Amino acid residues 32EGDL35 are essential for the anti-IFN activity of NV Amino acids 32EGDL35 within the NV were found to be required for the efficient growth of IHNV in RTG-2 cells (Figure 5B). Therefore, it is possible that this amino acid sequence is essential for the ant-IFN activity of NV. We first determined whether 32EGDL35 are required for efficient growth of IHNV in poly I:C-treated RTG-2 cells. In RTG-2 cells pre-treated with 25 mg/ml of poly I:C for 24 h, the growth of both rIHNV-NVPRT and rIHNV-NVPRT-DEGDL were blocked (Figure 8A). However, in RTG-2 cells treated with poly I:C at 24 h after viral infection, the growth of wild-type rIHNV-NVPRT was not significantly affected by poly I:C treatment, while that of rIHNV-NVPRT- DEGDL was severely decreased (Figure 8B). Thus amino acids 32EGDL35 are required for efficient growth of IHNV in RTG-2 cells treated with poly I:C after virus infection. We also determined the expression levels of IFN1 and Mx1 in RTG-2 cells which were infected with rIHNV-NVPRT or rIHNV-NVPRT-DEGDL and then treated with 25 mg/ml of poly I:C at 24 h p.i. Cells infected with rIHNV-NVPRT or rIHNV-NVPRT-DEGDL produced de- creased levels of IFN1 (18 and 35 copies, respectively) and Mx1 (290 and 503 copies, respectively) compared with mock-infected poly I:C treated control (IFN1, 53 copies; Mx1, 680 copies). However, cells infected with rIHNV-NVPRT-DEGDL produced 1.9- and 1.7-fold higher amount of IFN1 and Mx1, respectively, than those infected with rIHNV-NVPRT (Figure 8C, 8D). To determine whether 32EGDL35 are required to inhibit the host IFN system, RTG-2 cells were infected with rIHNV-NVPRT or rIHNV-NVPRT-DEGDL and the expression levels of IFN1 and Mx1 were analyzed using real-time PCR at 24 h p.i. Their mRNA copy numbers were normalized per 1000 copies of ARP mRNA. While rIHNV-NVPRT induced 3- and 43-fold increases in IFN1 (9 copies) and Mx1 (295 copies) expressions compared with mock- infected control (IFN1, 3 copies; Mx1, 7 copies), respectively, rIHNV-NVPRT-DEGDL induced 9-fold and 73-fold increases of IFN1 (26 copies) and Mx1 (515 copies) expressions, respectively (Figure 8E, 8F). In addition, RTG-P1 cells treated with super- natants from rIHNV-NVPRT-DEGDL-infected RTG-2 cells showed 3.0-fold higher level of luciferase activity than those treated with supernatants from rIHNV-NVPRT-infected RTG-2 cells (Figure 8G). Collectively, these data demonstrate that the amino acid sequence 32EGDL35 is essential for the efficient anti- IFN activity of NV. Discussion NV is a non-virion protein whose gene is unique to the genus Novirhabdovirus. Although NV is known to be required for the efficient growth of IHNV in rainbow trout [6], the exact role of the NV in IHNV infection is still unclear. In this study, we investigated the cellular localization of NV by transient expression of a NV- GFP fusion protein and showed that the NV protein can be actively imported into the nucleus, since it can direct nuclear accumulation of a cytoplasmic protein, GFP, in transiently transfected CHSE-214 and RTG-2 cells. This demonstrates that, even though it is smaller than the diffusion limits of the nuclear pore complex (NPC), the 111-aa NV protein of IHNV is able to exploit cellular mechanisms for active nuclear import. The region of NV protein responsible for nuclear localization was mapped to amino acid residues 32EGDL35. A survey of IHNV NV sequences present in GenBank revealed that these residues were conserved among IHNV strains and especially amino acid residues 33GDL35 were strictly conserved among all IHNV strains of which NV has been sequenced (data not shown). We eliminated the possibility NLS of IHNV NV Is Necessary for IHNV Growth that this short amino acid region comprises a portion of a larger signal disrupted by removal of these amino acids, since the addition of amino acids 32EGDL35 to GFP resulted in the accu- mulation of fluorescent label in the nucleus. To determine the role of this motif in IHNV replication, we constructed a mutant rIHNV expressing a 32EGDL35-deleted NV protein (rIHNV- NVPRT-DEGDL) and compared its growth in rainbow trout cells with that of an NV-knockout mutant rIHNV (rIHNV-32/87- DNV-GFP). Even though there was a slight difference in their growth, the growth of both mutant rIHNVs was highly attenuated in RTG-2 cells relative to rIHNV with an intact NV gene, indicating that 32EGDL35-mediated nuclear localization of NV is important for NV function. Importin b (karyopherin) proteins bind nuclear localization signals (NLSs) to facilitate the nuclear import of NLS-containing proteins [41]. Interestingly, amino acid sequence analysis revealed that the IHNV NV does not contain any classical NLS. Therefore it is necessary to consider the mechanism by which the NV protein enters the nucleus. One possibility is that the amino acid sequence 32EGDL35 is a novel NLS which can mediate the nuclear transport of NV via binding to importins. Generally, NLSs which bind either importin a or importin b have basic amino acid residues such as lysine at their cores [41,44]. However, 32EGDL35 do not contain any basic residues but, on the contrary, possess acidic residues such as glutamic acid (E) and aspartic acid (D). Thus, it is unlikely that 32EGDL35 bind directly to importins. Another possibility is that this motif mediates the nuclear import of NV by binding to other proteins containing NLSs. Even though the amino acid sequence 32EGDL35 is highly conserved among IHNV strains, it does not show any similarity to other known motifs or domains. Thus, at present, it is difficult to predict candidates for NLS-containing proteins to which 32EGDL35 can bind. Further characterization of the NV-interacting proteins, including importins, will be needed to elucidate the molecular mechanism through which the NV protein enters the nucleus. Type I IFNs are well-known to block virus multiplication by inducing upregulation of antiviral genes [8,9]. Recently, IFN genes have been identified in a number of species of teleost fish including rainbow trout [10]. Thus, IHNV should possess strategies to block the induction of IFN systems in trout RTG-2 cells which would otherwise suppress virus growth early in infection [7]. The nonstructural proteins of RNA viruses have been reported to be implicated in down-regulation of the host innate immune response [7,45]. In this study, we provided evidences that the IHNV NV possesses capability for down-regulation of the host IFN system: (a) NV-knockout mutant rIHNV induced greater expressions of the IFN1 and Mx1 genes than the wild-type rIHNV in RTG-2 cells; (b) RTG-2 cells infected with NV-knockout rIHNV produced a higher level of IFN1 activity in cell culture supernatant relative to wild-type rIHNV; (c) wild-type rIHNV more efficiently blocked the expressions of IFN1 and Mx1 than the NV-knockout mutant rIHNV in RTG-2 cells treated with poly I:C after viral infection. These results suggest that the NV protein can support IHNV growth through inhibition of host IFN systems. It is next necessary to determine the role of nuclear localization of the NV as it pertains to its inhibitory function. IHNV is an RNA virus that is believed to replicate in the cytoplasm like other RNA viruses. Many RNA viruses whose primary site of replication is the cytoplasm produce viral proteins that localize to the nucleus during virus infection in order to inhibit IFN systems [46]. Thus, it is possible to speculate that the IHNV NV protein localizes to the nucleus to block the induction of IFN systems in RTG-2 cells. If this is true, we would expect that the mutant rIHNV expressing an 32EGDL35-deleted NV protein would less effectively block the IFN PLoS ONE | www.plosone.org 10 July 2011 | Volume 6 | Issue 7 | e22362