HTLV-1 Tax and Drosha Interactions Figure 2. Nuclear colocalization of HTLV-1 Tax with cellular proteins. HeLa cells were grown on coverslips in the presence or absence of HTLV-1 Tax. Forty-eight hours post transfection of pcTax (5 mg), cells were stained for BRG1 (A), Drosha (B, C), Rb (D), and GIT2 (E) and were visualized with confocal microscopy. Cellular protein staining is represented in green, Tax staining in red, and yellow foci represent colocalization of dual stained cells. Nuclei were stained with DAPI (blue). GIT2 serves as a cytoplasmic protein control. doi:10.1371/journal.pone.0040662.g002 blot is included as an indication that Drosha is being ubiquitinated in the presence of Tax, however, not in the presence of a Tax mutant (compare lane 5 to lane 7). A summary of relative Drosha binding affinity to Tax constructs from multiple experiments is depicted in Figure 3C. Based on these data, Tax binds to Drosha via the N-terminus (aa 1–244), given that the strongest binding was shown to occur within this region, and can also be identified in complex with Drosha. Collectively, these data indicate that Tax and Drosha interact and can be found in complex in vitro. Previously, we have shown that the binding of Tax and Rb results in a targeted degradation of Rb via the proteasome pathway [47]. To investigate whether Tax utilizes a similar mechanism to decrease Drosha protein levels, we designed experiments using proteasome inhibitors. We treated HTLV-1 infected C81 cells with proteasome inhibitors PSI and ALLN (0.1, 1.0, 10 mM). At 48 hours post-treatment, the cells were collected and western blotted for the presence of Drosha. Figure 3D shows the western blot of Drosha normalized to b-Actin. Both PSI and ALLN treated cells showed an increase in Drosha protein levels concurrent with increasing concentration of proteasome inhibitor. Indeed, the 10 mM-treated cells show almost a 30% increase in Drosha protein levels compared to C81 cells alone. All treated cells exhibited no toxicity across three independent experiments (data not shown). This data further indicates that Drosha protein levels PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e40662