Fig 6. CHIKV SIE is not mediated by a single viral protein.

(A) Schematic representation of the tetracycline-inducible construct. PuroR, puromycin resistance. LTR, long terminal repeat; HIV-1 ψ, packaging signal of human immunodeficiency virus type 1; PGK, mouse phosphoglycerate kinase 1; PuroR, puromycin resistance (puromycin N-acetyltransferase); AmpR, ampicillin resistance (β-lactamase); cPPT/CTS, central polypurine tract and central termination sequence of HIV-1; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; RRE, Rev response element of HIV-1; MCS, multiple cloning site, where genes of interest were cloned; IRES, internal ribosome entry site; ori, origin of replication. (B–D) 3T3 cells stably co-expressing the transactivator protein (3T3 transactivator) and the tetracycline-inducible construct described in (A) were treated with doxycycline (1 μg/mL) or left untreated for 16 h, then infected with CHIKV-GFP for 8 h at an MOI of 4, and subsequently harvested and analyzed by flow cytometry. Representative flow cytometry plots (B), percentages of cells expressing the transgene (C) and percentage of infected cells (D) and are displayed. SP, structural protein. (E, F) 3T3 cells stably expressing the transactivator protein (3T3 transactivator) were transfected with the construct containing nsP2 gene. Twenty-four hours post-transfection, cells were plated and treated or not with 1 μg/mL of doxycycline for 16 h, then challenged with CHIKV-GFP at an MOI of 4. Eight hours post-infection, they were harvested and analyzed by flow cytometry. Representative flow cytometry plot (E) and percentage of infected cells in the mCherry⁺and mCherry⁻populations (F) are shown. Bars indicate mean ± SD of biological triplicates and data are representative of two independent experiments. ***p < 0.001 (two-way ANOVA followed by Sidak’s post-test (C) or unpaired t-test (D)).