PHYSICAL AND FUNCTIONAL-CHARACTERIZATION OF THE DOUBLE-STRANDED-RNA BINDING-PROTEIN ENCODED BY THE VACCINIA VIRUS E3 GENE

The vaccinia Virus E3 gene encodes a 190-amino acid double-stranded (d s) RNA-binding protein that antagonizes cellular antiviral response pa thways triggered by dsRNA and interferon. The physical and functional properties of the E3 protein were determined using recombinant E3 prod uced in bacteria and purified to homogeneity. We show by sedimentation and chemical crosslinking that E3 is a dimer in solution al high ioni c strength. E3 self-associates to form higher order oligomers as ionic strength is reduced from 1 to 0.1 M NaCl. Structure probing by limite d proteolysis suggests that E3 consists of amino- and carboxyl-termina l domains separated by a trypsin-sensitive bridge at residues Lys-92 a nd Arg-95. The carboxyl-domain of E3 contains a conserved dsRNA bindin g motif (dsRBM) found in many other proteins that interact with dsRNA. That the C-terminal domain per se binds to dsRNA was verified by stud ies of recombinant E3(100-190) purified from bacteria. The affinity of the C-terminal domain for dsRNA was comparable to that of the full-le ngth E3 protein (K-D similar to 7 to 9 nM). E3(100-190) did not bind t o DNA-DNA duplexes or to DNA-RNA hybrids, suggesting that the dsRBM sp ecifically recognizes an A-form helix E3(100-190) is a dimer in soluti on; however, unlike the full-sized E3 protein, E3(100-190) does not fo rm higher order multimers at low ionic strength. (C) 1996 Academic Pre ss, Inc.