miRNA, however, has been experimentally shown to both interfere with HTLV-1 infected cell growth, as well as stimulate infected cell growth upon overexpression [90]. Overexpression of miR146a appears to favor the proliferation of an HTLV-1 infected cell population, however this effect is not always reproducible [67]. Due to the nature of the control of miRNA production in HTLV-1 infection, it is not surprising to observe an increase in viral replication in the absence of Drosha, due likely to the downreg- ulation of a cellular miRNA that would otherwise suppress viral replication. In order to investigate the control of miRNA production in HTLV-1 cells, we analyzed the pool of published downregulated miRNAs for those that target cellular proteins known to interact with Tax and regulate transcription. Our rationale here was that if HTLV-1 infection downregulates a particular miRNA which targets a cellular protein necessary for Tax transactivation, we could mimic this interaction by introduc- ing an antagomiR into uninfected cells, therefore disabling the endogenous miRNA and increasing protein expression. Based on three recent studies which examined expression changes of cellular miRNAs in HTLV-1 infected cell lines, we selected the downregulated miRNAs, miR-199a, miR-132, and miR-Let7i for further analysis [67,89–90,92]. In ovarian cancer cells, miR- 199a-3p has been shown to regulate IKK-b to affect NF-kB activity as well as targeting the SWI/SNF subunit BRM in a variety of human cancers [93–94]. Upon KSHV (Kaposi’s sarcoma-associated herpesvirus) infection, as well as HSV-1 (herpes simplex virus-1) and HCMV (human cytomegalovirus) infection, miR-132 is upregulated and regulates the transcriptional co-activator p300 [95]. Following microbioal infection of Crypto- sporidium parvum, miR-Let7i forms an inducible silencing complex with the NF-kB p50 subunit [96]. Confirming this rationale in the scope of HTLV-1 infection, we observed a increase in IKK-b and BRM protein levels in the presence of Tax and an opposite effect upon overexpression of functional Drosha. We observed an increase in cellular levels of IKK-b, p65, p50, GSK-3b, and BRM upon treatment with antagomiRs against the above downregulated miRNAs as compared to cells alone, indicating that the antagomiRs mimic the cellular protein expression seen in Tax-expressing cells. Tax plays a major role in the activation of cellular NF-kB, however, HTLV-1 infected cells also develop Tax- independent activation of NF-kB. This delicate balance between activation and suppression of these cellular genes via the manipulation of the RNAi pathway by HTLV-1 infection strongly supports the notion that dysregulation of Drosha and other core components of the RNAi machinery control the rate of infection and T-cell transformation. Materials and Methods Cell Culture Uninfected T-cell lines H9, Jurkat, and CEM cells as well as HTLV-1 infected, Tax-positive T-cell lines C81, MT2, and MT4 were obtained from the NIH AIDS Research and Reference Reagent Program. HTLV-1 infected, Tax-negative cell lines were a generous gift from Dr. Cynthia Pise-Masison (NCI/NIH, Bethesda, MD). HeLa cervical carcinoma cell line and 293T endothelial kidney cells were obtained from ATCC (Manassas, VA). Primary CD4+ T-cells (#2W-200, Lonza, Allendale, NJ) infection were a generous gift from Dr. Pooja Jain (Drexel University College of Medicine, Doylestown, PA). Suspension cell lines were maintained in RPM1-1640 media containing 10% FBS, 1% L-glutamine, and 1% streptomycin/penicillin (Quality Bio- logical) at 37uC in 5% CO2. Adherent