Identification of a conserved motif that is necessary for binding of the vaccinia virus E3L gene products to double-stranded RNA.
The E3L gene of vaccinia virus encodes the double-stranded (ds) RNA binding proteins p20 and p25 that exhibit inhibitory activity for the IFN-induced, P1/elF-2 alpha protein kinase. A region in the E3L encoded proteins (residues 156-180) shares a high degree of similarity with several proteins that bind double-helical RNA including the P1/elF-1 alpha kinase, bacterial and yeast RNase III, and a human transactivator response element/Rev response element binding protein. In this study, mutants of E3L proteins were constructed in order to determine the region of the proteins required for dsRNA binding and kinase inhibitory activity. Our data indicate that both the region necessary for dsRNA binding and for kinase inhibitory activity are located at the carboxyl terminus of the protein. The E3L proteins with 7 amino acids deleted from the carboxyl terminus (184-190) could bind to dsRNA, but with lower affinity than could the full-length protein. This protein did not detectably inhibit kinase in vitro. Deletion of 26 amino acids from the carboxyl terminus of the E3L proteins (165-190) abolished dsRNA binding activity and kinase inhibitory activity. In addition, mutations at amino acid 164, 167, or 174 severely inhibited binding to dsRNA. On the other hand, deletion of 83 amino acids from the amino terminus did not affect the proteins' ability to bind dsRNA or inhibit kinase. These results suggest that a region of sequence between amino acids 164 and 183 is necessary for E3L proteins' dsRNA binding activity. This region lies within the homologous domain that the E3L proteins share with other dsRNA binding proteins.
Chang
H W
HW
Department of Microbiology, Arizona State University, Tempe 85287-2701.
Jacobs
B L
BL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D005814:Genes, Viral / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017354:Point Mutation; D011131:Polyribonucleotides / Q000378:metabolism; D014176:Protein Biosynthesis; D047428:Protein Kinase Inhibitors; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012087:Reoviridae / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D019892:eIF-2 Kinase
C081374:E3L protein, Vaccinia virus; D011131:Polyribonucleotides; D047428:Protein Kinase Inhibitors; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014764:Viral Proteins; D019892:eIF-2 Kinase