Fig 1.

During CHIKV RNA replication, nsP3 localizes to punctate cytoplasmic foci concurrently with G3BP. (A) Mock-transfected Vero cells were left untreated or were treated with sodium arsenite (AsNaO₂). Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline for 10 min at room temperature and permeabilized with ice-cold acetone-methanol (1:1) for 10 min at −20°C. SGs were stained with G3BP antibody (G6046; Sigma). (B, top) Vero cells transfected with in vitro-transcribed CHIK replicon RNA (CHIKrep) and stained with J2 anti-dsRNA and G3BP antibodies. (B, bottom) CHIKrep-transfected cells (left) and cells from the same sample that remained untransfected (right). (C) Schematic representation of the CHIKrep-nsP3mC-FlucEGFP replicon expressing nsP3 with an internal mCherry (mC) fusion and an FlucEGFP fusion protein from the subgenomic promoter. (D) Vero cells were transfected with a pRL-TK plasmid constitutively expressing Renilla luciferase (Rluc) together with either CHIKrep-FlucEGFP or CHIKrep-nsP3mC-FlucEGFP in vitro-transcribed RNA. Cells were lysed at 16 hpt, and Fluc/Rluc activities were measured. Luciferase activity in mock-transfected cells was also measured. The Fluc measurements were normalized to Rluc to compensate for differences in transfection efficiency. The values depicted are averages of triplicate samples and are expressed as the percentage of normalized Fluc activity relative to that of CHIKrep-FlucEGFP. Error bars represent standard deviations. (E) CHIKrep-nsP3mC-FlucEGFP in vitro-transcribed RNA was transfected into Vero cells, and at 16 hpt, cells were fixed and stained with G3BP antibodies. Where indicated, samples were treated with sodium arsenite (0.5 mM) for 30 min at 16 hpt.