Nuclear localization of NV-GFP protein in RTG-2 cells.

<p><i>A</i>, Schematic diagram of the GFP fusion proteins used to transfect RTG-2 cells. <i>B</i>, RTG-2 cells were transiently transfected with pEGFP-N1, pEGFP/NV(1–111), pEGFP/NV(³²EGDL³⁵), or pEGFP/NV(ΔEGDL). 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.</p

Requirement of NV for inhibition of IFN systems in IHNV-infected RTG-2 cells.

<p><i>A</i> and <i>B</i>, IFN1 and Mx1 expression in RTG-2 cells infected with rIHNV-32/87 or rIHNV-32/87-ΔNV-GFP. RTG-2 cells were harvested at 24 h p.i., and total RNA was analyzed with real-time PCR for IFN1 (<i>A</i>) and Mx1 (<i>B</i>). 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 µg/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 ± SD of three independent experiments (**P<0.01; ***P<0.001). <i>C</i>, Assay for functional IFN1 released from RTG-2 cells infected with rIHNV-32/87 or rIHNV-32/87-ΔNV-GFP. The culture supernatants were collected at 24 h p.i. and IFN activities were measured by adding the supernatants to RTG-P1 cells and analyzing luciferase activity in RTG-P1 cells as described in the Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022362#s2" target="_blank">Methods</a>. 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 µg/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 ± SD of three independent experiments (*P<0.05; ***P<0.001).</p

Requirement of NV for inhibition of poly I∶C-induced IFN response in RTG-2 cells.

<p><i>A</i>, Effect of poly I∶C pre-treatment on the growth of rIHNVs. RTG-2 cells were pre-incubated with poly I∶C at 25 µg/ml for 24 h. The cells were then infected with rIHNV-32/87 or rIHNV-32/87-ΔNV-GFP 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 of samples taken from duplicate infections. <i>B</i>, Effect of poly I∶C treatment after virus infection on the growth of rIHNVs. RTG-2 cells were infected with rIHNV-32/87 or rIHNV-32/87-ΔNV-GFP at an MOI of 0.01 PFU/cell. After incubation for 24 h, cells were washed three times and treated with 25 µg/ml of poly I∶C or serum-free media. At 0 and 24 h after poly I∶C treatment, samples of the supernatant medium were collected and titrated in duplicate. 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 ± SD of three independent experiments (***P<0.001). ns, not significant. <i>C</i> and <i>D</i>, Analysis of IFN1 and Mx1 expressions in RTG-2 cells treated with poly I∶C after virus infection. RTG-2 cells were infected with rIHNV-32/87 or rIHNV-32/87-ΔNV-GFP at an MOI of 1 PFU/cell. After incubation for 24 h, cells were incubated with culture media containing 25 µg/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 (<i>C</i>) and Mx1 (<i>D</i>). 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 µg/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 ± SD of three independent experiments (**P<0.01; ***P<0.001).</p

The amino acids ³²EGDL³⁵ are essential for the IFN1 inhibitory function of NV.

<p><i>A</i>, Effects of poly I∶C pre-treatment on the growth of rIHNV-NV_PRT and rIHNV-NV_PRT-ΔEGDL in RTG-2 cells. RTG-2 cells were pre-incubated with poly I∶C at 25 µg/ml for 24 h. Cells were then infected with rIHNV-NV_PRT or rIHNV-NV_PRT-ΔEGDL 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. <i>B</i>, Effects of poly I∶C treatment after viral infection on the growth of rIHNV-NV_PRT and rIHNV-NV_PRT-ΔEGDL. RTG-2 cells were infected with rIHNV-NV_PRT and rIHNV-NV_PRT-ΔEGDL at an MOI of 0.01 PFU/cell. After incubation for 24 h, cells were washed and incubated with fresh culture media containing 25 µg/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 ± SD of three independent experiments (**P<0.01). ns, not significant. <i>C</i> and <i>D</i>, 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-NV_PRT, or rIHNV-NV_PRT-ΔEGDL at an MOI of 1 PFU/cell. After incubation for 24 h, cells were incubated with culture media containing 25 µg/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 (<i>C</i>) and Mx1 (<i>D</i>). 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 µg/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 ± SD of three independent experiments (*P<0.05; **P<0.01; ***P<0.001). <i>E</i> and <i>F</i>, IFN1 and Mx1 expression in RTG-2 cells infected with rIHNV-NV_PRT, or rIHNV-NV_PRT-ΔEGDL. RTG-2 cells were harvested at 24 h p.i., and the total RNA was analyzed with real-time PCR for IFN1 (<i>E</i>) and Mx1 (<i>F</i>). 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 µg/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 ± SD of three independent experiments (**P<0.01; ***P<0.001). <i>G</i>, Assay for functional IFN1 released from RTG-2 cells infected with rIHNV-NV_PRT, or rIHNV-NV_PRT-ΔEGDL. The culture supernatants were collected from RTG-2 cells infected with rIHNV-NV_PRT, or rIHNV-NV_PRT-ΔEGDL at 24 h p.i. The IFN1 activities in the supernatants were measured as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022362#pone-0022362-g007" target="_blank">Figure 7C</a>. 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 µg/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 ± SD of three independent experiments (***P<0.001).</p

Nuclear localization of NV-GFP proteins in CHSE-214 cells.

<p>CHSE-214 cells were transiently transfected with plasmids pEGFP-N1 or pEGFP/NV(1–111) and incubated for 48 h. <i>A</i>, The fluorescent signals were examined using confocal microscopy. Each image is representative of the majority of the cells observed in several fields. <i>B</i>, Cells were harvested and nuclear and cytoplasmic fractions were prepared as described in the Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022362#s2" target="_blank">Methods</a> 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.</p

Growths of rIHNV-32/87, rIHNV-32/87-ΔNV-GFP, rIHNV-NV_PRT, and rIHNV-NV_PRT-ΔEGDL in RTG-2 cells.

<p><i>A</i>, Schematic representation of the recombinant IHNVs used in this study. rIHNV-NV_PRT, and rIHNV-NV_PRT-ΔEGDL were generated as described in the Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022362#s2" target="_blank">Methods</a> section, and rIHNV-32/87 and rIHNV-32/87-ΔNV-GFP were used as controls. NV_PRT refers to the NV gene from the IHNV PRT strain. All other genes are from IHNV 32/87 strain. <i>B</i>, Comparative growths of rIHNV-32/87, rIHNV-32/87-ΔNV-GFP, rIHNV-NV_PRT, or rIHNV-NV_PRT-ΔEGDL in RTG-2 cells. The cells were infected with rIHNVs at an MOI of 0.01 PFU/cell, and samples of the supernatant medium were collected at 0, 24, and 48 h p.i. The samples were titrated in duplicate using a plaque assay on EPC cells. The results are presented as the means ± SD of three independent experiments (**P<0.01). C. Effect of NV expression on the growth of rIHNV-32/87-ΔNV-GFP in RTG-2 cells. RTG-2 cells were transfected with pcDNA6/V5-NV_PRT or empty vector pcDNA6/V5. After incubation for 24 h, the level of NV protein was determined by Western blotting with anti-V5 antibody (top panel). RTG-2 cells transfected with pcDNA6/V5-NV_PRT or empty vector were infected with rIHNVs at an MOI of 0.01 PFU/cell and samples of the supernatant medium were collected at 0, 24, and 48 h p.i. The samples were titrated in duplicate using a plaque assay on EPC cells. The results are presented as the means ± SD of three independent experiments (**P<0.01).</p