Fig 1.

RVFV-induced IFN-β responses are dependent on cytoplasmic RIG-I and are independent of TLRs. (A) HEK293XL cells were transfected with a luciferase construct for the IFN-β promoter and stimulated with medium only (M) or RVFV rMP-12 or NSs del strains at an MOI of 1 or 5 for 18 h. (B) IFN-β activation in HEK293XL cells transfected with 0, 10, 50, or 100 ng of RIG-I dominant-negative construct (RIG-I Dn) and stimulated with medium (M) or NSs del at an MOI of 5 for 18 h. Cells were stimulated with control ligands Sendai virus (SV) and 400 ng of transfected poly(I·C) [Trans poly(I:C)] for 18 h with or without the addition of RIG-I dominant-negative construct. (C and D) HEK293XL cells were stably transfected with TLR7 (C) or TLR8 (D) and transiently transfected with MyD88 dominant-negative construct (MyD88 Dn) at 0, 10, 50, or 100 ng. The cells were transfected with IFN-β reporter construct and stimulated with medium (M) or NSs del strain at an MOI of 5. Controls were performed using the NF-κB luciferase reporter and the TLR7-specific ligand Gardiquimod (C) or the TLR7/8 ligand R848 (D) for 18 h. The data represent mean values ± the standard deviations based on triplicate wells from a representative experiment. Each experiment was performed at least three times. Significance: ***, P ≤ 0.001; **, P ≤ 0.01. (E) Western blot confirming the expression of RIG-I Dn in HEK293XL cells and of MyD88 Dn in TLR7 and TLR8 cells. The cells were either (i) not transfected, (ii) transfected with IFN-β luciferase reporter, Renilla, and pcDNA 3.1 (plasmids), (iii) transfected with the dominant-negative construct, reporter, and Renilla (MyD88 Dn + plasmids), or (iv) the dominant-negative construct alone (MyD88 Dn).