IHNV replication is highly sensitive to the IFN-induced antiviral responses of host cells. It is now evident that most viruses have evolved means to down- regulate IFN responses. In many cases they use nonstructural viral proteins for that purpose. A good example is the nonstructural protein NS1 of the influenza A virus. NS1 binds to RIG-I/IPS-1 complexes [16,17,18] and blocks downstream signaling [19,20,21], resulting in attenuation of type I IFN and inflammatory cytokine expression. These anti-interferon activities of NS1 are possible due to a complex regulation of NS1 cellular localization mediated by two nuclear localization signals (NLSs) and one nuclear export signal (NES), which allow the transport of this protein between the cytoplasm and nucleus of the infected cell [22,23]. Rhabdoviruses also have the ability to limit IFN production and IFN signaling [24]. However, they have been reported to use structural proteins for this purpose. In mammalian rhabdoviruses, the M protein of vesicular stomatitis virus (VSV) and the P protein of rabies virus (RV) have been shown to inhibit host IFN-mediated responses using different molecular mechanisms [25,26,27,28,29,30,31,32, 33,34,35]. However, until now, it is not known how IHNV interacts with the host IFN system. In this study, we showed that the 111-amino acid (aa) NV protein of IHNV can localize to the nucleus, and identified amino acid residues, 32EGDL35, within the NV protein that are re- sponsible for the nuclear localization of the NV protein. We then investigated the roles of the NV protein and the 32EGDL35 residues in IHNV growth using a series of various recombinant IHNV. Collectively, our data suggest that the NV can support the growth of IHNV in host cells through inhibition of the induction of IFN systems, and that the amino acid sequence 32EGDL35, responsible for nuclear localization of the NV, plays important role in the inhibitory activity of NV. Methods Cells and viruses CHSE-214 (chinook salmon embryo) (ATCC CRL-1681), RTG- 2 (rainbow trout gonad) (ATCC CCL-55), and EPC (epithelioma papulosum cyprini) (ATCC CRL-2872) cells were grown at 18uC in Eagle’s minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). The transgenic cell line, RTG-P1 [36], was purchased from ATCC (CRL-2829) and was cultivated in Eagle’s MEM supplemented with 10% FBS and 200 mg/ml Neomycin (G418, Sigma, St. Louis, MO). The IHNV-PRT strain used in this study is a 1991 isolate from rainbow trout Pyongchang Korea [37] at passage level 35. The virus was propagated in EPC cells at 16uC and quantified in terms of plaque forming units (PFU/ ml) using the standard plaque assay [38]. Effect of poly I:C on IHNV growth To determine the effect of pretreatment of cells with poly I:C on the growth of IHNV, 25 cm2 culture flasks of RTG-2 cells were inoculated with poly I:C (final concentration 25 mg/ml) (P1530, Sigma) and incubated for 24 h. The medium was removed and replaced with fresh medium without poly I:C, and the cells were challenged with IHNV at an MOI of 0.01 PFU/cell. Samples of the supernatant were collected at 0, 24, and 48 h after virus infection and were stored at 280uC until use. To assess the effect of poly I:C treatment after viral infection, RTG-2 cells were challenged with IHNV at an MOI of 0.01 and were incubated for 24 h at 16uC. The medium was