JCV Small t Protein Functions Results JCV tAg Interacts with PP2A The ability of polyomaviruses to induce S phase progression of a host cell is critical to the establishment of a productive infection. The early proteins of these viruses target a number of cellular factors to overcome normal cell cycle control mechanisms. The cellular phosphatase, PP2A, represents one such target as it regulates several important cell signal transduction pathways and is critical to cellular proliferation. Recently, Sariyer and co- workers [40] demonstrated an in vitro interaction between bacterially-produced JCV tAg fused to GST and PP2A present in extracts of U-87MG tumor cells. To demonstrate that this interaction occurs in vivo, lysates of Rat2, MEF and 3T3 cells expressing untagged JCV proteins were subjected to co-IP/WB analysis. A physical interaction between PP2A subunit C and JCV tAg was detected when anti-PP2A-C antibody was used for the IP step and WB was performed using anti-T protein antibodies (Fig. 1); the interaction was also detected when the order of antibodies was reversed (data not shown). These interactions between PP2A and tAg were detected in rat and mouse cells expressing all five JCV early proteins (Fig. 1, lanes 9–11), as well as in mouse cells expressing tAg only (Fig. 1, lane 12). Mutant JCV tAgs Interact with PP2A The unique C-terminal region of SV40 tAg contains a sequence, including two cysteines and a proline, which contributes to the tAg-PP2A interaction [23]. Alteration of any of the three residues reduces PP2A binding; substitution of alanine for the proline reduces binding to the greatest extent [23]. In addition, two motifs identified in the unique tAg sequence contain clusters of three cysteines that are involved in binding zinc ions. Mutation of any of the six cysteines in these two clusters reduces tAg stability, resulting in a half-life of 30–-60 minutes [41]; these motifs may also influence PP2A binding. The three sets of cysteine-containing sequences in the SV40 protein are conserved in the JCV tAg; CxxxPxC (amino acids 95–101) and CxCxxC (amino acids 109– 114; Cluster 1 and 136–141; Cluster 2) (Fig. 2). A recent study performed with JCV tAg deletion mutants found that amino acids 82 to 124 are critical to PP2A binding in vitro [40]. Based on this information, we substituted an alanine for the conserved proline at residue 99 in the JCV tAg. This residue is also immediately Figure 1. Wild type tAg interacts with cellular phosphatase PP2A in cells expressing JCV early proteins. JCV early proteins, expressed in Rat 2 (R2) or MEF cells transformed with pSR:T+/t+/T’+ or in G418-selected 3T3 cells transfected with pSR:T+/t+/T’+ (encodes all 5 JCV early proteins) or pSR:T2/t+/T’2 (encodes JCV tAg only) were incubated with anti-T monoclonal antibody PAb 962 (a-T; lanes 4–7) or anti-PP2A antibody (a-PP2A; lanes 9–12). The amount of total cell protein subjected to IP in lanes 9–12 was four times that employed in the corresponding samples in lanes 4–7. Immunoprecipitated proteins were separated on a 20% SDS-polyacrylamide gel, and WB analysis was performed using a cocktail of anti-T monoclonal antibodies to detect JCV early proteins either expressed in the different cell lines (lanes 4–7) or expressed and bound to PP2A (lanes 9– 12). Untransfected 3T3 and Rat 2 cells were included as negative controls (no JCV T proteins are present; lanes 1, 2), and a-mouse IgG was used in the IP step with the R2:T+/t+/T’+ cell extract to test for non-specific binding (lanes 3, 8). 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.g001 PLoS ONE | www.plosone.org 4 May 2010 | Volume 5 | Issue 5 | e10606