Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA
(A) Schematic stem-loop structure of HBV ε RNA. Ribonucleotide sequences (nt 1847–1991, genotype D, subtype ayw) are presented with base paring indicated by dotted line. (B) Verification of the purified recombinant 6×His-tagged ISG20 by SDS-PAGE Coomassie staining. (C) EMSA assay of ISG20-ε binding. The indicated amount of ISG20 proteins were incubated with 100 ng 32P-end-labeled ε RNA in binding buffer to form nucleoprotein complexes. Monoclonal anti-His antibody was used for supershifting of the His-ISG20/ HBV ε complex. Excessive amount of cold unlabeled HBV ε RNA (10×, 20×, 40×) were used to compete with the binding of ISG20 to 100 ng radiolabeled HBV ε. The nucleoprotein complexes were separated by native PAGE and the shifted bands were detected by autoradiography.