HTLV-1 Tax and Drosha Interactions Figure 1. The RNAi machinery is dysregulated with HTLV-1 infection. A) Uninfected (H9, Jurkat, CEM), HTLV-1 infected, Tax-positive (C81, MT2, MT4), and HTLV-1 infected, Tax-negative (ED-, MT1) T-cell lines were screened for endogenous protein levels of Drosha, DGCR8, Dicer, and Ago2. Seventy-five micrograms of total lysates were used for western blots. Multiple bands for DGCR8 are indicative of isoforms at 86 and 65 kDa. b-Actin serves as a loading control. B) Total lysates of HTLV-1 infected, Tax-negative cell lines ED- and MT1 were titrated (50, 75 and 100 mg) and screened for endogenous protein levels of Drosha, DGCR8, Dicer, and Ago2. Multiple bands for DGCR8 are indicative of isoforms at 86 and 65 kDa. b-Actin serves as a loading control. C) Whole cell lysates (50 mg) from HTLV-1 infected primary CD4+ T-cells were run on a 10% Tris-glycine SDS-PAGE gel and western blotted for Drosha. Data is representative of three independent experiments. b-Actin serves as a loading control. D) Total RNA was isolated with TRIzol from CEM, MT2, ED-, and MT1 cell lines. RT-PCR was performed for cellular Drosha transcript levels. GAPDH serves as a housekeeping gene loading control. E) Uninfected 293T cells were transfected with pcDNA (0.1, 1.0, and 10 mg), pACH (0.1, 1.0, and 10 mg), pcTax (0.1, 1.0, and 10 mg), or pACH.M22 (0.1, 1.0, and 10 mg) and were screened for protein levels of Drosha, Dicer, Ago2, Ago1, and Tax. Seventy-five micrograms of total lysates was used for western blots. b-Actin serves as a loading control. F) Total RNA was isolated with TRIzol from the transfected cells in panel E. RT-PCR was performed for cellular Drosha transcript levels. GAPDH serves as a housekeeping gene loading control. doi:10.1371/journal.pone.0040662.g001 collected 48 hours post-transfection and were western blotted for the presence of Drosha, Dicer, Ago2, Ago1, Tax and b-Actin. Figure 1E shows endogenous protein levels of core RNAi machinery components in 293T cells transfected with pcDNA and other vectors. Comparing Drosha proteins levels in cell expressing pACH, pcTax, and pACH.M22 to the pcDNA control (lanes 6, 7, 8 and 9, 10, 11 and 12, 13, 14 to lanes 2, 3, 4, respectively) results in a 27.5% decrease in pACH (10 mg) expressing cells (lane 8), a 67.3% decrease in pcTax (10 mg) expressing cells (lane 11) and only a 10.4% decrease in pACH.M22 (10 mg) expressing cells. These results imply that in the pACH.M22 mutant Tax expressing cells, we observe close to endogenous levels of Drosha, indicating the downregulation of Drosha may be the result of a functional Tax. Overall, relative expression levels of Dicer, Ago2, and Ago1 reproduce the data from Figure 1A. The Tax western blot serves as a positive control for Tax expression (lanes 2 and 6–14). The b-Actin western serves as a positive loading control. Again we confirmed that this decrease in Drosha protein levels is due to Tax expression and not due to an effect on total transcript levels (Figure 1F). Collectively, these data indicate that HTLV-1 infection in the presence of Tax downregulates the RNAi enzyme Drosha in chronically infected cell lines, transfected cells, and primary cells infected with HTLV- 1 virus. Colocalization of Tax with Drosha in the Nucleus The viral protein Tax has been well documented as interacting directly with multiple critical cellular proteins that control oncogenesis. We sought to determine whether or not Tax interacts with Drosha in order to downregulate and/or signal the Drosha protein for possible degradation. To investigate the potential interaction of Tax and Drosha, we transfected pcTax into HeLa cells and stained the cells 48 hours post-transfection for confocal imaging. We labeled both untransfected and Tax-transfected HeLa cells with antibodies against BRG1, Drosha, Rb, GIT2, and Tax. BRG1 and Rb serve as positive Tax-interacting proteins as we have previously shown their intracellular interaction [1,47]. BRG1 is constitutively expressed in HeLa cells in the absence of Tax, however