HTLV-1 Tax and Drosha Interactions Figure 4. Tax prevents primary miRNA cleavage by Drosha. 293T cells were transfected with Tax expression vectors (1 mg): Tax WT, TD1 (D1– 37), TD55 (D55–92), TD99 (D99–142), TD150 (D150–198), TD254 (D254–287), or TD319 (D319–353). A) Forty-eight hours post transfection, total RNA was isolated by TRIzol extraction and expression of miR326 was determined by quantiMiR PCR kit using primers specific to miR326. Values shown are normalized to U6 and shown relative to control. B) Primary transcripts encoding miR326 were detected by RT-qPCR against a region upstream of the miRNA hairpin. Quantities were normalized to GAPDH and efficiency of Drosha processing was determined by charting the expression of mature miRNA over the expression of the primary transcript. Data is shown with the efficiency in the control cells set to 100%. C) A graphical depiction of the Tax mutant constructs TD1 and TD55 with deleted regions and the corresponding domains and motifs of full length Tax. The function of each domain is indicated in brackets and arrows, as well as the associated interacting cellular proteins below each region. The proposed Drosha binding region is indicated by the grey shaded box. doi:10.1371/journal.pone.0040662.g004 chemokines, inhibit the viral DNA polymerase BALF5, and downregulate the cellular protein PUMA (p53 upregulated modulator of apoptosis) [72–75]. This complex interaction network between viruses and the RNAi pathway can be best described as reciprocal due to host cell miRNAs targeting both cellular and viral transcripts as well as viral miRNAs targeting both cellular and viral transcripts. Additionally, viruses can produce RNA molecules and express proteins which disrupt the cellular RNAi pathway, resulting in either induction or suppres- sion. EBV induces the expression of the cellular miR-155 which assists in the expansion of infected B-cells and promotes their transformation whereas HIV-1 generates both RNA sequences and proteins which suppresses the RNAi pathway. Indeed, HIV-1 has been shown to not only encode its own TAR (trans-activation response region)-derived miRNA which acts as an RNAi decoy, but it also utilizes the viral transactivator Tat as an RNAi suppressor [14,21–22,76–80]. In this study, we characterize the effect HTLV-1 infection has on the cellular RNAi pathway. We have demonstrated that Drosha is downregulated in HTLV-1 infected cell lines, HTLV-1 transfected cells, and infected primary cells. We have also shown that Dicer is downregulated in HTLV-1 infected cell lines only, suggesting that this loss of Dicer could be due to a cell line specific phenomenon and not so much HTLV-1 infection or early stage replication. Figure 5. Loss of Drosha increases viral replication. A) 293T cells were transfected with HTLV-1 pACH (5 mg) and 24 hours later were transfected with siRNAs against Luciferase (150 nM), Drosha (50, 150, and 300 nM), DGCR8 (50, 150, 300 nM), and Ago2 (50, 150, 300 nM) or were treated with 0.1, 1.0, or 2.5 mM Acriflavine (ACF). At 72 hours post-transfection, supernatants were collected and assessed for viral replication by RT (reverse transcriptase) assay. Data was collected in triplicate from at least 2 independent experiments. B) 293T cells from panel A were assayed by western blot for the efficiency of siRNA knockdown of the target proteins. b-Actin serves as a positive loading control. doi:10.1371/journal.pone.0040662.g005 PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e40662