mutant (T+/t2/T’2, muT+/t+/T’+, T2/t+/T’2, T+/t2/T’+, or muT+/t2/T’+) JCV genomes. At each time point, viral DNA was extracted by the Hirt procedure [39], purified, digested with DpnI and EcoRI, electrophoresed on a 0.8% agarose gel, transferred to nitrocellulose membranes and hybridized to a [P32]dCTP-labeled JCV DNA probe. Bands were visualized with a Typhoon phosphorimager and quantitated with ImageQuant software. The marker (M) shown in the first lane of each blot is 1 ng of linear JCV DNA (5130 bp). The duplicate, independent samples representing each DNA construct were analyzed, and the position of Dpn1-resistent replicating genomes on the Southern blots is denoted by an arrow at days 10, 14 and 21 p.t. Dpn1- and EcoRI-sensitive input DNAs are noted at the day 0 time point. Hirt extracts of uninfected PHFG cells (PHFG) were subjected to the same Dpn1 assay. doi:10.1371/journal.pone.0010606.g005 construct might be considered a logical choice to test this prediction, the JCV early proteins in this chimeric DNA do not productively interact with the SV40 origin sequences to mediate viral DNA replication [38]. To overcome this obstacle, PHFG cells were co-transfected with two DNAs, the first was a construct containing the JCV origin of replication (pM1o; [38]) and the second was one of three constructs containing wild type (T+/t+/ T9+) or mutant (T+/t2/T9+ or T+/t2/T92) JCV early coding regions under the control of a CMV promoter. This DpnI assay differed from those described above in at least three ways: (i) the CMV promoter is more active than the JCV transcriptional signals and the T proteins are expressed at higher levels, (ii) the constructs contain the JCV early coding region linked to plasmid sequences rather than the intact JCV genome and (iii) the JCV proteins and replication origin are not resident on the same DNA molecule. Following co-transfection of PHFG cells, replicated viral DNA was isolated at days 0, 3, 4, and 5 p.t. Replication of the origin plasmid pM1o was detected at days 3, 4 and 5 p.t. in the presence of pCMV:T+/t+/T9+ (Fig. 8). At days 4 and 5 p.t., the small t construct, pCMV:T+/t2/T9+, mediated replication of the pM1o vector at less than 10% the level observed in the presence of all 5 JCV early proteins. No replication was detected at any time point in the presence of the construct expressing TAg only (pCMV:T+/ t 2 /T92), suggesting that at least some contributions of the tAg and the T9 proteins to DNA replication are unique. Discussion A substantial number of biological differences have been documented between the primate polyomaviruses JCV and SV40, yet the sequence and the organization of the two viral genomes are remarkably similar [2]. Early studies employing chimeric viruses indicated the transcriptional control regions of each virus, including their viral promoter and enhancer elements, have a significant impact on the individual trans- forming and lytic behaviors of JCV vs. SV40 [35,38,48,49]. PLoS ONE | www.plosone.org 8 May 2010 | Volume 5 | Issue 5 | e10606