Rabies Virus Nucleoprotein Functions To Evade Activation of the RIG-I-Mediated Antiviral Response

Comparison of the gene expressions of SYM-I cells infected with Ni, Ni-CE, and CE(NiN) strains using a DNA microarray. SYM-I cells were infected with Ni, Ni-CE, and CE(NiN) strains at an MOI of 2. After 24 h, the total cellular RNA was extracted and used for DNA microarray analysis. The data were normalized by GeneSpring GX software. (A) Cluster analysis of genes of SYM-I cells infected with each virus. The expression pattern of genes involved in “host-pathogen interaction” is represented as a hierarchical clustering, using Cluster and Java TreeView. Genes shown in red are upregulated, and those shown in green are downregulated relative to mock-infected cells. (B) Expression levels of 10 host immunity-related genes, most of which were differentially expressed in Ni-CE- and CE(NiN)-infected cells. Each bar represents the fold change in expression compared to the expression level of each gene in mock-infected cells.

Validation by real-time RT-PCR of DNA microarray results for IFN-β (A), IFN-λ1 (B), CXCL10 (C), and CCL5 (D). The assay was performed with the same total RNA used in the DNA microarray experiment. Expression levels of genes were normalized to mRNA levels of GAPDH. Each bar represents the mean (± the SD) of three independent replicates. *, Significant difference (P < 0.01); ND, no detection.

Infection of CE(NiN) strain inhibits activation of IRF-3-dependent but not NF-κB-dependent IFN-β promoter. SYM-I cells were cotransfected with pRL-TK and pNF-κB-Luc (NF-κB-responsive reporter plasmid) (A) or 4×IRF-3-Luc (IRF-3-responsive reporter plasmid) (B). After 24 h, the cells were mock infected or infected with each strain at an MOI of 2. The luciferase activities were measured 24 h after transfection. The data represent firefly luciferase activity normalized to Renilla luciferase activity and are presented as means (± the SD) of three independent replicates. *, Significant difference (P < 0.01); ns, no significant difference.

Ni and CE(NiN) infections prevent nuclear translocation and dimerization of IRF-3. (A) SYM-I cells were inoculated, in suspension, with each virus strain at an MOI of 2 FFU/cells and seeded. After 24 h, cells were transfected with the GFP-IRF-3 expression plasmid (green). At 24 h posttransfection, the cells were fixed and stained with an anti-N monoclonal antibody (red). (B) Assessment of the rate of GFP-IRF-3 nuclear translocation in GFP-IRF-3-expressing and virus-infected calls. Each value is the average (± the SD) of three independent experiments in which 100 cells were counted. *, Significant difference (P < 0.01). (C) Subcellular localization of endogenous IRF-3 in SYM-I cells infected with each strain. SYM-I cells were mock infected or infected with Ni, Ni-CE, or CE(NiN) strains at an MOI of 2. After 24 h, cells were fixed and examined by double immunofluorescence staining using anti-IRF-3 polyclonal antibody (green) and anti-N monoclonal antibody (red). (D) Extracts from SYM-I cells infected with Ni, Ni-CE, and CE(NiN) strains for 24 h were analyzed by native gel electrophoresis, followed by Western blotting, to detect IRF-3. N protein and tubulin of same samples were detected by SDS-PAGE, followed by Western blotting.

Effects of transient expression of Ni or Ni-CE N protein on IRF-3-dependent promoter activities in SYM-I cells infected with NDV or Ni-CE strain. SYM-I cells were cotransfected with pRL-TK, 4×IRF-3-Luc, and 1 μg of each plasmid driving the expression of the indicated viral protein or empty vector. At 24 h posttransfection, the cells were infected with NDV at an MOI of 1 and incubated for 12 h (A) or infected with Ni-CE strain at an MOI of 2 and incubated for 24 h (B). Then, the cells were lysed, and the luciferase activities were measured. The data represent firefly luciferase activity normalized to Renilla luciferase activity and are presented as means (± the SD) of three independent replicates. *, Significant difference (P < 0.01); ns, no significant difference.

N protein does not affect expression level of and activity of viral P protein to inhibit the IRF-3 pathway. (A) SYM-I cells were infected with the Ni, Ni-CE, or CE(NiN) strain at an MOI of 2. After 24 h, the cells were lysed and N, P, and tubulin were detected by Western blotting. (B) SYM-I cells in a 24-well plate were cotransfected with 1 μg of each plasmid driving the expression of the indicated viral protein or empty vector. At 48 h posttransfection, the cells were lysed, and N, P, and tubulin were detected by Western blotting. (C) SYM-I cells in a six-well plate were cotransfected with 4 μg of pCAGGS-CEP and 4 μg of pCAGGS-NiN or pCAGGS-CEN. Cell extracts were prepared at 48 h posttransfection and directly subjected to Western blotting with anti-N antibody, anti-P antibody, or anti-tubulin antibody (top). The same cell extracts were subjected to coimmunoprecipitation analysis with anti-N antibody (middle) or normal mouse IgG (bottom). The immunoprecipitated samples were examined by Western blotting. (D) SYM-I cells were cotransfected with pRL-TK, 4×IRF-3-Luc, and 1 μg of each plasmid driving the expression of the indicated viral protein or empty vector. At 24 h posttransfection, the cells were infected with NDV at an MOI of 1 and incubated for 12 h. Then the cells were lysed and luciferase activities were measured. The data represent firefly luciferase activity normalized to Renilla luciferase activity and are presented as means (± the SD) of three independent replicates.

Ni N protein, but not Ni-CE N protein, functions to evade activation of RIG-I-mediated antiviral response. (A) SYM-I cells were cotransfected with pRL-TK, 4×IRF-3-Luc, and 1 μg of pEF-Flag-RIG-I or empty vector. At 24 h posttransfection, the cells were infected with Ni, Ni-CE, and CE(NiN) strains at an MOI of 2 and incubated for 24 h. Then, the cells were lysed and luciferase activities were measured. *, Significant difference (P < 0.01); ns, no significant difference. (B) SYM-I cells were inoculated, in suspension, with each virus strain at an MOI of 2 FFU/cells and seeded. After 24 h, cells were transfected with pRL-TK, 4×IRF-3-Luc, and 1 μg of pEF-Flag-RIG-IC. After 24 h, the cells were lysed and luciferase activities were measured. *, Significant difference (P < 0.01); ns, no significant difference. (C) SYM-I cells were inoculated, in suspension, with each virus strain at an MOI of 2 FFU/cells and seeded. After 24 h, cells were transfected with pRL-TK, 4×IRF-3-Luc, and 0.5, 1, or 2 μg of pEF-Flag-RIG-IC. After 24 h, the cells were lysed, and the luciferase activities were measured. *, Significant difference versus mock-transfected cells (P < 0.01). (D) SYM-I cells were inoculated, in suspension, with each virus strain at an MOI of 2 FFU/cells and seeded. After 24 h, cells were transfected with pRL-TK, 4×IRF-3-Luc, and 1 μg of pEF-Flag-RIG-IN. After 24 h, the cells were lysed, and the luciferase activities were measured. The data are presented as means (± the SD) of three independent replicates. ns, No significant difference.