Fig. 5. Recovery and characterization of RVFV encoding tetracysteine (tc)-NSs.

a Schematic depiction of NSs N-terminally tagged with a tc peptide (tc-NSs). b Titration of the recombinant RVFV coding for tc-NSs (RVFV tc-NSs) in a monolayer of Vero cells by plaque-forming assay. After 5 days of incubation at 37 °C, plaques were colored with crystal violet. RVFV and its mutant lacking the full sequence coding for NSs (RVFV ΔNSs) were used as controls. wt, wild type. c Titer of the genetically engineered RVFV tc-NSs after rescue and five passages in Vero cells. Points represent titers of independent virus productions (n = 3). Center line, mean. pfu plaque-forming units. d The interferon-β (IFN-β) mRNA levels were quantified by real-time quantitative reverse transcription PCR (qRT-PCR) in L-929 cells infected at MOI ~3 with the indicated viruses for up to 20 h. Points represent replicates (n = 2). Results are representative of three independent experiments. Center line, mean. e Vero cells were exposed to RVFV and RVFV tc-NSs (MOI ~3) and subjected to immunofluorescence staining against NSs (green) 16 h pi. Tc peptide and nuclei were stained with ReAsH (red) and Hoechst (blue), respectively, prior to imaging by confocal microscopy. Images are representative of three independent experiments. Scale bar, 15 µm. f, g RVFV tc-NSs (MOI ~5) was added to Vero cells for 3 h, and after replacing the input virus by phenol red-free medium containing the ReAsH dye, infected cells were imaged in real-time with a wide-field microscope at 37 °C for up to 14 h. Images were taken every 5 min, and each frame was analyzed as described in Supplementary Fig. 2. The number of NSs fibrillary aggregates per cell (n = 50) and the average length of NSs filaments (n = 120) are showed as points in f and one line in g, respectively. Results are representative of three individual experiments.