The N-terminal 85 amino acids of the barley stripe mosaic virus gammab pathogenesis protein contain three zinc-binding motifs.
Barley stripe mosaic virus RNAgamma encodes gammab, a cysteine-rich protein that affects pathogenesis. Nine of the eleven cysteines are concentrated in two clusters, designated C1 (residues 1 to 23) and C2 (residues 60 to 85), that are arranged in zinc finger-like motifs. A basic motif (BM) rich in lysine and arginine (residues 19 to 47) resides between the C1 and C2 clusters. We have demonstrated that gammab binds zinc and that the C1, BM, and C2 motifs have independent zinc-binding activities. To evaluate the requirements for binding, mutations were introduced into each region. Cysteine residues at positions 7, 9, 10, 19, and 23 in the C1 motif were replaced with serines. In the BM, asparagines were substituted for lysines at positions 26 and 35, glutamine for arginine at position 25, and glycines for arginines at positions 33 and 36. The C2 mutations included cysteine replacements with serines at positions 60, 64, 71, and 81, and a histidine-to-leucine change at position 85. These mutations destroyed zinc-binding activity in each of the isolated motifs. gammab derivatives containing mutations in only two of the motifs retained the ability to bind zinc, whereas a gammab derivative containing mutations inactivating all three motifs destroyed the ability to bind zinc. Plants inoculated with transcripts containing combinations of the C1, BM, and C2 mutations elicited a "null" phenotype in barley characteristic of gammab deletion mutants and also delayed the appearance and reduced the size of local lesions in Chenopodium amaranticolor. These results show that zinc binding of each of the motifs is critical for the biological activity of gammab.
Bragg
Jennifer N
JN
Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
Lawrence
Diane M
DM
Jackson
Andrew O
AO
D002846:Chromatography, Affinity; D001467:Hordeum / Q000821:virology*; D009154:Mutation; D010935:Plant Diseases / Q000821:virology; D018515:Plant Leaves / Q000821:virology; D012328:RNA Viruses / Q000472:pathogenicity*; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*; D016335:Zinc Fingers
D017361:Viral Nonstructural Proteins; C091602:gamma(b) protein, Barley stripe mosaic virus; D015032:Zinc