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

Barley stripe mosaic virus (BSMV) encodes a 17-kDa cysteine-rich protein (CRP) protein, designated γb, that is expressed from the 3′-proximal cistron of RNAγ (Fig. ​(Fig.1A).1A). The γb protein is expressed early and remains at elevated levels throughout the course of BSMV infection (6). Although γb is dispensable for both viral replication and movement in the ND18 strain of the virus, in the γΔ2.1 deletion mutant, which removes the bulk of the γb 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 γb has differential effects on RNA accumulation and protein expression and that it may regulate the synthesis of proteins encoded on RNAβ. Interestingly, in the type strain of BSMV, the γb protein is essential for systemic symptom development (21), and the requirement for γb is linked to a 372-nucleotide (nt) direct repeat in the 5′ region of RNAγ that overlaps the start codon and increases the size of the γa 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 γb protein. FIG. 1. The tripartite genome of BSMV and subgenomic RNAs (sgRNAs) used for expression of the seven proteins encoded by the RNAs and motifs located within γb. (A) The filled circles represent the 5′ cap structure present on all of the BSMV genomic ... Nine of the eleven cysteine residues in the γb protein are concentrated in two clusters toward the N terminus of the protein. The C1 (amino acids 1 to 23) and C2 (amino acids 60 to 85) clusters are arranged in zinc finger-like motifs (with one histidine residue included in C2 at position 85). The γb protein also contains a basic motif (BM) between amino acids 19 to 47 and six heptad repeats of a coiled-coil motif between residues 95 to 140 (Fig. ​(Fig.1B).1B). Extensive analyses of mutations introduced into the C1, BM, and C2 regions of γb have demonstrated that these motifs each have important virulence functions (4). Subcellular fractionation of infected barley extracts has revealed that the site-specific mutations in these regions also have striking effects on the solubility of the protein and that mutant derivatives are often shifted from the soluble fraction to the cell wall and membrane-containing fractions (4). Furthermore, single-stranded RNA-binding activity has been demonstrated for a recombinant GST-γb fusion protein in in vitro gel shift studies. Lysine and arginine residues within the BM were demonstrated to mediate this binding, whereas cysteine and histidine substitutions in the C1 and C2 regions had little effect on in vitro RNA-binding activity (5). Small cysteine-rich proteins (CRPs) are also encoded by the 3′-terminal open reading frame (ORFs) in a number of other plant viruses including members of the Tobravirus, Carlavirus, Furovirus, and Pecluvirus genera (17). Although these CRPs do not share a substantial amount of sequence identity with γb, they do share limited regions of sequence and predicted structural similarity. For example, amino acids 26 to 134 of the BSMV γb protein, which encompass the majority of the BM, C2, and putative coiled-coil regions of the protein, can be aligned with the corresponding amino acids of the CRPs of the other hordeiviruses, Poa semilatent virus (PSLV) and Lychnis ringspot virus, and also with the CRPs of Soilborne cereal mosaic virus (SBCMV), Soilborne wheat mosaic virus (SBWMV), Peanut clump virus (PCV), Indian peanut clump virus, Chinese wheat mosaic virus (CWMV), Sorghum chlorotic spot virus (SCSV), and Oat golden stripe virus (OGSV) (10). One predicted zinc binding motif (CCCH) is conserved among all of the proteins of this family, as is the arrangement of the basic, zinc-binding, and coiled-coil motifs. Three additional CRPs that affect replication (Beet necrotic yellow vein virus (BNYVV) p14, Tobacco rattle virus (TRV) p16, and Pea early browning virus (PEBV) p12) each contain cysteine and histidine residues arranged in putative zinc-binding motifs adjacent to a region of clustered basic arginine and lysine residues (15, 18) but lack a predicted coiled-coil region. Understanding the functions of these proteins during virus infections is of particular interest because, like γb, a number of them have been demonstrated to be viral pathogenicity determinants and are suggested to play regulatory roles during infection (4, 7, 12, 13, 18). Deletion of BSMV γb (5, 21) and TRV p16 (18), both of which have RNA-binding activity, or PCV p15 (7) causes a dramatic decreases in viral RNA levels that can be rescued by supplying the appropriate CRP in trans. Furthermore, the TRV p16 protein can be functionally replaced by the BSMV γb, SBWMV p19, or Cucumber mosaic virus (CMV) 2b proteins, suggesting that these proteins may share some common biological functions (18). The TRV p16, BSMV γb, and SBWMV p19 proteins are all CRPs but, interestingly, the CMV 2b protein does not encode a cysteine-rich region. The 2b protein is most commonly recognized as a suppressor of RNA silencing (20). The PCV CRP, p15, also acts as a suppressor of RNA silencing (8) and more recently, a similar role has been suggested for the BSMV and PSLV γb proteins (30). In these experiments, Nicotiana benthamiana plants were inoculated with a TMV derivative (TMV-GFP) that expresses green fluorescent protein (GFP). RNA silencing, preventing systemic movement and fluorescence of TMV-GFP, was induced by coinoculation with a Potato virus X (PVX) vector encoding a truncated GFP sequence that does not fluoresce (PVX-GF). However, when TMV-GFP was coinoculated with PVX-GF that also expresses either the BSMV or PSLV γb proteins, systemic movement of TMV-GFP was rescued, and GFP fluorescence was detected in the systemically infected leaves (30). The C1 and C2 motifs of γb have long been predicted to be zinc-binding regions and are comparable in size (20 to 40 amino acids) to the majority of identified zinc-binding motifs (1). The γb C1 and C2 motifs have the patterns CX2/3CX8CX3C and CX3CX16CX3H (or CX3CX6CX9CX3H), respectively, and whereas the spacings are unique, the identity and arrangement of the ligands are characteristic of structural sites in which the metal stabilizes the protein and is required for its proper folding (1, 2). To provide more insight into the role the γb protein plays in BSMV infections, we analyzed the zinc-binding activity of wild-type (wt) γb obtained from infected plants, as well as that of recombinant γb fusion protein derivatives containing either large deletions or site-specific mutations within the C1, BM, and C2 regions. Mutations that affected γb zinc-binding activity were incorporated into biologically active cDNA clones and were used to evaluate the phenotypic effects of the substitutions, as well as their effects on RNA and protein accumulation during BSMV infections.