Figure 2.

D471G amino acid substitution in LCMV-NP does not affect its interaction with LCMV-Z. (A) VLP assay: Human 293T cells were co-transfected with 2 μg of the indicated pC wt or mutant NPs HA-tagged, together with 2 μg of pC-LCMV-Z FLAG-tagged expression plasmid. Empty pC plasmid was included to normalize the total amount of transfected DNA. At 72 hpt, cell lysates were prepared and analyzed for protein expression levels for NP (α-HA) and Z (α-FLAG) (i). GAPDH was used as a loading control. TCS from same transfected cells were used for isolation of VLP to evaluate NP incorporation (α-HA) into Z-mediated VLP (α-FLAG) (ii). Numbers at the bottom of each WB lane represent the quantification of band intensities normalized to NP and Z wt expression levels. (B) M2H assay: Human 293T cells (6.5x10⁵) were co-transfected in triplicate (12-well plate format) with 2 μg of the indicated pC-VP16-tagged wt or mutant NPs, together with 2 μg of pC-GAL4-tagged Z expression plasmid as described in Figure 1B. At 72 hpt, LCMV NP-Z interaction was determined by GFP expression (i) and by luciferase activity (ii). pC-VP16 expression plasmid was used as negative control. Reporter gene activation (FFL) is shown as percentage of wt interaction (pC-NP-VP16 and pC-GAL4-Z) after normalization of transfection efficiencies with the Renilla luciferase expression plasmid pRL SV40. Expression levels of wt and mutant NP were determined by WB using an anti-VP16 polyclonal antibody (iii). GAPDH was used as a loading control. Numbers at the bottom of each WB lane represent the quantification of band intensities normalized to wt NP lane as described in material and methods.