Two nuclear localization signals regulate intracellular localization of the duck enteritis virus UL13 protein

Figures

• 

  Figure 1. Map of the UL13 recombinant plasmids. (A) Construction of the UL13 NLS deletion plasmids with the EGFP-N1 vector. (B) Construction of the UL13 NLS and EGFP fusion plasmids with the pc-DNA 3.1(+)vector.

• 

  Figure 2. Localization of the UL13 and UL13-ΔNLS proteins in transfected DEFs. (A) Verification of UL13 and UL13-ΔNLS fusion protein localization via IFA. The pEGFP-N1- UL13 and UL13-ΔNLS plasmids were transfected into DEFs. Sixteen hours post transfection, the cells were fixed, permeabilized and incubated with rabbit anti-UL13 antiserum. The nuclei were stained with DAPI (blue). The expression and distribution of GFP protein (green) was monitored by fluorescence microscopy. Images were taken in separate channels using a 40× objective and merged in SPOT software. (B) Nuclear cytoplasmic fluorescence ratio. The ratio of nuclear and cytoplasmic fluorescence distribution in the recombinant plasmid transfected group was calculated by Image-Pro Plus. Fifty cells from each group were analyzed. The ratio of nuclear and cytoplasmic fluorescence in the pEGFP-UL13-ΔNLS1, pEGFP-UL13-ΔNLS2, and pEGFP-UL13-ΔNLS1 and ΔNLS2 recombinant plasmid transfected groups was analyzed by GraphPad Prism 6.0 software. ∗P < 0.05; ∗∗P < 0.01.

• 

  Figure 3. Construction of NLS-GFP recombinant plasmids and localization analysis of the fusion proteins. (A) Localization of the NLS-GFP fusion proteins in transfected DEFs. NLS-GFP recombinants were transfected into DEF cells, which were then fixed 16 h posttransfection. Images of the GFP reporter (green) and nuclei stained with DAPI (blue) were taken in separate channels using a 40× objective and merged in SPOT software. (B) Comparison of the nuclear and cytoplasmic fluorescence ratios of GFP and the NLS-GFP fusion protein. The mean nuclear and cytoplasmic fluorescence ratios were quantified using Image-Pro Plus software; differences in the nuclear and cytoplasmic fluorescence ratios of GFP and the NLS1-GFP and NLS2-GFP, NLS1&NLS2-GFP proteins was analyzed using ANOVA with GraphPad Prism 6.0 software. N = 50 for each group.

• 

  Figure 4. Western blot analysis of the UL13 NLS. (A) The nuclear and cytoplasmic distribution of pUL13-NLS1 and pUL13-NLS2 was analyzed by western blotting. DEFs were transfected with the NLS1 and NLS2 plasmids, and after 48 h, the cells were harvested. (B) The nuclear and cytoplasmic distribution of the pUL13-NLS1& NLS2- deleted plasmids were analyzed b through western blot. DEF were transfected with the NLS1 and NLS2 plasmids, and after 48 h, the cells were harvested.

• 

  Figure 5. Effect of nuclear import inhibitors on the localization of the DEV pUL13. (A) Localization of the UL13-GFP and NLS1&NLS2-GFP proteins in cells treated with or without ivermectin. Sixteen hours posttransfection, 25 μmol/L ivermectin was added 1 h before fixation. Images of the GFP reporter (green) and nuclei stained with DAPI (blue) were taken in separate channels using a 40× objective and merged in SPOT software. (B) Comparison of the nuclear and cytoplasmic fluorescence ratios of proteins in cells treated with or without ivermectin. The mean nuclear and cytoplasmic fluorescence ratios were quantified using Image-Pro Plus software; the differences in the nuclear and cytoplasmic fluorescence ratios of GFP-NLS1and NLS2-GFP protein in cells treated with or without ivermectin were analyzed using ANOVA with GraphPad Prism 6.0 software. N = 50 for each group.

• 

  Figure 6. Construction and identification of DEV CHv-UL13 ΔNLS and DEV CHv-UL13 ΔNLS R recombinant viruses. (A) Schematic representation of the DEV genome in the UL13 region and parts of the DEV CHv-UL13ΔNLS and revertant DEV CHv-UL13ΔNLS R sequences. (B) Restriction Fragment Length Polymorphism analysis. Lane 1 and lane 3, DEV CHv-G plasmids digested with SalI restriction endonuclease; lane 2, DEV CHv-UL13ΔNLS-G plasmids were digested with the restriction endonuclease SalI; lane 4, DEV CHv-UL13ΔNLS R-G plasmids digested with the restriction endonuclease SalI. Digestion of the deletion and revertant products resulted in shifts of the 5,714-bp fragment of the parental virus (lane 1 and lane 3) to 5,765 bp and 5,798 bp (lane 2 and lane 4), respectively. No extraneous alterations were evident in either clone. M, 1 kb plus DNA ladder. ∗different bands.

• 

  Figure 7. Analysis of the characteristics of the UL13-deletion mutant virus in cultured cells. (A) Plaque assay. The figures represent the phenotypes of the plaques formed by infection with DEV CHv, DEV CHv-UL13ΔNLS, and DEV CHv-UL13ΔNLS R. The histogram shows the analysis of the mean are of viral plaques formed by the various viruses following infection. The digital images were analyzed using Adobe Photoshop, and statistical analyses were performed using GraphPad Prism 6.0 software. N = 50 for each group. (B) Growth curve. DEFs were infected with DEV CHv, DEV CHv-UL13ΔNLS, or DEV CHv-UL13ΔNLS R at a titer of 200 TCID50 and harvested 6, 12, 24, 48, 72, and 96 h after infection. The curves were generated based on the viral titers in cells harvested at different time points by determining the TCID50 using Excel. Significant difference between DEV CHv- and DEV CHv-ΔUL13-infected cells were analyzed using GraphPad Prism 6.0 software.