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. 2020 Jan 15;12(1):105. doi: 10.3390/v12010105

Figure 3.

Figure 3

NP position 111 significantly affects oligomerization of NP. (A) Schematic of the NP oligomerization assay. We co-expressed NP fused to NanoLuc (NLuc, donor) and HaloTag (acceptor) in HEK 293FT cells. NP–NP binding and oligomerization brought the tags into close spatial proximity, producing bioluminescence resonance energy transfer (BRET) emission at 625 nm. To calculate BRET signal in milliBRET units, we normalized 625 nm BRET luminescence against NP-NLuc luminescence at 465 nm and subtracted spectral spillover from NP-NLuc into the 625 nm channel; (B) BRET oligomerization assay controls. Absence of either tag, free NLuc (not fused to NP), or deletion of the NP oligomerization domain (∆OD, residues 20–38) reduced BRET signal; (C) EBOV VP35 NP-binding peptide (NPBP) disrupted NP oligomerization. In addition to NP-NLuc and NP-HaloTag, we co-expressed varying amounts of VP35[NPBP] in cells. To quantify VP35[NPBP] expression, we fused it to enhanced green fluorescent protein (eGFP), separated by a ‘self-cleaving’ porcine teschovirus 1 2A peptide (P2A). We fitted oligomerization versus eGFP fluorescence units (FU) to an inverse function (Equation (1); n = 3 biological replicates per VP35[NPBP] plasmid amount). Shading indicates 95% confidence intervals based on 999 bootstrap pseudoreplicates; (D) Donor saturation assay with NP mutants. We expressed a constant amount of NP-NLuc (donor) and expressed varying amounts of NP-HaloTag (acceptor) in cells to generate donor saturation curves (Equation (2); n = 6 biological replicates per NP-HaloTag plasmid amount). We fitted data to saturation curves, calculated maximum oligomerization (Max) and ratio of NP-HaloTag to NP-NLuc plasmid needed to reach half Max ± standard error (BRET50 ± SE) for each NP mutant. eGFP (black dots near x-axis) did not produce data suitable for curve fitting. We assessed statistical significance of differences in BRET50 between NP mutants by ANOVA with Dunnett’s test to correct for multiple hypothesis testing. Shading indicates 95% confidence intervals based on 999 bootstrap pseudoreplicates.