cell lines were maintained HTLV-1 Tax and Drosha Interactions in DMEM containing 10% FBS, 1% L-Glutamine, and 1% streptomycin/penicillin (Quality Biological) at 37uC in 5% CO2. Antibodies and siRNA Antibodies used for confocal microscopy are as follows: a-BRG1 (sc-10768), a-Rb (sc-74562), and a-GIT2 (sc-5416) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), a-Drosha (ab12286) was obtained from Abcam (Cambridge, MA), a-Tax was generated in house, and AlexaFluor 488 Goat-anti-Rabbit (A11008), AlexaFluor 660 Donkey-anti-Rabbit (A21083), and AlexaFluor 568 Goat-anti-Mouse (A11004) were obtained from Invitrogen (Carlsbad, CA). Antibodies used for western blotting are as follows: a-Drosha (ab12286), a-DGCR8 (ab82876), a-Dicer (ab14601), a-Ago2 (ab57113), and a-Ago1 (ab5070-100) from Abcam (Cambridge, MA), a-Tax was generated in house, a-Actin (sc-1615), a-Ubiquitin (sc-9133), a-p65 (sc-7151), a-p300 (sc-585), and a-IKK-b (sc-7329) were obtained from Santa Cruz Biotech- nology, Inc. (Santa Cruz, CA), and a-GSK-3b (27C10) was obtained from Cell Signaling (Beverly, MA). SiRNA against Luciferase (D-002050-01-20) was obtained from Thermo Scientific (Rockford, IL) and siRNA against siDrosha (GS29102), siDGCR8 (GS54487), and siAgo2 (GS27161) were obtained from Qiagen (Valencia, CA). Electroporations/Transfections 293T cells were transfected with plasmid DNA according to Attractene lipid reagent recommended protocols (Qiagen, Valen- cia, CA). Briefly, 293T cells at 2.56105 cells were plated in DMEM +/+/+ (500 ml, with serum, with L-glutamine, with penicillin/streptomycin) in a 24-well plate. A reaction mixture containing pcDNA, pACH, pcTax, or pACH.M22 (0.1, 1.0, or 10 mg) plasmid DNA was incubated with Attractene reagent (3 ml) in DMEM2/2/2 (100 ml, without serum, without L-glutamine, without penicillin/streptomycin). The reaction mixture was incubated for 20 min at 25uC and then added dropwise onto the cells. Cells were collected 48 hours post-transfection. Lipid transfections of pcTax, pACH, and siRNA were performed according to Attractene and Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA) lipid reagent recommended protocols (Qiagen, Valencia, CA). Briefly, 2.56105 cells were plated in DMEM +/+/+ (500 ml) in a 24-well plate. A reaction mixture containing ACH DNA (5 mg) was incubated with Attractene reagent (3 ml) in DMEM 2/2/2 (100 ml). The reaction mixture was incubated for 20 min at 25uC and then added dropwise onto the cells. At 24 hours post-transfection, a reaction mixture containing siLuc (150 nM), siDrosha (50, 150, 300 nM), siDGCR8 (50, 150, 300 nM) or siAgo2 (50, 300 nM), was incubated with RNAiMax reagent (1.5 ml). The reaction mixture was incubated for 20 min at 25uC and then added dropwise onto the cells. Lipid transfections of the Tax expression vectors (Tax WT, TD1 (D1–37), TD55 (D55–92), TD99 (D99–142), TD150 (D150–198), TD254 (D254– 287), or TD319 (D319–353)) were performed in 6-well plates according to Lipofectamine 2000 recommended protocols (Invi- trogen, Carlsbad, CA). Lipid transfections of pACH.Tax (5 mg), pACH.M22 (5 mg), and pGFP-Drosha (10 mg) were performed with Attractene as indicated above. The wild-type pEGFP- C1_Drosha expression vector was kindly provided by Dr. Bharat Ramratnam (Warren Alpert Medical School, Brown University, Providence, RI). Confocal Microscopy HeLa cells were grown on coverslips for 48 hrs post pcTax transfection. Cells were fixed with 4% paraformaldehyde for one hour. Cells were permeabilized with Triton X-100 in PBS (0.5%) PLoS ONE | www.plosone.org 11 July 2012 | Volume 7 | Issue 7 | e40662