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. 2012 Aug;86(15):8185–8197. doi: 10.1128/JVI.07240-11

Fig 4.

Fig 4

Residues contributing to the anti-IFN activity of LCMV-NP are critical for inhibition of SeV-mediated transcriptional activation of NF-κB. (A and B) Inhibition of SeV-mediated induction of an NF-κB-dependent promoter by LCMV-NP C-terminal deletion (A) and single-amino-acid (B) mutants. 293T cells (12-well format, triplicates) were cotransfected as described for Fig. 2 using two doses (10 and 100 ng) of C-terminal HA-tagged LCMV-NP wild type (WT) and the indicated mutants. NF-κB reporter gene activation (i) is expressed as fold induction over the level seen with the empty vector-transfected and mock-infected control cells. Values were assessed by a two-tailed paired Student's t test versus SeV-infected empty plasmid-transfected cell results (*, P = 0.01 [A]; *, P = 0.049; **, P = 0.002 [B]). Lysates (100 μg of total protein) from same transfected cells were analyzed for NP expression levels by Western blotting using an anti-HA polyclonal antibody (ii). Protein expression levels were normalized with respect to GAPDH as described for Fig. 2. Molecular mass markers (kDa) are indicated on the left. LCMV-NP (wild-type and mutants) and GAPDH are indicated on the right. (C) Effect of LCMV-NP single amino acid substitutions in nuclear translocation of GFP-p65. 293T cells were cotransfected with GFP-p65 together with HA-tagged LCMV-NP wild type or indicated NP mutants as described for Fig. 3B. As a negative control, cells were transfected with an HA-tagged LCMV-Z pCAGGs expression plasmid. At 24 h p.t., cells were infected with SeV (MOI = 3), and subcellular localization of GFP-p65 was assessed at 12 to 16 h p.i. Representative images of GFP-p65 NF-κB (green), LCMV-Z and LCMV-NP wild type (WT) and mutants (red), cellular nuclear staining (blue), and the corresponding merged images are illustrated. (D) Percentage of GFP-p65 nuclear translocation. GFP-p65 nuclear translocation was assessed as described for Fig. 3C. Results were evaluated using a two-tailed paired Student's t test versus SeV-infected LCMV-Z (*, P = 0.171; **, P = 0.034).