Figure 6.

Effects of D471 substitution on LCMV-NP functions. (A) NP-NP interaction: Human 293T cells were co-transfected as described in Figure 1B with 2 μg of the indicated pC wt or mutant NP-VP16 fusion proteins, together with 2 μg of NP-GAL4 expression plasmids. At 72 hpt, cell extracts were prepared to determine the strength of the interaction. VP16 expression plasmid was used as negative control. Reporter gene activation (FFL) is shown as percentage of wt interaction (pC-NP-VP16 and pC-NP-GAL4) after normalization of transfection efficiencies based on levels of Renilla luciferase activity driven by plasmid pRL SV40. Cell lysates were used to detect expression of wt and mutant NPs by WB using an anti-VP16 polyclonal antibody. GAPDH was used as a loading control. (B) NP-Z interaction: Human 293T cells were co-transfected as described in Figure 2B with 2 μg of the indicated pC wt or mutant NP-VP16, together with 2 μg of GAL4-Z expression plasmids. At 48 hpt, cell extracts were prepared to determine the strength of the interaction and protein expression. Reporter gene activation (FFL) is shown as percentage of wt interaction (pC-NP-VP16 and pC-GAL4-Z) after normalization of transfection efficiencies based on Renilla luciferase values. Cell lysates were used to detect expression of wt and mutant NPs by WB using an anti-VP16 polyclonal antibody. GAPDH was used as a loading control. (C) Replication and transcription activity: BHK-21 cells were co-transfected with the LCMV MG as described in Figure 3 together with expression plasmids for the viral polymerase (L) and wt or indicated mutant NPs, and pSV40-Cluc expression vector to normalize transfection efficiencies. At 48 hpt, TCS were collected for luciferase assay and cell lysates were prepared for protein detection. Empty pC was used as a negative control. RLUs (%) were calculated based on the replication and transcription activity mediated by wt NP, after normalization by Cluc luminescence values. Expression levels of wt and mutant NPs were determined by WB using an anti-HA antibody. GAPDH was used as a loading control. (D) Inhibition of induction of IFN-I: Human 293T cells were co-transfected as described in Figure 4 with 0.1 μg of the indicated pC-NP HA-tagged expression vectors together with the IFNβ reporter plasmids. At 16 hpt, cells were infected with SeV (moi=3) to induce activation of the IFNβ promoter, and 24 hours later cell lysates were prepared for luciferase assay and detection of protein expression. Luciferase values were normalized with respect to those obtained in cells transfected with Empty pC and infected with SeV, after adjusting for renilla luminescence values. Expression of wt and mutant NPs were determined by WB using an anti-HA antibody. GAPDH was used as a loading control.