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. 2020 Feb 18;11:178. doi: 10.3389/fmicb.2020.00178

FIGURE 5.

FIGURE 5

Anthralin inhibits the cap-snatching activity of viral RdRp. (A) Binding of vRNA to vRdRp. The polymerase complexes were incubated at 37°C for 30 min in the binding buffer with 5′ FAM-labeled vRNA. The protein-RNA complexes were resolved from free RNA by electrophoresis on 4% non-denaturing gels. (B) Cap-binding analysis in a gel shift assay. Following 30 min reaction for the binding of an unlabeled oligoribonucleotide containing the vRNA, the 20-nucleotide-long capped RNAs marked by 5′-FAM were added, and then incubated in the presence or absence of anthralin for 30 min at 37°C. The complexes were separated from free RNA by electrophoresis on 4% non-denaturing gels. (C,D) Endoribonuclease assays. 5′ FAM-labeled Capped RNA, vRNA, diluted series anthralin and vRdRp (C) or PA subunit alone (D) were mixed and incubated with 0.1 μg of tRNA as an additional inhibitor of non-specific nucleases at 37°C water bath for 60 min and samples were analyzed on a 20% acrylamide, 7 M urea denaturing gel.