Fig. 6
Functionality of LSTOP and L3mut in an EBOV transcription/replication plasmid-based system (mini-genome) in cell culture. a, b Schematic diagrams of the conserved domains (grey boxes), functional motifs (purple) and variable regions or hinges (discontinuous green line) in EBOV L3mut and LSTOP. This diagram is based on data for filovirus and mononegavirus models for the L protein. Conserved blocks I–III constitute a RdRp, which is closely associated with a capping domain (Cap). Block VI has methyltransferase (MTase) activity, and downstream of this is located in a small C-terminal domain (CTD). Red highlighted the amino acid position where the a three most frequently found amino acid changes in the L protein at positions 572, 98s and 2061 are located and b the truncated protein due to the stop codon in L. c EBOV mini-genome system activity at different ratios between EBOV L and LSTOP and d at equal EBOV L but different LSTOP amounts. Results are shown as the mean ± S.D. from one experiment performed in triplicate. ***P < 0.001; **P < 0.01; *P < 0.05. Western blot for luciferase (LUC), EBOV L/LSTOP and house-keeping GAPDH protein abundance in cells transfected with the mini-genome system. e EBOV mini-genome system activity in the presence of the L, no L and L3mut and at different ratios between L and L3mut. Blotting showed LUC and GAPDH abundance in cells transfected with the mini-genome system plasmids. f VP35-eGFP was used in a co-immunoprecipitation (coIP) assay to examine its interaction with EBOV LSTOP. Blotting showed the presence of eGFP and viral proteins VP35/eGFP, VP35, L and NP in the cell lysates (input (I)) and coIP fraction (eluate (E)). g Proposed model of the competition between EBOV L and LSTOP for the viral RdRp co-factor VP35 and the potential reduction in the EBOV RNA synthesis observed in patients with lower viral load (inset panel)