JCV Small t Protein Functions Figure 2. Comparison of JCV and SV40 tAg amino acid sequences. The JCV tAg sequence containing 172 amino acids is indicated in red letters, and the 174 amino acid SV40 tAg is shown in black letters. The sequences following the vertical line comprise the unique C-terminal region of each protein. Two conserved cysteine (CxCxxC) clusters (Cluster 1, 2) that contribute to SV40 tAg binding to zinc ions, and possibly to PP2A, are noted in both sequences, as are two recently recognized LxCxE motifs in JCV tAg. Three amino acid residues are highlighted with a blue letter and arrow. Histidine 42 (H42) is a conserved residue in the HPDKGG hexapeptide motif of the J domain that spans most of the N-terminal region of tAg as well as the other JCV and SV40 early proteins; H42 is required for functional interaction with Hsc70. Proline 99 is within a conserved CxxxPxC sequence that influences SV40 tAg binding to PP2A. Cysteine 157 is the central residue of the newly recognized second LxCxE motif in JCV tAg. doi:10.1371/journal.pone.0010606.g002 adjacent to an LxCxE domain found in the JCV, but not SV40, tAg (Fig. 2). In addition, a cysteine (residue 157) present in a second LxCxE motif in the JCV tAg (residues 155–159) was changed to an alanine. Both tAg point mutants were examined for their ability to bind to PP2A and to members of the Rb family of tumor suppressor proteins. 3T3 cells were stably transfected with DNA constructs encoding the wild type, P99A or C157A tAgs. Lysates were prepared from cells expressing all five JCV early proteins or tAg only, and total protein amounts used in co-IP experiments were adjusted to yield comparable tAg levels. These protein extracts were immunopre- cipitated either with anti-T or anti-PP2A antibody and blotted with anti-T-protein antibodies (Fig. 3). Relative to PP2A-tAg interactions observed in two independently-derived cell lines expressing wild type JCV early proteins (Fig. 3A, lanes 5, 6), binding of PP2A to the mutant P99A tAg in two different lines was nearly undetectable (Fig. 3A, lanes 7, 8). The binding of mutant C157A tAg to PP2A in one cell line was similar to that of the wild type tAg (Fig. 3B, compare lane 8 with lanes 6, 7), but binding of the tAg in the second C157A line was reproducibly higher than that of wild type tAg (Fig. 3B, compare lane 9 with lanes 6, 7). Binding to PP2A was again observed in cells producing wild-type tAg in the absence of the other four JCV early proteins (Fig. 3B, lane 10). As observed in the first set of experiments (Fig. 1), the tAg-PP2A interaction in this 3T3 cell line was less robust than that observed in cells expressing all five JCV early proteins. JCV tAg Physically Interacts with Members of the Rb Family of Proteins Bollag and co-workers [7] demonstrated that the LxCxE motif present in the JCV TAg and T9 proteins binds members of the Rb family of tumor suppressor proteins. While performing these co- IP/WB experiments, a 20 kDa band was observed to interact with p107 and p130. Although JCV tAg is 20 kDa, it was ruled out because an LxCxE motif had not been identified in any of the polyomavirus tAgs. Upon reexamination of the JCV sequence, we recognized two LxCxE domains in the unique C-terminal coding region of JCV tAg (Fig. 2). The first of these, residues 93–97, is common to JCV and BKV but not to SV40 or the other 3 human polyomaviruses (WUV, KIV, MCV); the second, residues 155– 159, is unique to JCV tAg. To determine whether JCV tAg interacts with the Rb family of proteins, we performed co-IP/WB upon lysates from cells expressing all five JCV early proteins or tAg alone, and with total protein amounts adjusted to comparable levels of tAg expression in each cell line. Lysates were incubated with Molt 4 extracts overnight to compensate for the low levels of expression of Rb proteins in the rat and mouse cells [7,8], immunoprecipitated with anti- p107 antibody and immunoblotted with a cocktail of antibodies that recognize the JCV early tumor proteins. All five JCV early proteins bound p107. The interaction between T9165 and p107 is difficult to detect, not only because T9165 binding is inefficient [7], but also because it is expressed at low levels relative to the other early viral proteins. Wild type JCV tAg present in both rat and mouse cells expressing the intact JCV early region was found to interact comparably with p107 (Fig. 4, lanes 7–9), while the binding of tAg to p107 without co-expression of the other four early proteins was only slightly decreased (Fig. 4, lane 11). The C157A tAg which carries a mutation that disrupts one LxCxE domain, also interacted with p107 (Fig. 4, lane 10). Comparable binding of the wild type and mutant tAgs to p130 was also detected (data not shown). As noted with the other JCV early PLoS ONE | www.plosone.org 5 May 2010 | Volume 5 | Issue 5 | e10606