A Nuclear Localization of the Infectious Haematopoietic Necrosis Virus NV Protein Is Necessary for Optimal Viral Growth Myeong Kyu Choi1,5., Chang Hoon Moon1,7., Myoung Seok Ko1., Unn-Hwa Lee1, Wha Ja Cho2, Seung Ju Cha2, Jeong Wan Do 3 , Gang Joon Heo4, Soo Geun Jeong 5 , Yoo Sik Hahm5, Abdallah Harmache6, Michel Bremont6, Gael Kurath7, Jeong Woo Park1* 1 Department of Biological Sciences, University of Ulsan, Ulsan, Korea, 2 Biomedical Research Center, Ulsan University Hospital, College of Medicine, University of Ulsan, Ulsan, Korea, 3 South and West Sea Fisheries Research Institute, National Fisheries Research and Development Institute, Yeosu, Korea, 4 College of Veterinary Medicine, Chungbuk National University, Cheongju, Korea, 5 Ulsan Institute of Health and Environment, Ulsan, Korea, 6 Unite de Virologie and Immunologie Moleculaires, INRA CRJ, Domaine de Vilvert, Jouy en Josas, France, 7 US Geological Survey, Western Fisheries Research Center, Seattle, Washington, United States of America Abstract The nonvirion (NV) protein of infectious hematopoietic necrosis virus (IHNV) has been previously reported to be essential for efficient growth and pathogenicity of IHNV. However, little is known about the mechanism by which the NV supports the viral growth. In this study, cellular localization of NV and its role in IHNV growth in host cells was investigated. Through transient transfection in RTG-2 cells of NV fused to green fluorescent protein (GFP), a nuclear localization of NV was demonstrated. Deletion analyses showed that the 32EGDL35 residues were essential for nuclear localization of NV protein, and fusion of these 4 amino acids to GFP directed its transport to the nucleus. We generated a recombinant IHNV, rIHNV- NV-DEGDL in which the 32EGDL35 was deleted from the NV. rIHNVs with wild-type NV (rIHNV-NV) or with the NV gene replaced with GFP (rIHNV-DNV-GFP) were used as controls. RTG-2 cells infected with rIHNV-DNV-GFP and rIHNV-NV-DEGDL yielded 12- and 5-fold less infectious virion, respectively, than wild type rIHNV-infected cells at 48 h post-infection (p.i.). While treatment with poly I:C at 24 h p.i. did not inhibit replication of wild-type rIHNVs, replication rates of rIHNV-DNV-GFP and rIHNV-NV-DEGDL were inhibited by poly I:C. In addition, both rIHNV-DNV and rIHNV-NV-DEGDL induced higher levels of expressions of both IFN1 and Mx1 than wild-type rIHNV. These data suggest that the IHNV NV may support the growth of IHNV through inhibition of the INF system and the amino acid residues of 32EGDL35 responsible for nuclear localization are important for the inhibitory activity of NV. Citation: Choi MK, Moon CH, Ko MS, Lee U-H, Cho WJ, et al. (2011) A Nuclear Localization of the Infectious Haematopoietic Necrosis Virus NV Protein Is Necessary for Optimal Viral Growth. PLoS ONE 6(7): e22362. doi:10.1371/journal.pone.0022362 Editor: Ding Xiang Liu, Nanyang Technological University, Singapore Received January 10, 2011; Accepted June 22, 2011; Published July 21, 2011 Copyright: 2011 Choi 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: This project was partially supported by a Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean government (MEST), grant code: 2009-0060042 and BRL-2009-0087350, http://www.nrf.re.kr/html/en/. The funders had no role in study design, data collection and analysis, decision publish, or preparation of the manuscript. to Competing Interests: The authors have declared that no competing interests exist. * E-mail: jwpark@ulsan.ac.kr . These authors contributed equally to this work. Introduction Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus belonging to the genus Novirhabdovirus of the Rhabdoviridae family. IHNV causes an acute disease in wild and hatchery-reared salmonid fish in North America, Europe, and Asia [1,2]. Similar to other rhabdoviruses, the IHNV genome encodes five structu- ral proteins that make up the virions: a nucleoproetin (N), a polymerase-associated phosphoprotein (P), a matrix protein (M), a unique glycoprotein (G), and a RNA polymerase protein (L) [3,4]. The Novirhabdovirus genome possesses an additional non-virion (NV) gene located between the viral glycoprotein (G) and the polymerase (L) genes [3]. While the functions of most IHNV proteins are known, that of the NV protein remains unclear. Recently, Biacchesi et al [5] demonstrated that a recombinant IHNV (rIHNV) knockout with no NV gene was severely impaired for growth in cell culture and had reduced pathogenicity in rainbow trout [6], suggesting a crucial role of NV for optimal IHNV replication. However, the molecular basis for the role of NV is not known. Virus-infected cells synthesize and secrete type I interferons (IFNs) which are considered the first line of host defense against viral infection [7,8]. Secreted IFNs stimulate susceptible cells to express more than 300 IFN-stimulated genes (ISGs), whose concerted action leads to limiting further viral growth and spread [8]. The myxovirus resistance gene Mx, the PKR protein kinase stimulated by dsRNA, and the 29-59 oligoadenylate synthetase (OAS) are among the best-characterized antiviral ISGs [9]. The IFN system has also been found in teleost fish including Atlantic salmon and rainbow trout. Teleost fish IFNs can induce the expressions of several ISGs with antiviral activity [10,11]. The IFN system induced by poly I:C treatment [12,13] limited the growth of IHNV in cell culture [14] and recombinant IFN treatment protected rainbow trout from IHNV infection [15], suggesting that PLoS ONE | www.plosone.org 1 July 2011 | Volume 6 | Issue 7 | e22362