JCV Small t Protein Functions Figure 4. Wild type and mutant JCV tAgs bind the Rb protein p107. JCV early proteins expressed in Rat 2 (R2) or MEF cells transformed with pSR:T+/t+/T’+ (encodes all 5 JCV early proteins) or in G418-selected 3T3 cells stably transfected with pSR:T+/t+/T’+, pSR:T2/t+/T’2 (encodes tAg only) or pSR:T+/C157At+/T’+ (encodes tAg mutated at residue 157 and the other 4 JCV early proteins) were incubated with anti-T monoclonal antibody PAb 962 (a-T; lanes 2–6) or anti-p107 antibody (a-p107; lanes 7–11). Negative controls included EBC lysis buffer immunoprecipitated with anti-T monoclonal antibody PAb 962 (Con, lane 1) and Molt 4 extract with anti-p107 antibody (Molt 4, lane 12). The amount of total cell protein subjected to IP in lanes 7–11 was 10 times that employed in the corresponding samples in lanes 2–6. Proteins were separated on an 18% SDS-polyacrylamide gel and WB analysis was performed using a cocktail of anti-T monoclonal antibodies. The asterisks denote antibody light and heavy chains. This figure represents 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.g004 on the time point). As reported earlier [45], JR:T+/t+/T92 DNA replication was reduced more than 10-fold (6–8% of wild type). DNA replication of the tAg mutant was not detected at any time point, confirming the results presented in Figure 5 indicating that JCV tAg has a significant influence upon this critical activity. Expression of JCV tAg in trans does not Relieve the Replication Defect of JR:T+/t2/T9+ An SV40 H42Q mutant altered at a critical residue of the shared J domain of the three early proteins is replication-defective [16]. We created an H42Q mutant of JCV tAg to see if this alteration impacted tAg’s ability to support DNA replication. Because the mutation affected the other four JCV early proteins as well, we introduced the H42Q mutation into a construct that only expresses tAg to generate JR:T2/H42Qt+/T92. This DNA, or a DNA expressing wild type tAg only, was co-transfected into cells with a construct expressing wild type TAg and the three T9 proteins, but not tAg (JR:T+/t2/T9+), to determine if tAg expression complemented replication of the latter DNA in trans. Dpn1-resistent DNA was not detected using either combination of co-transfected DNAs at any time point, although replication was observed with the wild type control construct (JR:T+/t+/T9+) and to a lesser extent with the construct lacking the T9 proteins (JR:T+/t+/T92) (Fig. 7). The absence of detectable complemen- tation with the wild type tAg precludes a determination of whether the H42Q mutation in tAg impacts this protein’s replication function. Elevated Expression of JCV TAg and T9 Proteins Promotes DNA Replication in the Absence of tAg The levels of viral proteins expressed from the JCV transcrip- tional signals are low relative to the levels induced by the SV40 signals [35,47]. We hypothesized that if JCV early protein expression was increased, viral DNA replication might be elevated to detectable levels for some replication-defective JCV constructs, including the tAg deletion mutant. Although the SR:T+/t2/T9+ PLoS ONE | www.plosone.org 7 May 2010 | Volume 5 | Issue 5 | e10606