FIG 4.

VP3 PDE activity is required for inhibiting the OAS-RNase L pathway in the full-length VP3 protein. (A) HEK293 cells were transfected with the GFP empty vector or GFP-tagged WT and VP3 mutants from the simian RV RRV strain. At 24 h, cell lysates were harvested and either immunoprecipitated with an anti-GFP antibody and probed for GFP in a Western blot (top) or directly examined by Western blotting for GAPDH (bottom). (B) HEK293 cells were transfected with the GFP empty vector or the GFP-tagged WT and VP3 mutants from the simian RV RRV strain. At 24 hpt, cells were fixed with 4% paraformaldehyde, stained for COXIV (mitochondrial marker [mito]), and imaged with a Zeiss LSM710 confocal microscope. Inset images are labeled with red boxes in the merged channel. Yellow signals, indicative of VP3 and COXIV colocalization, are pointed out with white arrows. (C) HEK293 cells were transfected with GFP and the indicated RRV VP3 WT and mutants for 24 h and transfected with 1 μg/ml poly(I·C) for another 6 h. Total RNA was then extracted and resolved by an RNA chip assay. (D) Quantification of intact 18S and 28S rRNA bands and RNase L cleavage products in panel C. The arithmetic means ± standard deviations from three independent experiments are shown.