NLS of IHNV NV Is Necessary for IHNV Growth Figure 8. The amino acids 32EGDL35 are essential for the IFN1 inhibitory function of NV. A, Effects of poly I:C pre-treatment on the growth of rIHNV-NVPRT and rIHNV-NVPRT-DEGDL in RTG-2 cells. RTG-2 cells were pre-incubated with poly I:C at 25 mg/ml for 24 h. Cells were then infected with rIHNV-NVPRT or rIHNV-NVPRT-DEGDL at an MOI of 0.01 PFU/cell, and samples of the supernatant medium were collected at 0, 24, and 48 h p.i. All data points represent the average titers of samples taken from duplicate infections. B, Effects of poly I:C treatment after viral infection on the growth of rIHNV-NVPRT and rIHNV-NVPRT-DEGDL. RTG-2 cells were infected with rIHNV-NVPRT and rIHNV-NVPRT-DEGDL at an MOI of 0.01 PFU/cell. After incubation for 24 h, cells were washed and incubated with fresh culture media containing 25 mg/ml of poly I:C. At 0 and 24 h after poly I:C treatment, samples of the supernatant medium were collected. The virus titer in the supernatant medium collected at 0 h after poly I:C treatment was defined as one. The results are presented as the means 6 SD of three independent experiments (**P,0.01). ns, not significant. C and D, Analysis of IFN1 and Mx1 expressions in RTG-2 cells treated with poly I:C after viral infection. RTG-2 cells were infected with rIHNV-NVPRT, or rIHNV-NVPRT-DEGDL at an MOI of 1 PFU/cell. After incubation for 24 h, cells were incubated with culture media containing 25 mg/ml of poly I:C. At 24 h after poly I:C treatment, total RNA was extracted from the cells and analyzed with real-time PCR for IFN1 (C) and Mx1 (D). The negative control was mock-infected RTG-2 cells without poly I:C treatment. The positive control was mock-infected RTG-2 cells stimulated with 25 mg/ml poly I:C. The levels of IFN1 and Mx1 are expressed as mRNA copy number normalized to 1000 copies of ARP mRNA. The results are presented as the means 6 SD of three independent experiments (*P,0.05; **P,0.01; ***P,0.001). E and F, IFN1 and Mx1 expression in RTG-2 cells infected with rIHNV-NVPRT, or rIHNV-NVPRT-DEGDL. RTG-2 cells were harvested at 24 h p.i., and the total RNA was analyzed with real-time PCR for IFN1 (E) and Mx1 (F). The negative control was mock- infected RTG-2 cells without poly I:C treatment. The positive control was mock-infected RTG-2 cells stimulated with 25 mg/ml poly I:C. The levels of IFN1 and Mx1 are expressed as mRNA copy number normalized to 1000 copies of ARP mRNA. The results are presented as the means 6 SD of three independent experiments (**P,0.01; ***P,0.001). G, Assay for functional IFN1 released from RTG-2 cells infected with rIHNV-NVPRT, or rIHNV-NVPRT- DEGDL. The culture supernatants were collected from RTG-2 cells infected with rIHNV-NVPRT, or rIHNV-NVPRT-DEGDL at 24 h p.i. The IFN1 activities in the supernatants were measured as described in the legend of Figure 7C. The negative control was mock-infected RTG-2 cells without poly I:C treatment. The positive control was mock-infected RTG-2 cells stimulated with 25 mg/ml poly I:C. The luciferase activity in the RTG-P1 cells treated with supernatants from the negative control cells was defined as one. The results are presented as the means 6 SD of three independent experiments (***P,0.001). doi:10.1371/journal.pone.0022362.g008 systems. Consistent with our expectation, an rIHNV mutant with an 32EGDL35-deleted NV induced more up-regulations of IFN1 and Mx1 gene expressions than the wild-type rIHNV in RTG-2 cells, and cells infected with this mutant produced a higher level of IFN1 activity in cell culture supernatant relative to wild-type rIHNV-infected cells. In addition, this mutant failed to block the inductions of IFN1 and Mx1 in RTG-2 cells treated with poly I:C after viral infection. The 32EGDL35 deletion mutant mimicked the NV knockout rIHNV in all experiments conducted here, sug- gesting that the 32EGDL35-mediated nuclear localization of NV is critical for the inhibitory function of NV. The existence of the NV gene is restricted only to the genus Novirhabdovirus and, thus, it is likely that rhabdoviruses