NLS of IHNV NV Is Necessary for IHNV Growth Figure 3. Identification of an amino acid motif responsible for nuclear localization of NV. A, Schematic diagram of the GFP fusion proteins used to identify the motif responsible for nuclear localization of NV. The names of the constructs are shown on the left. B, CHSE-214 cells were transiently transfected with various types of plasmid constructs and, after incubation for 48 h, fluorescent signals were examined using confocal microscopy. Each image is representative of the majority of the cells observed in several fields. doi:10.1371/journal.pone.0022362.g003 Amino acids 32EGDL35 are required for the efficient growth of IHNV in RTG-2 cells To determine whether the amino acids 32EGDL35 are involved in the growth of IHNV in cell culture, two rIHNVs based on the genomic background of rIHNV-32/87 were generated by replacing the NV of rIHNV-32/87 strain with the wild-type NV of the PRT strain (rIHNV-NVPRT) or mutant NV of PRT lacking amino acids 32EGDL35 (rIHNV-NVPRT-DEGDL) (Figure 5A). A parental rIHNV-32/87 and a NV-knockout mutant rIHNV-32/ 87-DNV-GFP [5] were used as controls. The IHNV-PRT and IHNV-32/87 strains were originally isolated from rainbow trout [5,37] and, thus, we examined growth of the rIHNVs in rainbow trout-derived RTG-2 cells. We infected RTG-2 cells with rIHNVs at an MOI of 0.01 and infectious virus titers shed into the media were determined via plaque assay. Figure 5B illustrates a comparison of the rIHNV yields at 0, 24, and 48 h post-infection (p.i.) in RTG-2 cells, presented as averages from three independent experiments. Replacement of the NV gene of rIHNV-32/87 with the NV gene from the PRT strain of IHNV did not result in any significant change in viral growth. Deletion of the 32EGDL35 resulted in a 9- and 5-fold decrease in growth (9.86104 and 7.86105 PFU/ml) relative to rIHNV- NVPRT (8.96105 and 3.96106 PFU/ml) at 24 and 48 h p.i., respectively. Deletion of the entire NV gene resulted in a 9- and 12-fold decrease in growth (9.86104 and 3.16105 PFU/ml) relative to the parental rIHNV-32/87 (8.66105 and 3.86 106 PFU/ml) at 24 and 48 h p.i., respectively. To determine whether expression of wild-type NV rescues the growth of NV- knockout rIHNV, we analyzed the growth of rIHNVs in RTG-2 cells transfected with pcDNA3/NVPRT. As shown in Fig. 5C, overexpression of NV significantly increased the viral yields of rIHNV-32/87-DNV-GFP. These results demonstrate that NV is essential for efficient growth of IHNV in RTG-2 cells and, importantly, that the amino acid sequence 32EGDL35 is critical for the functioning of NV. However, deletion of the entire NV leads to a greater decrease in viral growth than deletion of only the 32EGDL35. After 72 h p.i., even though the viral yield of all four rIHNVs increased, the difference in viral yield among the four rIHNVs decreased and at 120 h p.i., all four rIHNVs yielded a similar final infectivity titer (6.7,7.4 107 PFU/ml) (data not shown). The reduced difference after 72 h p.i. was likely because the RTG-2 cells infected with rIHNV-32/87 or rIHNV-NVPRT were largely dead by 72 h p.i. while most cells infected with rIHNV-32/87-DNV-GFP or rIHNV-NVPRT-DEGDL remained alive. NV is required for inhibition of poly I:C-induced IFN responses in RTG-2 cells Poly I:C is a well-documented inducer of IFN systems and viral resistance in animal cells [43]. To determine the effects of a poly I:C-induced IFN response on the growth of the rIHNVs, RTG-2 cells were treated with 25 mg/ml of poly I:C. As shown in the poly I:C positive controls in Figure 6C and 6D, this treatment significantly induced the expression of both IFN1 and the Mx1 gene in RTG-2 cells at 24 h after treatment. At 24 h after poly I:C treatment cells were infected with rIHNV-32/87 or rIHNV-32/ 87-DNV-GFP, samples of culture supernatant were collected at 0, 24, and 48 h p.i., and infectious titers were determined by plaque Figure 4. Nuclear localization of NV-GFP protein in RTG-2 cells. A, Schematic diagram of the GFP fusion proteins used to transfect RTG-2 cells. B, RTG-2 cells were transiently transfected with pEGFP-N1, pEGFP/NV(1–111), pEGFP/NV(32EGDL35), or pEGFP/NV(DEGDL). After incubation for 48 h, fluorescent signals were examined using confocal microscopy. Each image is representative of the majority of the cells observed in several fields. doi:10.1371/journal.pone.0022362.g004 PLoS ONE | www.plosone.org 6 July 2011 | Volume 6 | Issue 7 | e22362