Figure 1. Nuclear localization of NV-GFP proteins in CHSE-214 cells. CHSE-214 cells were transiently transfected with plasmids pEGFP- N1 or pEGFP/NV(1–111) and incubated for 48 h. A, The fluorescent signals were examined using confocal microscopy. Each image is representative of the majority of the cells observed in several fields. B, Cells were harvested and nuclear and cytoplasmic fractions were prepared as described in the Materials and Methods section. The resulting fractions were analyzed using 10% SDS-PAGE, followed by Western blotting with anti-GFP antibody. C denotes the cytoplasmic fraction, N denotes the nuclear fraction. doi:10.1371/journal.pone.0022362.g001 cytoplasmic extracts was generally in agreement with the sub- cellular distribution observed via fluorescence microscopy. The GFP encoded by the control plasmid without fusion to NV was not detected in the nuclei with Western blotting (Figure 1B). Although the fluorescence signal of the GFP alone appeared evenly dis- tributed throughout the cell, we suspect that the diffuse dis- tribution in the cytoplasm made it appear that this protein was distributed in the nucleus under fluorescence microscopy. These results suggest that the nuclear localization of NV is an intrinsic property of this protein and is independent of other viral factors. The amino terminus of NV is required for nuclear localization The sequence of the NV gene does not encode any canonical nuclear-localization signal (NLS) [41,42]. Thus to map the protein NLS of IHNV NV Is Necessary for IHNV Growth domains responsible for nuclear localization, three NV fragments, corresponding to amino acid residues (aa) 1 to 37 (NV(1–37)), aa 38 to 73 (NV(38–73)), and aa 74 to 111 (NV(74–111)) of NV, were tagged with GFP (Figure 2A) and were expressed in CHSE-214 cells. Figure 2B shows typical images of cells that are represen- tative of each truncated NV-GFP fusion protein at 48 h after transfection. While both the NV(38–73) and the NV(74–111) were evenly localized in the cytoplasm and the nucleus, the N-terminal fragment NV(1–37) had a nuclear-localization pattern similar to that of full-length NV(1–111) (Figure 2B), indicating that the NV(1–37) alone can mediate nuclear targeting. Amino acids 32EGDL35 are responsible for nuclear localization of NV protein To more precisely define and characterize the regions within the NV(1–37) responsible for nuclear localization, a series of truncations from either the C or N terminus of NV(1–37) were performed (Figure 3A). Each truncated NV fragment was tagged with GFP and expressed in CHSE-214 cells. Removal of 31 additional residues from the N terminus or two residues from the C terminus of NV(1–37) did not alter its nuclear localization pat- tern (Figure 3B). Collectively, these results suggest that 32EGDL35 of NV are sufficient for nuclear translocation since the 32EGDL35- GFP fusion protein accumulated in the nuclei (Figure 3B). To determine whether 32EGDL35 were essential for nuclear localization of NV, they were deleted from the NV-GFP fusion protein (Figure 3A). CHSE-214 cells were transfected with this construct, and the distribution of the NV(D32EGDL35)-GFP mutant proteins was examined 48 h after transfection. As shown in Figure 3B, NV(D32EGDL35)-GFP was not targeted to the nucleus, indicating that amino acid residues 32EGDL35 are critical for nuclear localization of NV. To determine which amino acids within 32EGDL35 are necessary for nuclear localization, each amino acid within 32EGDL35 was replaced with uncharged alanine and the resultant mutants were expressed as fusion proteins with GFP (Figure 3A). As shown in Figure 3B, all substitutions of amino acid residues within the 32EGDL35 decreased the accumulation of fluorescent label in the nucleus (Figure 3B). These results suggest that all 4 residues within 32EGDL35 may be critical for nuclear localization. The nuclear localization of NV was also confirmed in RTG-2 cells using confocal microscopy. While fluorescent signals of the NV-GFP and the 32EGDL35-GFP were accumulated within the nucleus, those of the NV(D32EGDL35)-GFP were diffused throughout the nucleus and cytoplasm just like those of the GFP (Figure 4). These results suggest that nuclear localization of IHNV NV mediated by the amino acids 32EGDL35 is common to both CHSE-214 and RTG-2 cells. Figure 2. Mapping of the NV region that supports nuclear localization. A, Schematic diagram of the full-length NV-GFP fusion protein and truncated mutants containing the N-terminal (NV(1–37)), middle region (NV(38–73)), and C-terminal (NV(74–111)) of the NV protein. B, CHSE-214 cells were transiently transfected with mutant NV constructs and examined live 48 h after transfection using confocal microscopy. doi:10.1371/journal.pone.0022362.g002 PLoS ONE | www.plosone.org 4 July 2011 | Volume 6 | Issue 7 | e22362