FIG. 1.

E3L allows VV to evade the antiviral activity of RNase L. Plaque assays (A) and one-step viral-growth curves (B) of wild-type VV and VVΔE3L (Copenhagen strain) are shown. (C) Diagram of RNase L and mutant RNase L_ΔEN. (D) Ponasterone induction of RNase L in comparison to β-actin as determined by probing a Western blot with antibodies. (E) Assay for RNase L and mutant RNase L_ΔEN levels by covalent cross-linking to a ³²P-labeled 2-5A analog before and after ponasterone induction (5 μM for 24 h). (F) Assay for RNase L activity in intact cells as measured by specific cleavages in rRNAs. Cells were incubated in the absence or presence of ponasterone (5 μM) for 24 h and transfected with 2-5A [p₃A(2′p5′A)₃] (1 μM) for 3 h. Total RNA was isolated, electrophoresed in a formaldehyde-1.2% agarose gel, stained with ethidium bromide (lower panel), and probed with ³²P-labeled 18S rRNA cDNA (upper panel). (G) RNase L^−/− cells containing inducible cDNAs for RNase L and RNase L_ΔEN were incubated in the absence or presence of ponasterone (5 μM) for 24 h and infected at an MOI of 6 with wild-type VV or VVΔE3L (Copenhagen strain). At 24 h postinfection, viruses were harvested and virus yields were determined by plaque titration on BHK21 cells.