belonging to genera other than the genus Novirhabdovirus use strategies different from that of IHNV; the rhabdovirus vesicular stomatitis virus (VSV) and rabies virus (RV) are good examples [24]. In the case of VSV, the matrix (M) protein contains NLSs [31] and is reported to suppress IFN systems through inhibition of the transcription factor TFIID [25,26] and blocking of nuclear transport [29,30,35]. On the contrary, RV uses phosphoprotein (P) to inhibit host IFN systems [24]. The RV P protein contains an NLS [47] and suppresses the IFN systems through inhibition of the nuclear accumulation of STAT1, the binding of STAT1 to the DNA, and the function of ISG products such as PML [34,48]. In this study, we did not determine the subcellular localization of IHNV M and P proteins and their potential functional roles in the inhibition of host IFN systems. However, it is possible to speculate that IHNV has evolved to use the NV protein instead of the M or P proteins to inhibit the host IFN systems. Among other species in the genus Novirhabdovirus there are inconsistent reports regarding the importance of NV. For Viral hemorrhagic septicemia virus (VHSV) recent reports demonstrate that NV is required for efficient viral growth in cell culture and pathogenicity in fish [49,50]. VHSV NV was reported to rescue the growth of NV-knockout mutant rIHNV [6], suggesting the possibility that VHSV NV supports the viral growth via inhi- bition of host IFN system as IHNV NV does. However, the underlying mechanisms of IFN inhibition of VHSV NV may be different from those of IHNV NV, since VHSV NV exhibits a low level of amino acid sequence similarity with IHNV NV and does not have EGDL motif. It is unclear whether all novirhabdoviruses use the NV to inhibit IFN systems, but if they do, a different NLS may be utilized. In the case of a different species, snakehead rhabdovirus (SHRV), an NV-knockout mu- tant virus showed the same pathogenicity as wild-type virus, suggesting that this virus may cope with the host antiviral response using proteins other than NV [51]. Pathogenicity of SHRV was tested using zebrafish instead of snakehead. Thus, it is possible that the NV of SHRV might play an essential role for the efficient growth and pathogenicity of the virus in the snakehead fish host from which it originated. Our data revealed that, in RTG-2 cells, poly I:C treatment before viral infection induced high levels of IFN1 and Mx1 and limited production of both wild-type and NV-knockout rIHNV. However, when poly I:C treatment occurred after infection, this treatment did not affect the growth of wild-type rIHNV but significantly limited that of the NV-knockout rIHNV. This suggests that, in RTG-2 cells treated with poly I:C after IHNV infection, wild-type IHNV but not NV-knockout mutant IHNV can cope with the IFN systems using the NV. However, in RTG-2 cells pre-treated with poly I:C, pre-existing antiviral activities may block the expression of viral genes and, thus, it may be impossible for IHNV to express a sufficiently large amount of NV protein adequate to inhibit IFN systems. In conclusion, our results show that the NV protein localizes to the nucleus, and that the amino acid sequence 32EGDL35 within the NV protein is responsible for nuclear localization. Even though the NV protein cannot cope with a pre-existing strong IFN response, it has the capability to inhibit IFN1 synthesis and 32EGDL35 are essential for this inhibitory activity. It remains unclear as to how 32EGDL35 mediate the nuclear localization of NV and how these residues inhibit IFN systems. It will be interesting to know which cellular proteins recognize these residues of the NV protein to mediate nuclear localization of NV and to inhibit IFN systems. Author Contributions Conceived and designed the experiments: JWP GJH SGJ YSH MB GK. Performed the experiments: MKC CHM MSK U-HL SJC JWD AH. Analyzed the data: WJC MSK MKC. Contributed reagents/materials/ analysis tools: AH MB. Wrote the paper: JWP MB GK. PLoS ONE | www.plosone.org 12 July 2011 | Volume 6 | Issue 7 | e22362