HTLV-1 Tax and Drosha Interactions Figure 3. HTLV-1 Tax interacts with Drosha in vitro. A) HeLa whole cell lysates (1 mg) were incubated with purified GST-fusion proteins (,5 mg) bound to Glutathione-Sepharose beads: GST-Alone, GST-Tax wild type, GST-Tax N-terminus, 1–244, GST-Tax C-terminus, 244–336, and GST-Tax truncated, 288–353. Beads were washed gently, proteins eluted, and western blotted for the presence of Drosha. CEM whole cell extract serves as a positive Drosha control. B) 293T cell lysates were prepared from cells alone or cells transfected with pACH.Tax (5 mg) or pACH.M22 (5 mg) in the presence of the proteasome inhibitor PSI (10 mM). Approximately 1 mg of lysate was incubated with a-IgG, a-Drosha, or a-Tax overnight at 4uC. The next day, 50 ml of a 30% Protein A + G slurry was used to IP Drosha or Tax and subsequently western blotted for the presence of Drosha and Ubiquitin. Multiple isoforms of Drosha as detected by the antibody are indicated as Drosha I (170 kDa), Drosha II (156 kDa), and Drosha III (115 kDa). C) Graphical depiction of the GST-Tax constructs and the regions which they span for the Tax protein. Relative Drosha binding is based on densitometry counts. D) C81 cells were treated with the proteasome inhibitors PSI and ALLN at increasing concentrations (0.1, 1.0, and 10 mM) for 48 hours. Cell lysates were western blotted for the presence of Drosha. b-Actin serves as a loading control. Drosha recovery as a percentage of CEM Drosha levels is indicated from densitometry counts normalized to b-Actin using ImageJ. doi:10.1371/journal.pone.0040662.g003 can be rescued from suppression by HTLV-1 infection with proteasome inhibitors, suggesting that Drosha is targeted by Tax to be degraded via the proteasomal pathway. Collectively, these data point to the regulation of Drosha by direct binding to the N- terminus of Tax and further degradation by the proteasomal pathway. Tax Prevents Primary miRNA Cleavage by Drosha We have shown that Tax and Drosha interact in vitro and that this complex results in a downregulation/degradation of the cellular Drosha protein. This suppression of Drosha is not completely efficient however, as there is still some endogenous Drosha remaining in HTLV-1 infected cells. Here we question whether the remaining Drosha is functional in Tax expressing cells. We utilized a system where we quantify levels of cellular miR326 in Tax transfected cells normalized to a U6 control. We then calculate Drosha functional efficiency as a measure of the relative level of miRNA present versus the relative level of primary transcript, where 100% is the rate of production in the control. To this end, we transfected 293T cells with expression vectors (1 mg) for wildtype Tax or the following iterative Tax deletion mutants: TD1 (D1–37), TD55 (D55–92), TD99 (D99–142), TD150 (D150– 198), TD254 (D254–287), or TD319 (D319–353). We collected the cells 48 hours post-transfection and isolated total RNA by TRIzol extraction. The expression of miR326 was determined by the quantiMiR PCR kit and is shown as a fold change in Figure 4A normalized to U6 and relative to mock transfected control. This data indicates that the Tax D1 mutant and the TaxD55 mutant increase miR326 expression 1.5 and 4 fold, respectively, above basal levels. Interestingly, the Tax mutants spanning from residue 99 to 353 have no functional implications for miR326 expression. These mutants are as functional as the Tax WT, indicating that these regions of deletion are not critical for Drosha function. The significance of the Tax D1 and D55 mutants therefore implies that the regions of Tax deleted in these mutants are necessary for Drosha interaction as well as suppression of function. In order to compare the increase in expression of miR326 to the overall transcript levels in the cell, therefore ruling out non-specificity, we detected primary transcripts encoding miR326 against a region upstream of the miRNA hairpin. These values were normalized to GAPDH and are shown in Figure 4B as a ratio of the relative level of miRNA present over the relative level of primary transcript. The mock transfected cells were set to 100% efficiency. This data indicates an increase in Drosha efficiency of 100% over the mock transfected cells with the Tax D1 mutant. The Tax WT vector decreased Drosha functionality by 25% as compared to the PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e40662