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 (ds) RNA-binding protein that antagonizes cellular antiviral response pathways triggered by dsRNA and interferon. The physical and functional properties of the E3 protein were determined using recombinant E3 produced in bacteria and purified to homogeneity. We show by sedimentation and chemical crosslinking that E3 is a dimer in solution at high ionic strength. E3 self-associates to form higher order oligomers as ionic strength is reduced from 1 to 0.1 M NaCl. Structure probing by limited proteolysis suggests that E3 consists of amino- and carboxyl-terminal domains separated by a trypsin-sensitive bridge at residues Lys-92 and Arg-95. The carboxyl-domain of E3 contains a conserved dsRNA binding motif (dsRBM) found in many other proteins that interact with dsRNA. That the C-terminal domain per se binds to dsRNA was verified by studies of recombinant E3(100-190) purified from bacteria. The affinity of the C-terminal domain for dsRNA was comparable to that of the full-length E3 protein (KD approximately 7 to 9 nM). E3(100-190) did not bind to DNA-DNA duplexes or to DNA-RNA hybrids, suggesting that the dsRBM specifically recognizes an A-form helix. E3(100-190) is a dimer in solution; however, unlike the full-sized E3 protein, E3(100-190) does not form higher order multimers at low ionic strength.
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Molecular Biology Program, Sloan-Kettering Institute, New York, 10021, USA.
Shuman
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D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012330:RNA, Double-Stranded / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012996:Solutions; D014357:Trypsin; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
C081374:E3L protein, Vaccinia virus; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012996:Solutions; D014764:Viral Proteins; D014357:Trypsin