proteins, which then aids in miRNA-mediated target recogni- tion. This guide effector protein complex assists in either degrading the targeted message or preventing its translation [10]. The dysregulation of this pathway is highly evident across a variety of cancers and viruses, including HIV-1, HTLV-1, Influenza, HCV, Ebola, Vaccinia, PFV-1, LACV, Adenovirus, and SARS-CoV although the mechanisms of action for most of these viruses remains to be determined [11–27]. Indeed, cellular miRNAs are able to silence endogenous retroviruses, sequences which typically share a high degree of homology to present day retroviruses, such as HTLV-1 and HIV-1. HTLV-1 Tax acts to transactivate the viral long terminal repeat (LTR) through Tax-responsive elements (TREs) in the U3 region. This occurs through transcriptional induction of TREs, posttranslational modifications of TRE-binding factors, and binding with transcription factors. Tax is known to interact with the transcription factors CREB, serum-responsive factor (SRF), and NF-kB as well as with the cell cycle related proteins Cyclin D2 and D3, mitotic checkpoint regulators (MAD1), cyclin-dependent kinases (cdks), cdk inhibitors p16INK4a and p21/waf1, and p53 [5,28–43]. Phosphorylation of Tax is necessary for Tax localization in nuclear bodies as well as activation of cellular gene expression through the NF-kB pathway. Tax activates HTLV-1 transcription through CREB and three CRE enhancer sequences on the LTR. Tax also interacts with CBP/p300, which is involved in the formation of the preinitiation complex as well as the p300/CBP-associated factor, P/CAF, which also plays a role in active HTLV-1 gene transcription [37,44–46]. Taken together, HTLV-1 Tax is heavily involved in direct interactions with critical cellular proteins that control not only viral gene expression but also cellular genes involved in tumor formation. We have previously shown that Tax interacts directly with the cellular Rb (Retinoblastoma) protein and targets Rb for degradation via the proteasome pathway, resulting in a decrease in Rb protein expression in HTLV-1 infected cells and a dysregulation of the cell cycle [47]. Due to the nature of Tax to manipulate and control cellular proteins such as Rb and overall cell cycle progression, we became interested in determining the effect of HTLV-1 infection and Tax expression on other cellular proteins regulating oncogenesis, including the RNAi pathway. HTLV-1 encodes an additional regulatory protein, called Rex, which has recently been implicated in having RNA silencing suppressor activity. Specifically, Rex interacts with both RNA and Dicer, suppressing Dicer’s enzymatic activity [15]. Recently, Tax has been shown to specifically mediate the downregulation of cellular miRNAs which are associated with the regulation of chromatin remodeling factors [48]. Specifically, Tax downreg- ulated miR-149 and miR-873, both of which directly target p300 and P/CAF [48]. This suggests that HTLV-1 infection and its viral transactivators may play a role in dysregulation of the RNAi pathway. Here we show that HTLV-1 infection in the presence of Tax, significantly downregulates Drosha protein expression. We show the in vitro interaction between Tax and Drosha, both in the nucleus and in complex in the presence of proteasome inhibitors. We observe a downregulation of Drosha functionality in the presence of Tax as measured by primary miRNA cleavage. Finally, the changes in cellular miRNA expression in HTLV-1 infected cells can be mimicked in uninfected cells supplementing excess functional Drosha or the addition of antagomiRs against the cellular miRNAs which are downregulated by the virus. HTLV-1 Tax and Drosha Interactions Results HTLV-1 Downregulates Core Components of the RNAi Machinery Tax acts as the HTLV-1 viral transactivator protein by interacting with various cellular and viral proteins including transcription factors, kinases, and chromatin remodeling proteins, ultimately altering cellular pathways such as apoptosis and the cell cycle to control viral replication and oncogenesis [6–8]. Recently, retroviruses have been shown to control the cellular RNAi pathway in order to evade viral inhibition and also to hijack both the enzymatic and catalytic components of the miRNA machin- ery. This viral response assists in promoting an active viral infection, or a latent state of infection [11,48–49]. Indeed, other retroviruses such as HIV-1 have been shown to impart a specific suppression of Dicer expression in monocyte-derived-macrophag- es as compared to T-cells [50]. In order to examine the role HTLV-1 infection has on possible control of the RNAi pathway, we initially screened for the protein levels of core components of the RNAi machinery in HTLV-1 infected cells as compared to uninfected cells. Total protein levels of Drosha, DGCR8, Dicer, Ago2, and b-Actin were detected in three uninfected T-cell lines, H9, Jurkat, and CEM, as well as in three HTLV-1 infected, Tax- positive T-cell lines, C81, MT2, and MT4 (Figure 1A, lanes 1–3 and lanes 4–6). Drosha protein levels were significantly downreg- ulated in all of the HTLV-1 infected, Tax-positive cells tested when compared to the uninfected cells. Specifically, Drosha is decreased by 62.6% in C81 cells, 97.3% in MT2 cells, and 91.5% in MT4 cells when compared to CEM cells (normalized to b- Actin). Dicer protein was also slightly downregulated in C81 cells, but more so in MT2 cells when compared to CEM cells (normalized to b-Actin). Conversely, DGCR8 and Ago2 showed no significant changes in protein level amongst all cell lines screened, suggesting that the downregulation seen for Drosha and Dicer is specific and not a consequence of the entire pathway being inhibited. Interestingly, the protein levels of the core components of the RNAi machinery were also detected in two HTLV-1 infected, Tax-negative cell lines (Figure 1A, lanes 7, 8). Drosha was downregulated by 77.1% in MT1 cells and 51.3% in ED- cells as compared to CEM cells (normalized to b-Actin). We titrated both MT1 and ED- cells in order to determine if this downregulation was an artifact and indeed, with increasing concentrations of protein extract (50, 75, and 100 mg), Drosha levels also increase (Figure 1B), suggesting that the relative decrease in Drosha is less in HTLV-1 infected, Tax-negative cells as compared to Tax-positive cells. We reproduced these results in HTLV-1 infected CD4+ primary T-cells (Figure 1C), where Drosha levels are dramatically downregulated as compared to uninfected cells (lane 2 compared to lane 1). In order to rule out the possibility that the loss of Drosha protein is due to a decrease in transcript levels, we performed an RT-PCR of total mRNA isolated from CEM, MT2, ED-, and MT1 cells for Drosha transcripts. In Figure 1D, we show that the Drosha transcript levels are unchanged as compared to GAPDH across all four of these cell lines. Next, we attempted to mimic this downregulation of Drosha and Dicer by introducing both Tax and HTLV-1 into uninfected 293T cells and screening for core RNAi machinery components. 293T cells were transfected with three concentrations (0.1, 1.0, 10 mg) of plasmids expressing pACH (full-length HTLV-1 clone), pcTax, pACH.M22 (mutated tax gene), or an empty vector control. The pACH.M22 plasmid contains a double amino acid substitution at positions 130 and 131 and eliminates the ability of Tax to transactivate the NF-kB pathway [51–54]. Cells were PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e40662