control. It is interesting that the Tax D1 mutant increased the efficiency of processing of Drosha 100%, however, only increased the relative miR326 expression 1.5 fold. These two values are not necessarily proportional as the extrapolation of an active, functional Drosha in the presence of this mutant, does not necessarily have to result in a direct effect on miR326. In Figure 4C we represent a more detailed schematic of the binding domains and motifs of Tax. We depict important binding and interactive regions of Tax with brackets, including the numbered residues. We also depict the cellular proteins with which Tax interacts below each sequence of interest. We propose that Drosha is binding to the N-terminus of Tax, however, more specifically to the region of Tax from 1–92, depicted as the grey shaded area. We have previously confirmed that Tax interacts with the proteasome at residues 23–62, which is also the site of the NLS, Zn finger, as well as the domain involved in CREB activation [47]. This data agrees with the interactions of Tax and Drosha in Figure 3, where the N-terminus of Tax is responsible for Drosha binding. Collectively, these data indicate that the Drosha in Tax-containing and HTLV-1 infected cells is mostly functionally inactive and the functional suppression of Drosha is dependent on its interaction with a small region of the N-terminus of Tax. Loss of Drosha and DGCR8 Increases Viral Replication We have shown above that Drosha is downregulated, degraded, and mostly inactive in HTLV-1 infected cells, however, it was not clear what effect this dysregulation of Drosha would have on viral replication. Here we investigated whether loss of cellular Drosha by siRNA knockdown could affect viral replication. In doing this, it is important to distinguish between a Drosha-specific dysregu- lation as opposed to an overall disruption of the RNAi pathway, therefore we used siRNA against other RNAi components as a control. We transfected 293T cells with HTLV-1 infectious pACH clone (5 mg), and 24-hours post transfection, with siRNAs against Luciferase (150 nM), Drosha (50, 150, 300 nM), DGCR8 (50, 150, 300 nM), and Ago2 (50, 150, 300 nM). At 72hours post- transfection, we collected cell culture supernatants and assayed for the presence of virus using an RT (reverse transcriptase) assay. Results of such an experiment are shown in Figure 5A. Here we observed that pACH clone was able to replicate well with the highest concentration of siDrosha and even more dramatic results were obtained with siDGCR8. Interestingly, the loss of DGCR8 across all three concentrations of siRNA resulted in a consistent activation of viral replication. A knockdown of Ago2 did not result in a significant increase in viral replication. Western blot confirmation of the siRNA knockdowns are shown in Figure 5B. In order to determine whether or not the inhibition of Ago2 would result in a decrease in viral fitness and an inhibition of replication, we adopted an alternative approach and treated 293T cells transfected with pACH with the RISC/Ago2 inhibitor Acriflavine (ACF) (0.1, 1.0, and 2.5 mM) [66]. At 72 hours post transfection, we collected supernatants and assayed for the presence of virus via RT assay. We observed that that the efficient, drug inhibition of Ago2 function decreases viral replication at levels comparable to siAgo2 transfection. Collectively, these data indicate that the loss of Drosha and DGCR8 in HTLV-1 infected cells results in an increase in viral replication and its release from the cell. Suppression of Cellular miRNAs by HTLV-1 can be Rescued by Over Expression of Drosha and Mimicked by antagomiRs We were next interested in the regulation and downstream effects of the RNAi pathway in HTLV-1 infected cells. HTLV-1 HTLV-1 Tax and Drosha Interactions infection results in a dramatic up (i.e. miR-130b, miR-18a, miR- 20b) and downregulation (i.e. Let-7i, miR-132, miR-199a) of many host cellular miRNAs [67]. The modulation of these host miRNAs has an effect on the expression of cellular proteins such as p300, NF-kB, and BRM, to name a few, which are all recruited by Tax and play a key role in activating HTLV-1 gene transcription [5,30–36,38–40,44–45,56,60,68–71]. To better define the func- tional significance of Drosha downregulation by Tax, we transfected 293T cells with pACH.Tax (5 mg) and 24 hours later added back pGFP-Drosha (10 mg). The cells were collected 72 hours later and were lysed (50 mg) for western blots of the corresponding downstream proteins, IKK-b, BRM, and b-Actin. Here the rationale was that Tax would decrease Drosha levels, resulting in downregulation of miRNA, such as Let7i, miR-199a- 3p and miR-132. Downregulation of these miRNAs would in turn effect the translation of mRNAs for genes such as IKK-b and BRM (possibly regulated by miR-199a-3p) and other proteins important for HTLV-1 gene expression. Data in Figure 6A shows that transfection of Tax downregulates both IKK-b and BRM as compared to the control Drosha add back experiment (compare Lanes 2 and 3). On the other hand, the evidence of Drosha downregulation in the presence of Tax implies the downregulation of cellular miRNAs in response to HTLV-1 infection and subsequent increase in target protein expression. Of particular interest are a set of cellular miRNAs, such as Let-7i, miR-199a-3p, and miR-132 that are downregulated after infection, resulting in an upregulation of cellular proteins that are known to be recruited and utilized by the HTLV-1 promoter. In order to mimic the effect Tax has on cells by sequestering the endogenous miR, therefore allowing for the basal, unchecked expression of these cellular proteins, we transfected 293T cells with either Tax or an antagomiR against endogenous cellular miRs which are down- regulated with HTLV-1 infection. AntagomiRs used include anti- hsa-Let-7i to target p50 and p65 miRNA, anti-hsa-miR-132 to target GSK-3b miRNA, and anti-hsa-miR199a-3p to target IKK- b and BRM miRNA. Cells were collected 72 hours post transfection and western blots were performed for IKK-b, p65, p50, GSK-3b, and b-Actin. Results in Figure 6B are shown as 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. These results indicate that there was a dramatic increase of protein expression of IKK-b, and p65 of approximately 300 and 250%, respectively. There was an approximate 10% increase in protein levels of p50, GSK-3b and BRM upon antagomiR treatment as compared to cells alone. Collectively, these data indicate that proteins, such as IKK-b, among others, may directly be regulated by the Tax/Drosha interaction in HTLV infected cells. Discussion HTLV-1 is an oncogenic retrovirus which targets and manipulates common cellular tumor suppressors and signaling pathways in order to establish and maintain a productive infection. Recently, the role of miRNAs in both tumor development and viral infections has provided evidence that the RNAi pathway itself is also targeted by this virus. Indeed, all DNA tumor viruses, with the exception of human papillomaviruses, encode viral miRNAs which modulate tumorigenesis, therefore indicating that the cellular RNAi regulatory pathway is important for viral patho- genesis and control. For example, Epstein Barr Virus (EBV) encodes 25 viral miRNA precursors which are expressed differentially amongst various stages of latency and act to suppress PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e40662