Localization and Sub-Cellular Shuttling of HTLV-1 Tax with the miRNA Machinery Rachel Van Duyne1,2, Irene Guendel1, Zachary Klase3, Aarthi Narayanan 1 , William Coley2, Elizabeth Jaworski1, Jessica Roman 1 , Anastas Popratiloff4, Renaud Mahieux5, Kylene Kehn-Hall1, Fatah Kashanchi1,2* 1 Department of Molecular and Microbiology, National Center for Biodefense & Infectious Diseases, George Mason University, Manassas, Virginia, United States of America, 2 Department of Microbiology, Immunology, & Tropical Medicine, The George Washington University Medical Center, Washington, D.C., United States of America, 3 Molecular Virology Section, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America, 4 Department of Anatomy and Regenerative Biology, The George Washington University, Washington, D.C., United States of America, 5 Retroviral Oncogenesis Team, INSERM-U758 Virologie Humaine, Lyon, France Abstract The innate ability of the human cell to silence endogenous retroviruses through RNA sequences encoding microRNAs, suggests that the cellular RNAi machinery is a major means by which the host mounts a defense response against present day retroviruses. Indeed, cellular miRNAs target and hybridize to specific sequences of both HTLV-1 and HIV-1 viral transcripts. However, much like the variety of host immune responses to retroviral infection, the virus itself contains mechanisms that assist in the evasion of viral inhibition through control of the cellular RNAi pathway. Retroviruses can hijack both the enzymatic and catalytic components of the RNAi pathway, in some cases to produce novel viral miRNAs that can either assist in active viral infection or promote a latent state. Here, we show that HTLV-1 Tax contributes to the dysregulation of the RNAi pathway by altering the expression of key components of this pathway. A survey of uninfected and HTLV-1 infected cells revealed that Drosha protein is present at lower levels in all HTLV-1 infected cell lines and in infected primary cells, while other components such as DGCR8 were not dramatically altered. We show colocalization of Tax and Drosha in the nucleus in vitro as well as coimmunoprecipitation in the presence of proteasome inhibitors, indicating that Tax interacts with Drosha and may target it to specific areas of the cell, namely, the proteasome. In the presence of Tax we observed a prevention of primary miRNA cleavage by Drosha. Finally, the changes in cellular miRNA expression in HTLV- 1 infected cells can be mimicked by the add back of Drosha or the addition of antagomiRs against the cellular miRNAs which are downregulated by the virus. Citation: Van Duyne R, Guendel I, Klase Z, Narayanan A, Coley W, et al. (2012) Localization and Sub-Cellular Shuttling of HTLV-1 Tax with the miRNA Machinery. PLoS ONE 7(7): e40662. doi:10.1371/journal.pone.0040662 Editor: Dong-Yan Jin, University of Hong Kong, Hong Kong Received February 1, 2012; Accepted June 11, 2012; Published July 10, 2012 Copyright: 2012 Van Duyne et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The confocal images and the GWU CMIA are supported by a grant from the NIH, National Center for Research Resources, 1S10RR025565. Further support came from grants from the George Mason university funds to FK and NIH grants AI078859 and AI074410-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: fkashanc@gmu.edu Introduction Human T-lymphotropic virus type 1 (HTLV-1) was originally discovered in 1980, identified as the first human retrovirus, and currently infects more than 20 million people worldwide [1–5]. HTLV-1 is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in infected individuals. Oncogenesis is due primarily to the viral transactivator protein, Tax, a 40-kDa phosphoprotein that regulates not only viral transcription, but acts to manipulate host cellular functions such as cell cycle progression, apoptosis, chromatin remodeling, and other signal transduction pathways [6–8]. Recently, much interest has developed in elucidating the cross-talk between tumor development and HTLV-1 infection as it relates to the innate host response, in particular the small RNA regulatory network. Human microRNA (miRNA) sequences derived from the genome have the ability to silence cellular genes and are currently considered a primary host immune defense against cellular invaders such as pathogens and viruses. In a host cell, miRNAs are the product of the RNA interference (RNAi) pathway, a regulatory and innate defense mechanism that is conserved in eukaryotes. This pathway utilizes short non-coding RNA sequences of 18–21 nucleotides to bind to mRNA sequences with complementary homology, subsequently restrict- ing the translation of these transcripts [9]. Following RNA Pol II transcription of a gene, the Pri-miRNA consists of a series of RNA hairpins protruding from an RNA message with a 59cap and poly-A tail. This Pri-miRNA is cleaved by a microprocessor complex of nuclear proteins, Drosha, an RNase II endonucle- ase, and DCGR8 (Pasha), an RNA-binding protein, to form a stem and loop RNA-structure called Pre-miRNA. This Pre- miRNA is shuttled out of the nucleus into the cytoplasm via Exportin5 and is further processed by an additional RNase III enzyme, Dicer, which cleaves the hairpin into a short miRNA duplex. These miRNA then associate with the RNA induced silencing complex (RISC), composed of Ago2 and TRBP PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e40662