Fig 8.

WEEV structural proteins inhibit PRR signaling in neuronal cells. (A) BE(2)-C/m ISRE promoter-reporter cells were cotransfected with a control HA-tagged β-galactosidase (β-gal) expression vector and a second vector encoding the indicated WNV (NS1, NS2A) or WEEV (capsid [C], capsid-envelope [C-E], nsP1, nsP2, or nsP3) genes. Cells were subsequently stimulated 48 h after transfection with 100 U/ml IFN-α-A/D, 50 μg/ml extracellular poly(I-C) (pIC), or 700 ng/ml transfected poly(I-C) (T-pIC), and reporter gene activity was measured 24 h after stimulation. Results are expressed relative to cells cotransfected with empty vector (dashed line). We observed no significant cytotoxicity from transient overexpression of any viral gene, as assessed by MTT assay (data not shown). *, P < 0.05; **, P < 0.005. (B) BE(2)-C/m NF-κB promoter-reporter cells were transfected and stimulated as described above for panel A except that 25 ng/ml TNF-α was used instead of IFN-α-A/D. (C) Lysates from BE(2)-C/m cells transfected with WNV (lanes 2 and 3) or WEEV (lanes 4 to 6) nonstructural protein expression vectors as described for panels A and B were analyzed by immunoblotting for HA-tagged β-gal, V5-tagged viral proteins, and GAPDH as a loading control. Note the cross-reactivity of the V5 antibody with an ∼50-kDa protein in all samples. MW, molecular weight (in thousands). (D) Lysates from BHK-21 cells (left blots) or BHK-21 cells stably expressing bacteriophage T7 RNA polymerase (BHK-21/T7; right blots) cotransfected with a control HA-tagged β-gal expression vector and a second vector encoding WEEV capsid (Cap; lanes 2 and 6), capsid-envelope (Cap-Env; lanes 3 and 7), or T7 promoter-driven capsid/YFP chimera WEEV replicon (Rep-YFP; lanes 4 and 8) were analyzed by immunoblotting for WEEV capsid, HA-tagged β-gal, and actin as a loading control.