Fig 8. VEEV nsP2 can shut off global host macromolecular synthesis and downregulate the antiviral state.

(A and B) MEF cells were treated with 100-150 IU/mL (10mL total) mouse IFN in a 60mm dish or 400 IU/mL (10mL total) mouse IFN in a 100 mm dish for 16h and transfected with indicated plasmids. Cells were labeled with 100μCi/ml of [³⁵S] Cys/Met for 2h at 24h post transfection. Lysates were collected and resolved on SDS-PAGE gels and visualized as described in Materials and Methods. (A) Representative images of nsP2 induced shutoff in IFN primed cells compared to Mock or GFP control. (B) Densitometry was performed to quantify the extent of shutoff. ***, P<0.0004 using t-test. Ns = not significant. Data is the average of four independent experiments. (C and D) MEF cells were treated with 125-150 IU/mL mouse IFN in a 60mm dish for 16h and transfected with indicated plasmids. Lysates were collected at 24h post transfection and western blots for ISGs were performed as described in Materials and Methods. (C) Representative blot of IFIT1 and TGTP levels in VEEV and SINV nsP2 transfected cells. (D) Densitometry was performed to normalize IFIT1 and TGTP levels to actin and compared to GFP transfected control. *, P<0.04 using t-test. (E) Huh7 cells were treated with 2000 IU/mL for 16h and transfected with plasmids expressing indicated proteins. Cells were infected with 17-D nLuc at 8h following transfection (MOI = 1.5) and lysates were collected for luciferase assay at 24h p.i. Data is expressed as RLU/μg in IFN-primed cells as a percentage of untreated cells for each plasmid. Results are average of three independent experiments. **, P<0.002 using t-test. Ns = not significant. All error bars are standard deviations.