HTLV-1 Tax and Drosha Interactions Figure 6. Suppression of endogenous miRNAs dysregulated by HTLV-1. A) 293Ts were transfected with pACH.Tax (5 mg) and 24 hours later were transfected with pGFP-Drosha (10 mg). Cells were collected 48 hours post transfection and were western blotted (50 mg) for IKK-b, BRM and b- Actin. B) 293T cells were transfected with Tax (5 mg) or antagomiRs (100 nM) targeting miR-199a-3p, miR-132, or Let7i. Cells were collected 72 hours post transfection, and western blotted (50 mg) for IKK-b, p65, p50, GSK-3b, BRM, and b-Actin. Densitometry counts of western blots for each indicated protein normalized to b-Actin, plotted as a percent change (measured as arbitrary counts) between antagomiR treated cells and 293T cells alone. Densitometry counts were calculated using ImageJ. doi:10.1371/journal.pone.0040662.g006 HTLV-1 gene expression is regulated not only by Tax, but also by the viral protein Rex. Rex induces the expression of HTLV-1 structural proteins post-transcriptionally, by binding to the RxRE (Rex-responsive element) on the U3 region of the HTLV-1 LTR (long-terminal repeat) [81–83]. Recently, Rex has been shown to suppress the RNAi pathway by interacting with cellular Dicer, inhibiting the conversion of shRNA to siRNA [15]. As we have shown that Drosha is downregulated not only with HTLV-1 infection, but specifically in the presence of Tax, therefore we hypothesized that Tax could be interacting directly with Drosha. Tax-transfected HeLa cells show distinct, punctate foci in the nucleus when stained with antibodies against both Tax and Drosha in independent experiments. We observed that Tax and Drosha colocalize distinctly, however it is important to note that this HTLV-1-dependent downregulation of Drosha is not com- pletely efficient, and that there is still some endogenous Drosha remaining in these cells. This is analogous and similar to the colocalization of cellular Rb and Tax in cells (Figure 2D) as we have previously shown that Tax binds directly to Rb and is a necessary interaction for the subsequent degradation of Rb in vitro [47]. We next demonstrated that Drosha can be found in complex with Tax at the N-terminus, specifically at the region spanning aa 1–244. Within this N-terminal region of Tax, we observed two potential motifs that could be utilized for this interaction, the Zinc finger motif and the leucine-zipper-like region. The N-terminus of Tax is most commonly known for the CREB (cAMP-responsive element-binding)-transactivation domain where the transcription factors p300/CBP, and p/CAF (p300/CBP-associated factor) bind and promote viral transcription from the LTR [56]. We have specifically shown the interaction of Drosha and Tax with an immunoprecipitation of Drosha from Tax-expressing cells in the presence of proteasome inhibitors. We indicate that this interac- tion is not seen when a cell is expressing the Tax mutant, ACH.M22. Interestingly, the N-terminus portion of Tax is also known to interact with the proteasome, therefore this dual binding allows Tax to bring Drosha in close proximity with the proteasome complex, resulting in the degradation of Drosha. This proteasomal degradation is confirmed by a 50% recovery of total Drosha protein levels in HTLV-1 infected cells when treated with proteasome inhibitors PSI (cbz-ile-glu-(O-t-Bu)-ala-leucinal) and ALLN (N-acetyl-leu-leu-norleucinal). These proteasome inhibitors have also been well documented as anticancer and antiagiogenic therapeutics. Furthermore, a number of colleagues in the field have shown that proteasome inhibitors such as PS-341 or bortezomib can in fact downregulate HTLV-1 levels, resulting in a decrease of T-cell leukemia cells [84–88]. This is consistent with our siDrosha and proteasome inhibitor data, where a drug/ peptide inhibitor can regulate miRNA machinery by increasing proteins such as Drosha resulting in an increased host cellular RNAi response against HTLV-1 activity. Three large studies have recently examined the dysregulation of cellular miRNAs in both HTLV-1 infected cell lines and ATLL samples, resulting in surprisingly little overlap [67,89–91]. These studies do however provide a large pool of upregulated and downregulated cellular miRNAs from which to pull functional data in the context of HTLV-1 infection. We show here, that in the presence of Tax, endogenous Drosha is less functional and not as efficient in processing miRNAs. Drosha efficiency, as measured by the processing of endogenous miR326 as compared to overall levels of transcript, was highest in the presence of N-terminal Tax deletion mutants from residues 1–92. This again indicates that the interaction between Tax, Drosha, and the proteasome occurs on the N-terminal region of Tax. We also show that in the absence of Drosha, HTLV-1 viral replication increases by at least two-fold. The knockdown of DGCR8 also resulted in a significant increase in HTLV-1 replication, suggesting a dysregulation of the microprocessing complex. The downregulation of HTLV-1 replication with Ago2 knockdown was validated with a comparable downregulation when cells were treated with the RISC inhibitor ACF. We suggest that HTLV-1 dysregulates the RNAi pathway, including up- and down-regulation of cellular miRNAs by inhibiting the function of and degrading Drosha, resulting in a modulation of tumor and viral suppressing cellular functions. One miRNA upregulated in HTLV-1 infection, miR-146a, can be activated by Tax through an NF-kB-dependent pathway. This PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e40662