post-Tax transfection, BRG1 is found almost exclusively in the nucleus and colocalizes with Tax (Figure 2A). Similarly, Rb is heterogeneously expressed in HeLa cells, however colocalizes with Tax in the nucleus of Tax-expressing cells (Figure 2D). Interestingly, in Tax expressing cells, Drosha is almost exclusively found in the nucleus at specific foci colocalizing with Tax (Figures 2B, C). In order to address possible nonspecific shuttling between the nucleus and the cytoplasm due to transfection conditions or overexpression of Tax, we utilized GIT2 to serve as a negative control for a protein that does not shuttle to the nucleus or colocalize in the presence of Tax (Figure 2E). Therefore, our imaging data show that Tax and Drosha colocalize in the nucleus of Tax-expressing cells. Drosha is Found in Complex with Tax The full length Tax protein contains a variety of domains that impart specific protein-protein interactions as well as interactions with sub-cellular components. The N-terminus of Tax contains both a nuclear localization signal (NLS) as well as a zinc-finger domain which is involved with interactions with cellular transcription factors [31,49,55–57]. Specifically, this domain of Tax is responsible for Tax-mediated CREB transactivation. The C-terminus of Tax is responsible for nuclear-cytoplasmic shuttling and contains a Golgi localization motif, a secretion motif, an LXCXE-like motif, and a PDZ-binding domain [5,47,58–64]. Additionally, the region of Tax between the N- and C-termini includes two leucine zipper motifs used for dimerization as well as activation of NF-kB [5,58–61]. We have previously shown that Tax is capable of binding to cellular Rb via its LXCXE-like motif [47]. To identify the domain of Tax that binds to Drosha, we performed an in vitro binding assay using full-length Tax and various fragments covering the entire Tax protein fused to GST. Figure 3A shows the results of a GST-Tax pulldown from HeLa whole cell extracts, followed by a western blot for the presence of Drosha. Drosha bound to wild type Tax, but much stronger binding was observed at the N-terminal domain that contained sequence 1–244. This is partly because the C-terminus may fold back and contain inhibitory activity in an in vitro binding assay. We have also confirmed the interaction of Tax and Drosha in vitro with an immunoprecipitation (IP). We transfected 293T cells with the HTLV-1 clone pACH.Tax (5 mg) or pACH.M22 (5 mg) mutant Tax in the presence of the proteasome inhibitor PSI (10 mM). Our rational for using a proteasome inhibitor was to be able to isolate complexes that were available for biochemical analysis prior to the targeted degradation via the proteosome pathway. We collected cells 48 hours post transfection and lysed whole cell extracts (,1 mg) for an overnight IP with a-Tax, a-Drosha, or a-IgG antibodies. The following day, Protein A + G beads were incubated with the extracts for 2 hours, the beads were washed with low salt buffer, proteins eluted in Laemmli buffer, and were subsequently western blotted for the presence of Drosha. As shown in Figure 3B, Drosha can be successfully IPed out of cells expressing wild type Tax with a-Tax in the presence of a proteasome inhibitor (lane 5). 293T cells alone serve as a positive control for the Drosha western blot (lane 1) as well as a positive control for the Drosha IP/western blot (lane 3). Interestingly, the mutant ACH.M22 Tax is unable to interact with Drosha (lane 7). This Tax mutant is only faintly ubiquitinylated and can’t interact with the proteasome, therefore indicating that the interaction of Tax and Drosha and the subsequent degradation requires interaction with the proteasome [65]. The a-Drosha antibody used for the western blot recognizes all three isoforms of Drosha, corresponding to the molecular weights of 170, 156, and 115 kDa, which accounts for the multiple Drosha bands. However, the strongest Tax binding is to the 170 kDa form. A ubiquitin western PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e40662