JCV Small t Protein Functions Figure 3. Interaction of mutant JCV tAgs with PP2A. Interactions between PP2A and wild-type tAg and (A) tAg-mutant P99A or (B) C157A were compared. 3T3 cells were stably transfected with DNA constructs expressing JCV early proteins under the control of SV40 promoter-enhancer signals. Lysates of these cells were subjected to IP with the anti-T monoclonal antibody PAb 962 (a-T) or anti-PP2A antibody (a-PP2A). The amount of total cell protein subjected to IP with anti-PP2A antibody (lanes 5–8, Panel A; lanes 6–10, Panel B) was five times that used with the anti-T antibody (lanes 1–4, Panel A; lanes 1–5, Panel B). Samples were electrophoresed on 18% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. WB was performed using a cocktail of anti-T monoclonal antibodies. Results for two independently-derived cells lines (#1, #2) containing DNA constructs that either encode all 5 JCV wild type early proteins (T+/t+/T9+; Panel A, lanes 1,2,5,6, Panel B, lanes 1,2,6,7) or wild type TAg, T9165, T9136 and T9135 plus tAg mutant P99A (T+/P99At+/T9+; Panel A, lanes 3,4,7,8) or C157A (T+/C157At+/T9+; Panel B, lanes 3,4,8,9) are shown. A single 3T3 cell line expressing tAg only (T2/t+/T92) was isolated and tested for PP2A binding (Panel B, lanes 5, 10). Panels A and B of this figure each represent proteins electrophoresed on a single gel and transferred to a membrane, which was then cut in half and each half developed for different lengths of time. doi:10.1371/journal.pone.0010606.g003 proteins [7,8], binding of wild type tAg to pRB was difficult to detect, most likely because levels of this tumor suppressor protein are low in these cells (data not shown; [7]). JCV tAg Contributes to Viral DNA Replication in Permissive Cells SV40 TAg is recognized as the primary DNA replication protein. It is the only viral protein required to initiate viral DNA replication in a cell-free system [42], and in cultured cells, SV40 genomes encoding TAg without tAg are viable, although virus titers may be reduced relative to parental SV40 [43]. JCV TAg is also necessary and sufficient to promote viral DNA replication in a cell-free system [44]. However, in vivo, the JCV T9 proteins contribute to replication potential, and the contributions of tAg are unknown [45]. To examine the individual roles of the JCV early proteins in viral DNA replication, the intact wild-type JCV genome (JR:T+/t+/T9+), a TAg only mutant (JR:T+/t2/T92), a TAg deletion mutant (JR:muT+/t+/T9+), a tAg only mutant, (JR:T2/t+/T92), a tAg mutant (JR:T+/t2/T9+), and a T9 only mutant (JR:muT+/t2/T9+) were each transfected into two plates of PHFG cells (Fig. 5). Low-molecular DNA was extracted from the cells at several times p.t., and DNA replication was measured using a DpnI assay [46]. At day 0, equivalent amounts of input DNA were detected for each sample; at all subsequent time points, days 10, 14, and 21 p.t., increasing levels of the replicated wild type JCV genome were detected. No replication of any DNA construct lacking either TAg or tAg was observed in vivo at any time point. To further examine tAg’s role in viral DNA replication, the Dpn1 assay was repeated with the same DNA constructs used in Figure 5 plus the two tAg substitution mutants (JR:T+/P99At+/ T9+ and JR:T+/C157At+/T9+) and a T9 mutant (JR:T+/t+/ T92) (Fig. 6). At day 0, only input DNA was detected (data not shown), but at later time points, increasing levels of Dpn1-resistant replicated JCV DNA were observed with the two tAg point mutants and the T9 deletion mutant; the other DNA constructs exhibited the same replication behavior observed in the previous experiment (Fig. 5). Genomes containing the P99A and C157A tAg mutations exhibited reduced replication potential relative to wild type JCV DNA (3–4% and 20–29%, respectively, depending PLoS ONE | www.plosone.org 6 May 2010 | Volume 5 | Issue 5 | e10606