Fig. 2. NSs aggregates exhibit amyloid properties.

a Vero cells were infected with RVFV and RVFV ΔNSs for 16 h. Infected cells were permeabilized with a Triton X-100-based buffer and then stained with the amyloid-binding dye Thioflavin-S (ThS, blue) and Abs against NSs (purple) before analysis by confocal microscopy. Images are representative of three independent experiments. Scale bar, 20 µm. b Vero cells were exposed to RVFV (MOI ~5) and nuclei isolated 16 h pi before being subjected to 2%-SDS treatments and incubated at the indicated temperatures for 15 min. Nuclear extracts were analyzed by 12.5% SDS-PAGE without stacking gel, and under nonreducing conditions followed by WB with Abs against NSs. Results are representative of three independent experiments. c Vero cells were infected with RVFV at a MOI of 5 for 16 h and lyzed. Cell lysates were then subjected to increasing amounts of SDS and incubated at the indicated temperature for 5 min. Proteins were analyzed by 1.5% semi-denaturing detergent-agarose gel electrophoresis (SDD-AGE) followed by capillary transfer and immunoblotting against NSs. Experiments were repeated independently three times with similar results. d Total lysates of Vero cells infected at a MOI of 5 for 16 h were treated with 10 mM DTT and 2% SDS before being incubated at the indicated temperatures for 5 min. Samples were subsequently analyzed by 1.5% SDD-AGE and WB against NSs. HMW, high molecular weight; wt, wild type. Results are representative of three independent experiments.