JCV Small t Protein Functions Figure 6. Mutation of tAg residues proline 99 and cysteine 157 reduce JCV DNA replication. PHFG cells in 60 mm plates were transfected in duplicate with 400 ng of wild type (T+/t+/T’+) or 8 different mutant (muT+/t2/T’+, T+/P99At+/T’+, T+/C157At+/T’+, T+/t+/T’2, T+/t2/T’2, muT+/t+/T’+, T2/t+/T’2, T+/t2/T’+) JCV genomes. Duplicate, independent samples representing each DNA construct were extracted by the method of Hirt [39] on days 0, 7, 10 and 14 p.t. and analyzed using the Dpn1 assay as described in the legend to Figure 5. The marker (M) shown in the first lane of each blot is 1 ng of linear JCV DNA (5130 bp), and the position of Dpn1-resistent replicating genomes is denoted by an arrow at days 7, 10 and 14 p.t. doi:10.1371/journal.pone.0010606.g006 These sequences have diverged to the greatest extent with only 44% sequence identity between JCV and SV40. Experiments with the hybrid viral genomes also revealed the early coding regions to be sources of biological variation [35,48,50]. Although the N-terminal region of the tAgs share 82% sequence identity, the unique C-terminal portions, with only 54% identity, represent the sequences with the least amount of similarity among the early proteins; these sequences were the primary focus of the current study. Very little work has been conducted with the JCV tAg, although numerous studies with its SV40 counterpart suggest interesting initial experiments to perform. The binding of the unique region of the SV40 tAg to PP2A provides the mechanism by which tAg induces most observed alterations to the behavior and morphology of cells [22,31,51–54]. The only other interaction predicted for SV40 tAg occurs with the molecular chaperone Hsc70 via its J domain located within the N-terminal 82 amino acid sequences shared with TAg and 17kT. JCV tAg may bind to these and additional cellular and viral proteins. Purified GST-JCV tAg prepared in bacteria binds PP2A in vitro [40], and JCV tAg expressed in mammalian cells binds PP2A in vivo (current study). Many of the outcomes reported to result from the SV40 tAg-PP2A interaction, including those involving the activation of the PI3K and MAPK pathways, are likely to occur following contact between JCV tAg and PP2A. The JCV tAg has been shown to interfere with the phosphatase activity of PP2A in studies involving a second known binding partner of tAg, the JCV agnoprotein [40]. We were surprised to identify two LxCxE motifs that had been overlooked in the unique region of the JCV tAg; one of these sites is also found in BKV but neither site resides in the corresponding SV40, WUV, KIV or MCV polyomavirus proteins. As predicted, JCV tAg binds members of the retinoblastoma protein family. These interactions are relevant to the potential contributions tAg may make to JCV oncogenic activities. While the tAg-PP2A interaction may activate signaling pathways critical to cell growth and survival, effects on Rb proteins may also influence these processes via cell cycle regulation. The LxCxE motif and the tripeptide HDP of the J domain of the polyomavirus tumor proteins make direct contacts with the Rb proteins and Hsc70, respectively; together they effect the release of members of the E2F family of transcription factors from their Rb partners to promote cell cycle progression [reviewed in 55]. There is less certainty about the identity of tAg amino acids that are in direct contact with PP2A. Mutations introduced into the two CxCxxC clusters in the C terminus of SV40 tAg yield an unstable protein and disrupt zinc binding [23]. Under these conditions, the role of these two clusters in PP2A binding could not be determined. Mutations to the two cysteines and one proline of the CxxxPxC motif located upstream of the CxCxxC clusters did reduce PP2A binding significantly. Additional studies that relied upon peptide inhibition of the tAg-PP2A interaction without destabilizing tAg suggested that the CxCxxC clusters might indeed influence tAg binding to the A subunit [23,25]. Two recent X-ray crystallographic studies indicate the CxCxxC clusters and the CxxxPxC sequence in the C terminus of SV40 tAg and the HDPKGG sequence of the N-terminal J domain contribute to tAg binding to the scaffolding A and catalytic C subunits of PP2A [53,54]. To begin to identify JCV tAg residues critical to PP2A and Rb interactions and to DNA replication activity, we introduced mutations into the conserved CxxxPxC sequence and the unique LxCxE motif within the C-terminus of JCV tAg. Using a co- immunoprecipitation approach, we confirmed our prediction that the P99A mutant tAg would exhibit a loss of PP2A binding activity. This proline is conserved in the tAgs of primate polyomaviruses as well as of mouse polyoma virus. PLoS ONE | www.plosone.org 9 May 2010 | Volume 5 | Issue 5 | e10606