removed and replaced with fresh medium with or without poly I:C (25 mg/ml). Supernatant samples were collected at 0, 24, and 48 h after poly I:C treatment and were stored at 280uC until use. NLS of IHNV NV Is Necessary for IHNV Growth Plasmids expressing NV and transfections The full-length cDNA of NV was amplified using RT-PCR from the RNA of CHSE-214 cells infected with IHNV-PRT using PCR primers as follows: pcDNA-NV: 59-GGATCCATGGAC- CACCGCGAAACAAAC-39, 59-CTCGAGTCTGGGATAAG- CAAGAAA-39; pEGFP-NV: 59-GCTAGCATGGACCACCGC- GAAACAAAC-39, 59-GGATCCAATCTGGGATAAGCAAGA- AA-39. The PCR products were subcloned into the BamHI/XhoI site of pcDNA6/V5 (Invitrogen, Carsbad, USA) or NheI/BamHI site of pEGFP-N1 (Clontech, Mountain View, CA) to create pcDNA6/V5-NVPRT and pEGFP/NV(1–111), respectively. A variety of deletion mutants of NV were PCR amplified using pEGFP/NV(1–111) as a template with the following primer pairs: NV(1–37): 59-GCTAGCATGGACCACCGCGAAACAAAC-39, 59-GGATCCAAGCCCCATACCAGGTCTCC-39; NV(38–73): 59-GCTAGCATGTGTGAAGAGGACGACGCA-39, 59-GGAT- CCAACTTGGTGATGCTGAGGTC-39; NV(74–111): 59-GCT- AGCATGGAGGGGCATCTACTTTTT-39, 59-GGATCCAA- TCTGGGATAAGCAAGAAA-39; NV(1–20): 59-GCTAGCAT- GGACCACCGCGAAACAAAC-39, 59-GGATCCTTGTATCG- CAGAACTTC-39; NV(21–35): 59- CTAGCATGAACGAGG- TGGCCGGACACGGCTTCCTCTTTAAGGAGGGAGACC- TGCCG-39, 59-GATCCGGCAGGTCTCCCTCCTTAAAGA- GGAAGCCGTGTCCGGCCACCTCGTTCATG-39; NV(DEGDL): 59-GCTAGCATGGACCACCGCGAAACAAAC-39, 59-CACA- CCATACCTTAAAGAGGAAGCCGTGTC-39, 59-CCTCTTT- AAGGTATGGTGTGAAGAGGACGA-39, 59-GGATCCAAT- CTGGGATAAGCAAGAAA-39. PCR products were inserted into the NheI/BamHI site of pEGFP-N1. To generate the plasmid constructs pEGFP/NV(31–35) and pEGFP/NV(32EGDL35) and to mutate the 32EGDL35 into 32AGDL35, 32EADL35, 32EGAL35, or 32EGDA35, the following oligonucleotides were synthesized at Integrated DNA Technolo- gies (Coralville, IA), annealed, and ligated into the NheI/BamHI site of pEGFP-N1 (CLONTECH, Inc.): NV(31–35): 59-CTAG- CATGAAGGAGGGAGACCTGCCG-39, 59-GATCCGGCAG- GTCTCCCTCCTTCATG-39; 32EGDL35: 59-CTAGCATGGA- GGGAGACCTGCCG-39, 59-GATCCGGCAGGTCTCCCTC- CATG-39; 32AGDL35: 59-CTAGCATGGCGGGAGACCTGCC G-39, 59-GATCCGGCAGGTCTCCCGCCATG-39; 32EADL35: 59-CTAGCATGGAGGCAGACCTGCCG-39, 59-GATCCGGC- AGGTCTGCCTCCATG-39; 32EGAL35: 59-CTAGCATGGA- GGGAGCCCTGCCG-39, 59-GATCCGGCAGGGCTCCCTC- CATG-39; 32EGDA35: 59-CTAGCATGGAGGGAGACGCGC- CG-39, 59-GATCCGGCGCGTCTCCCTCCATG-39. To express the proteins in vitro, CHSE-214 and RTG-2 cells were electroporated with various types of pEGFP/NV constructs using the NeonTM Transfection System (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Subcellular fractionation Forty-eight hours after transfection with the pEGFP/NV(1–111) constructs, CHSE-214 cells were harvested, and nuclear and cytosolic fractions were prepared from 56107 CHSE-214 cells using the Subcellular Proteome Extraction kit (ProteoExtractTM, Calbiochem, Darmstadt, Germany) according to the manufactur- er’s instructions. SDS-PAGE and Western blots Cells were washed twice with cold PBS, and 30–50 mg of protein was resolved using SDS-PAGE, transferred onto Hybond- P membranes (GE Healthcare, Piscataway, NJ), and probed with appropriate dilutions of the anti-GFP (sc-9996, Santa Cruz Biotechnology, Santa Cruz, CA) or anti-V5 antibody (Genentech, PLoS ONE | www.plosone.org 2 July 2011 | Volume 6 | Issue 7 | e22362