[reviewed in 9]. SV40 TAg also interacts with insulin receptor substrate 1 (IRS1) through its LxCxE domain, leading to activation of PI3 kinase (PI3K), which in turn up-regulates phosphorylation of Akt [10]. The third transformation domain of TAg is a C-terminal bipartite region that directly binds and inactivates the tumor suppressor protein p53 [11]. Binding of SV40 TAg to p53 promotes the recruitment of CBP/p300, which in turn influences TAg acetylation [12,13] and stability, and impacts oncogenic transformation of NIH-3T3 cells [14]. JCV TAg has also been reported to interact with b-catenin, contributing to cellular transformation. b-catenin, a key member of the Wnt pathway, is stabilized and imported to the nucleus through a physical interaction with JCV TAg where it up-regulates expression of proteins involved in cell growth and proliferation [15]. Although the oncogenic mechanisms of the primate polyoma- virus TAgs have received much attention, the essential activities of this multifunctional protein relate to its role in mediating viral DNA replication. Many of the TAg sequences required for initiation and elongation of replication reside in the unique C- terminal region of the protein [5]. However, N-terminal sequences shared with the other tumor proteins, including tAg and the TAg splice variants (17KT, T9 proteins), also influence viral DNA replication. For example, the J domain of TAg is required for efficient viral DNA replication [16], yet few data are available that address the replication functions of these same sequences in the other tumor proteins. JCV tAg has only recently become a focus of study; however, a number of functions of the related SV40 tAg are known. SV40 tAg cooperates with TAg to enhance transformation when TAg levels are low or quiescent cells are being tested [reviewed in 17,18]. SV40 tAg expression is dispensable for TAg-mediated transfor- mation of dividing rodent cells, but is required for transformation of human cells [19]. In addition, tAg stimulates, but is not essential to, viral replication in permissive cells [20]. This stimulation likely results from the ability of tAg to alter the activity of cellular protein phosphatase 2A (PP2A), which in turn may lead to changes in the phosphorylation state of critical serine and threonine residues regulating TAg replication functions [reviewed in 20,21]. SV40 tAg is also critical to the maintenance of high viral DNA copy numbers in persistent infections of human mesothelial cells; in the absence of tAg, cells are immortalized but not transformed, and only low copy numbers are maintained [22]. Unique sequences within the SV40 and murine polyomavirus tAgs are responsible for the binding of PP2A [23–25], a phosphatase that regulates numerous cell activities by dephos- phorylating a variety of substrates. Making up as much as 1% of total cell protein [26], PP2A is a modular holoenzyme consisting of three subunits. A large number of regulatory subunit B isoforms directs functional specificity when joined to the core enzyme composed of the scaffolding subunit A and the catalytic subunit C [25,26]. For example, pools of holoenzymes with specific B subunits are formed differentially during the course of the cell cycle, and the PP2A holoenzymes with cell-cycle-specific regulatory subunits orchestrate cell cycle progression [27]. Various viral proteins form complexes with the AC core [21], and some partially displace B subunits already complexed in the holoenzyme [24]. Through co-immunoprecipitation (co-IP) experiments, SV40 tAg was associated with the A and C subunits of PP2A [28,29]. Addition of SV40 tAg to the AC core alters the subcellular localization of PP2A [30] as well as the phosphatase activity of the holoenzyme, and modulates protein kinase signaling pathways [21,31], thus potentially influencing the cell cycle. JCV Small t Protein Functions SV40 tAg facilitates the transformation of cells by activating phosphorylation of PI3K in a PP2A-dependent fashion, which in turn up regulates the activity of multiple signal transduction pathways [reviewed in 32]. The cellular kinase Akt, part of the PI3K-Akt pathway, contributes to enhanced activity of telomerase, a necessary but not sufficient step in the oncogenic progression of human cells [33]. Akt has two phosphorylation sites: T308, in the activation loop of the kinase, and S473, at the carboxyl terminus. Depending on cell growth conditions, phosphorylation of T308 alone or T308 and S473 together may be stimulated through the PI3K pathway following binding of PP2A by tAg [34]. TAg has also been shown to effect modification of both residues in Akt through its binding to IRS1 [10]. Here, we have examined the interaction of wild-type and mutant JCV tAgs with PP2A and members of the Rb family in cultured cells, and have investigated tAg functions that may influence JCV transforming and DNA replication potential. Materials and Methods Generation of Viral DNA Constructs We have adopted a uniform naming scheme for the recombi- nant DNAs (rDNAs) constructed for this study. For clarity, plasmids previously described have been renamed according to this scheme, with the original name indicated in parentheses after the first mention of each construct in the text. Expression of TAg (T), tAg (t), or the three T’ proteins (T9135, T9136, T9165) is indicated by a plus (+); lack of expression is denoted by a minus (-) sign. An amino acid substitution, for example the alteration of proline at residue 99 of tAg to alanine, is indicated by the single letter abbreviation of the wild type (P) and altered (A) amino acid with the residue number (99) identified between these letters. The viral source of the transcription signals effecting gene expression is noted at the start of each name. JR, SR and CMV denote the JCV, SV40 and cytomegalovirus regulatory regions, respectively. Therefore, the rDNA, SR:T+/P99At+/T9+, expresses the JCV wild type TAg and T9 proteins and the mutant P99A tAg under the control of the SV40 promoter/enhancer signals. pCMV:T+/t+/T9+ (previously pCMV-JCVE; [8]) expresses all five JCV early proteins via the CMV early promoter in the pCR3 vector (Invitrogen). pCMV:T+/t2/T92 (previously pCMV-DT9; [8]) expresses TAg only. pCMV:T+/t2/T9+ (previously pCMV- JCT; [8]) expresses TAg and the three T9 proteins, but not tAg. pJR:T+/t2/T9+ was constructed by first digesting pCMV:T+/ t2/T’+ with NcoI and PstI to isolate the DNA fragment containing TAg cDNA coding sequences. This fragment was ligated to pJR:T+/t+/T9+ (previously pMRMMT; [35]) which had been digested with the same restriction enzymes to remove the corresponding sequence. To create a second construct expressing these same JCV proteins but under the control of SV40 transcription signals (pSR:T+/t2/T’+), the JCV regulatory region was removed from pJR:T+/t2/T9+ by NcoI cleavage and replaced with the SV40 regulatory sequences obtained by NcoI digestion of pSR:T+/t+/T’+ (previously pSRMMT; [35]). The expected structures of the pJR:T+/t2/T9+ and pSR:T+/t2/T’+ rDNAs were confirmed by DNA sequence analysis. PCR was performed to generate a JCV tAg expression plasmid. An mRNA template from PHFG cells transfected with JCV DNA was amplified with primers tAg cDNA R (59-GAG CTT ATG GAT TTA TTA GGC CTT GAT AGG TCT GCA TGG-39), nt 4980–4942, and tAg cDNA F (59-GCT ATC CAT AGG TTG GCA CCT TAA AGC TTT AGA TCC CTG TAG G-39), nt 4412–4426, 4494–4517 with 4 non-JCV nt at the 59 end. The product, a DNA fragment containing the tAg cDNA, was ligated PLoS ONE | www.plosone.org 2 May 2010 | Volume 5 | Issue 5 | e10606