The N-Terminal 85 Amino Acids of the Barley Stripe Mosaic Virus γb Pathogenesis Protein Contain Three Zinc-Binding Motifs

Abstract

Barley stripe mosaic virus RNA␥ encodes ␥b, 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 ␥b 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. ␥b derivatives containing mutations in only two of the motifs retained the ability to bind zinc, whereas a ␥b 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 ␥b 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 ␥b.Barley stripe mosaic virus (BSMV) encodes a 17-kDa cysteine-rich protein (CRP) protein, designated ␥b, that is expressed from the 3Ј-proximal cistron of RNA␥ (Fig. 1A). The ␥b protein is expressed early and remains at elevated levels throughout the course of BSMV infection (6). Although ␥b is dispensable for both viral replication and movement in the ND18 strain of the virus, in the ␥⌬2.1 deletion mutant, which removes the bulk of the ␥b gene, symptom onset is delayed in barley, and symptoms are attenuated, resulting in a null phenotype characterized by an erratic mosaic pattern (21). Further analysis of this mutant suggests that ␥b has differential effects on RNA accumulation and protein expression and that it may regulate the synthesis of proteins encoded on RNA␤. Interestingly, in the type strain of BSMV, the ␥b protein is essential for systemic symptom development (21), and the requirement for ␥b is linked to a 372-nucleotide (nt) direct repeat in the 5Ј region of RNA␥ that overlaps the start codon and increases the size of the ␥a gene. This gene encodes the polymerase component of the viral replicase and therefore, this strain-specific difference suggests that subtle effects on replication contribute to the phenotypic effects of mutations in the ␥b protein.Nine of the eleven cysteine residues in the ␥b protein are concentrated in two clusters toward the N terminus of the protein. The C1 (amino acid...

The C‐terminal region of the Barley stripe mosaic virusγb protein participates in homologous interactions and is required for suppression of RNA silencing

“…A second approach, farwestern blotting, was employed to demonstrate direct protein–protein interactions. In these studies, we used five MBP fusion derivatives (MBP:lacZ, MBP:γb, MBP:γb(–) C1C2, MBP:γb1–85 and MBP:γb86–152) that were initially constructed for zinc binding experiments of γb (Bragg et al ., 2004). Following SDS‐PAGE, replicate samples of the fusion proteins were blotted on to nitrocellulose and were overlayed with radiolabelled, in vitro translated γb protein derivatives (Fig.…”

“…Despite its apparent solubility, various biochemical manipulations during extraction from plants and protoplasts often result in aggregation of γb. Site‐specific mutations in the C1, BM and C2 regions also have striking effects on the solubility of γb, and many mutant derivatives are shifted from the soluble fraction to the cell wall and membrane‐containing fractions (Bragg et al ., 2004; Donald and Jackson, 1994). Studies of TRV p16 suggest that the conditional solubility of γb may not be unique among the viral CRPs because the p16 protein can only be extracted from infected tissue using highly denaturing conditions.…”

Soilborne wheat mosaic virus (SBWMV) 19K protein belongs to a class of cysteine rich proteins that suppress RNA silencing

“…Whether these other plant viral CRPs are also suppressors of silencing can not be concluded at this point for two reasons: insufficient study and only weak sequence similarity relationships. Sequences of CRPs that affect virus replication and are encoded by members of other virus genera were also determined to be unrelated [25]. …”

“…The N-terminus has several conserved Cys residues that likely comprise a zinc finger motif. In fact, the ability of the gamma b protein of BSMV to bind Zn(II) was recently demonstrated [25]. …”

Double-Stranded RNA Binding May Be a General Plant RNA Viral Strategy To Suppress RNA Silencing

“…Interestingly, although BSMV ␥B and HC-Pro are very dissimilar, both suppressors contain a cysteine-rich region that might form a zinc finger motif (1,7,17,29,52). It is tempting to speculate that zinc finger domains play important roles in the ds-sRNA binding of BSMV ␥B and HC-Pro.…”