San Francisco). Immunoreactivity was detected using an ECL detection system (GE Healthcare). Autoradiography films were exposed at multiple time points to ensure that the images were not saturated. Confocal microscopy After transfection with 10 mg of various types of pEGFP/NV constructs, cells were seeded on confocal dishes (SPL-200350, SPL Life Sciences, Pocheon, Korea). On the following day, cells were observed with a FluoViewTM 500 confocal microscope (Olympus, Tokyo, Japan). Viral growth analysis Infections were carried out in 25 cm2 culture flasks containing confluent monolayers of RTG-2 cells at a MOI of 0.01 unless otherwise stated. At the end of 1 h adsorption period at 16uC, the cells were washed three times with MEM containing no serum. Five milliliters of MEM containing 5% FBS was then added to each culture flask and the flasks were incubated at 16uC. Samples of 200 ml of medium were collected at 0, 24, and 48 h post- infection (p.i.), and stored at 280uC. Viral titers were determined for all samples, in duplicate, using a plaque assay on EPC cells as described above. Mx promoter-reporter assay To confirm the presence and to quantify IFN in culture supernatants, we used the transgenic RTG-P1 cell line that contains a stable insertion of the IFN-responsive Mx promoter linked to a luciferase reporter gene [36]. Culture supernatant samples were collected from RTG-2 cells at 24 h p.i. and then incubated at 37uC for 2 h to inactivate the virus. One milliliter of culture supernatant sample was added to each of three wells in a 24-well plate containing the transgenic RTG-P1 cells, and incubated for 48 h. As a positive control, RTG-P1 cells were treated with poly I:C (25 mg/ ml) for 24 h. Cells from the wells were rinsed with EMEM-0 and trypsinized. The detached cells were collected using centrifugation, and the pellet was stored at 280uC. To assay the luciferase activity cells were lysed with lysis buffer (Promega, Madison, WI) and mixed with luciferase assay reagent (Promega) and the chemiluminescent signal was measured in a Wallac Victor 1420 Multilabel Counter (EG&G Wallac, Turku, Finland). Plasmid construction and recovery of recombinant virus A full-length antigenomic IHNV cDNA construct plasmid (pIHNV) derived from a French IHNV strain (IHNV 32/87) [5] was used to generate recombinant IHNVs (rIHNV). Coding regions (ORFs) of wild-type NV and NV-DEGDL, in which the nucleotides encoding32EGDL35 were deleted from the NV of the IHNV-PRT strain, were amplified using RT-PCR from pEGFP/ NV(1–111) and pEGFP/NV(DEGDL), respectively. RT-PCR was performed using the following primer pair: CACGTAAAGTAC- CAGGTCATC, ACACACCCACAGTATCACT; Unique SpeI and SmaI restriction enzyme sites surrounding the NV ORFs of pIHNV were created by site-directed mutagenesis with the Quick Change kit (Agilent Technologies, Inc., Santa Clara, CA). The entire NV32/87 ORF of pIHNV was deleted using SpeI-SmaI digestion and replaced with amplified DNA fragments containing the NVPRT or NVPRT-DEGDL ORFs to generate pIHNV/ NVPRT or pIHNV/NVPRT(DEGDL), respectively. The presence and positions of the inserted genes were confirmed by RT-PCR and restriction enzyme analysis as well as sequencing of the inserted sites in the plasmids. Recombinant viruses were recovered from EPC cells as previously described [5]. To summarize briefly, NLS of IHNV NV Is Necessary for IHNV Growth EPC cells were infected with a vaccinia virus recombinant that expresses T7 RNA polymerase (vTF7-3) [39] and was transfected with the full-length cDNA clones and three support plasmids that expressed the N, P, and L proteins required for RNA encapsida- tion and replication. Infectious viruses were recovered from the supernatant medium, and were amplified by passage on EPC cells at a low multiplication of infection. Viral titers of recombinant virus stocks were quantified using the plaque assay. We also used two additional rIHNVs as controls: a parental rIHNV generated from a French IHNV strain (IHNV 32/87) and a rIHNV-DNV- GFP in which the entire NV ORF was deleted and replaced with a green fluorescent protein (GFP) ORF [5]. Quantitative real-time and semi-quantitative RT-PCR for Mx1, IFN1 and IHNV G expressions in RTG-2 cells Total RNA was isolated from infected cells using the Trizol (Invitrogen) extraction method and DNase I-treated total RNA was reverse transcribed using oligo-dT and Superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s instruc- tions. Quantitative real-time PCR was performed using SYBRH Green PCR Master Mix (Applied Biosystems, Foster City, CA) and the ABI Prism 7900 HT (Applied Biosystems). Specificities of each primer pair were confirmed via melting curve analysis and agarose-gel electrophoresis. The housekeeping gene, acidic ribo- somal phosphoprotein P0 (ARP) was used as a normalizing gene [40], and PCR primer pairs were as follows: Mx1: 59-GG- TTGTGCCATGCAACGTT-39, 59-GGCTTGGTCAGGATG- CCTAAT-39; IFN1: 59-GCGAAACAAACTGCTATTTACAA- TGTATA-39, 59-TCACAGCAATGACACACGCTC-39; IHNV-G: 59-GCGCACGCCGAGATAATATCAA-39, 59-TCCCGTGAT- AGATGGAGCCTTT-39; ARP: 59-GAAAATCATCCAATT- GCTGGATG-39, 59-CTTCCCACGCAAGGACAGA-39. Statistical analysis Differences in the expression of IFN1 and Mx1, as well as Mx1 promoter activity and the viral growth among rIHNVs-infected cells, were evaluated by an unpaired Student’s t-test (two-tailed). A P value,0.05 was considered to indicate statistical significance. Results Nuclear localization of NV-GFP The NV of IHNV has been reported to be essential for efficient growth and pathogenicity of IHNV [6]. In an effort to gain further insight into the functions of the NV in viral replication, we first examined the intracellular distribution of the NV. To date there is no commercially available anti-IHNV NV antibody. Therefore, we tagged the NV with GFP by cloning the full-length cDNA of the NV gene into a pEGFP-N1 expression vector, transfected the resulting plasmid [pEGFP/NV(1–111)] into CHSE-214 cells and investigated the subcellular localization of the NV-GFP fusion protein using confocal microscopy. Representative images of cells transfected with plasmids encoding NV-GFP and GFP alone are shown in Figure 1A. As expected, diffuse fluorescence was observed throughout both the cytoplasm and the nucleus in the pEGFP-N1-transfected cells (Figure 1A). However, fusion of the NV protein to GFP altered the distribution of GFP, resulting in the accumulation of fluorescent label in the nucleus and decreased accumulation in the cytoplasm (Figure 1A). To confirm the confocal microscopy observations, nuclear and cytoplasmic fractions of CHSE-214 cells transfected with the pEGFP-N1 and pEGFP/NV(1–111) plasmids were analyzed using Western blot analysis with anti-GFP antibody (Figure 1B). The presence of NV-GFP fusion protein in both the nuclear and PLoS ONE | www.plosone.org 3 July 2011 | Volume 6 | Issue 7 | e22362