and tested. It is also possible that neither LxCxE motif is required for Rb binding, as a number of cellular proteins lacking this sequence do functionally interact with pRB, p107 and p130 [58]. Furthermore, sequences in addition to the LxCxE motif contribute to the ability of some viral oncoproteins to bind Rb proteins [59,60]. SV40 TAg has long been recognized at the key replication protein of this virus, and in vivo and in vitro studies have confirmed this role for JCV TAg as well. Regulation of SV40 DNA replication involves the incompletely understood coordination of TAg phosphorylation and dephosphorylation. Phosphorylation of serines 120 and 123 negatively regulates TAg functions, whereas phosphorylation of threonine 124 is required for TAg-mediated replication [reviewed in 20,21]. Modifications to the correspond- ing residues in JCV TAg, serines 121 and 124 and threonine 125, have been proposed, and threonine 125 has been demonstrated to be essential to replication activity [61,62]. A cell-free DNA replication system utilizing SV40 TAg indicated that addition of PP2A enhances DNA replication and that SV40 tAg inhibits this effect by blocking dephosphorylation of serines 120 and 123 [63,64]. However, SV40 tAg appears to stimulate SV40 replication to a limited extent in vivo; SV40 tAg deletion mutants produce slightly lower numbers of virions per infected cell [43] and addition of tAg protein to cells infected with SV40 tAg-defective genomes stimulates DNA replication [20]. Recently, tAg was shown to have a significant effect on DNA replication of the human polyomavirus MCV [65]. In the absence of tAg, TAg- mediated replication is reduced nearly 10-fold in 293 cells. Nesper et al. [44] developed a cell-free replication system to demonstrate that TAg is the only JCV protein required to mediate in vitro replication of a plasmid containing the JCV origin of replication. Using an in vivo approach with permissive PHFG cells, it was shown that JCV TAg is required for DNA replication, and JCV T9 proteins contribute to replication efficiency [45]. Further, a truncated JCV tAg mutant, encoding amino acids 1–89, replicated with similar efficiency to wild type JCV at days 7 and 14 p.t., but significant inhibition was observed at a later time point [40]. The authors speculated that reduced replication resulted from the inability of the mutant tAg to inhibit PP2A, thereby leading to dephosphorylation of the late JCV agnoprotein and inefficient DNA replication. In the current study we found that a JCV genome missing only tAg failed to replicate to detectable levels in PHFG cells. By modifying the replication assay and increasing expression of the JCV early proteins under the control of more potent CMV transcriptional signals, low levels of JCV DNA replication (,10% of wild type; Fig. 8) were observed; a construct lacking tAg and the three T9 proteins, however, still failed to replicate under these conditions. The inability of the CMV:T+/ t 2 /T92 construct to drive replication of the JCV origin plasmid suggests that not all contributions of the tAg and the T9 proteins to DNA replication are redundant. These data indicate that JCV tAg, unlike its SV40 counterpart but similar to MCV tAg, makes a significant contribution to viral DNA replication activity, and furthermore, that its effect on agnoprotein phosphorylation status does not completely explain its role in replication, since agnoprotein was not present in our assay. We speculate that interactions of tAg with PP2A and Rb proteins alter JCV TAg phosphorylation status and promote cell cycle progression, leading to conditions more favorable to viral DNA replication. References JCV Small t Protein Functions We also tested whether the H42Q, P99A and C157A mutations in JCV tAg had any effect on viral DNA replication. Because the latter two mutations reside in the unique coding sequences of tAg, this protein could be altered without affecting the other four early proteins. However, the H42Q mutation affects the shared N terminus of all five early proteins. To determine if the H42Q mutation affected DNA replication behavior, we co-transfected the JR:T+/t2/T9+ genome into PHFG cells with either the wild type or H42Q mutant tAg construct. We predicted this approach would allow us to determine whether H42Q tAg complemented the tAg-defective genome (i.e. JR:T+/t2/T9+). While no replica- tion was observed in this case, the control which employed the wild type tAg also failed to complement the tAg-defective DNA, and no conclusion could be reached regarding the influence of this J domain residue upon JCV DNA replication. This experiment does; however, provide new information indicating that expression of tAg in trans fails to correct the replication defect of a tAg- deficient JCV genome. It is possible that the levels of tAg produced from the JCV transcriptional signals are insufficient to permit detection of a complementing activity. It should be noted that a similar mutation to the J domain of the MCV tAg did not alter DNA replication behavior, while the same mutation in the MCV TAg markedly reduced this activity [65]. We demonstrated that the P99A mutant, and to a lesser extent the C157A mutant, exhibits a significant reduction in DNA replication relative to that observed with wild-type JCV. Thus, proline 99 and cysteine 157 are likely to either affect tAg’s ability to cooperate with the other T proteins or with cellular proteins that influence DNA replication potential and/or cell proliferation and survival. We have shown that the P99A mutation alters one such interaction, i.e. binding to PP2A. Significantly, Kwun and co- workers [65] reported that a mutation to a CxCxxC motif in the MCV tAg, abrogated the ability of this protein to stimulate DNA replication. The authors suggested that loss of binding to PP2A by the MCV mutant tAg affected the phosphatase’s ability to modify cellular or viral targets that impact replication either directly or indirectly. JCV tAg possesses unique features among polyomavirus small t proteins. Unlike SV40 tAg, it plays a significant role in the replication of viral DNA in vivo. SV40 tAg has been found to interact with PP2A, and it is predicted to bind Hsc70. In addition to these two cellular proteins, JCV tAg binds to the viral agnoprotein and to the Rb proteins, pRB, p107 and p130, most likely via two unique LxCxE motifs. Cooperation between the LxCxE and J domains of JCV tAg are expected to influence cell cycle progression. The study of JCV tAg has only recently been pursued, and it is expected that a greater understanding of this viral tumor protein will provide new insights into the pathogenic and oncogenic potentials of this human virus. Acknowledgments We thank R. Kartikeyan for helpful comments and for her assistance with the preparation of the manuscript. Author Contributions Conceived and designed the experiments: BB CAH MMRM RF. Performed the experiments: BB CAH MMRM. Analyzed the data: BB CAH MMRM RF. Wrote the paper: BB CAH RF. 1. Kean JM, Rao S, Wang M, Garcea RL (2009) Seroepidemiology of human polyomaviruses. PLoS Pathog 5: e1000363. 2. Frisque RJ, Bream GL, Cannella MT (1984) Human polyomavirus JC virus genome. J Virol 51: 458–469. PLoS ONE | www.plosone.org 11 May 2010 | Volume 5 | Issue 5 | e10606