Brome Mosaic Virus Coat Protein Research Articles

The intrinsically disordered N-terminal arm of the brome mosaic virus coat protein specifically recognizes the RNA motif that directs the initiation of viral RNA replication.

In the brome mosaic virus (BMV) virion, the coat protein (CP) selectively contacts the RNA motifs that regulate translation and RNA replication (Hoover et al., 2016. J. Virol. 90, 7748). We hypothesize that the unstructured N-terminal arm (NTA) of the BMV CP can specifically recognize RNA motifs. Using ion mobility spectrometry-mass spectrometry, we demonstrate that peptides containing the NTA of the CP were found to preferentially bind to an RNA hairpin motif that directs the initiation of BMV RNA synthesis. RNA binding causes the peptide to change from heterogeneous structures to a single family of structures. Fluorescence anisotropy, fluorescence quenching and size exclusion chromatography experiments all confirm that the NTA can specific recognize the RNA motif. The peptide introduced into plants along with BMV virion increased accumulation of the BMV CP and accelerated the rate of minus-strand RNA synthesis. The intrinsically disordered BMV NTA could thus specifically recognize BMV RNAs to affect viral infection.

Phosphorylation of the Brome Mosaic Virus Capsid Regulates the Timing of Viral Infection.

The four brome mosaic virus (BMV) RNAs (RNA1 to RNA4) are encapsidated in three distinct virions that have different disassembly rates in infection. The mechanism for the differential release of BMV RNAs from virions is unknown, since 180 copies of the same coat protein (CP) encapsidate each of the BMV genomic RNAs. Using mass spectrometry, we found that the BMV CP contains a complex pattern of posttranslational modifications. Treatment with phosphatase was found to not significantly affect the stability of the virions containing RNA1 but significantly impacted the stability of the virions that encapsidated BMV RNA2 and RNA3/4. Cryo-electron microscopy reconstruction revealed dramatic structural changes in the capsid and the encapsidated RNA. A phosphomimetic mutation in the flexible N-terminal arm of the CP increased BMV RNA replication and virion production. The degree of phosphorylation modulated the interaction of CP with the encapsidated RNA and the release of three of the BMV RNAs. UV cross-linking and immunoprecipitation methods coupled to high-throughput sequencing experiments showed that phosphorylation of the BMV CP can impact binding to RNAs in the virions, including sequences that contain regulatory motifs for BMV RNA gene expression and replication. Phosphatase-treated virions affected the timing of CP expression and viral RNA replication in plants. The degree of phosphorylation decreased when the plant hosts were grown at an elevated temperature. These results show that phosphorylation of the capsid modulates BMV infection. How icosahedral viruses regulate the release of viral RNA into the host is not well understood. The selective release of viral RNA can regulate the timing of replication and gene expression. Brome mosaic virus (BMV) is an RNA virus, and its three genomic RNAs are encapsidated in separate virions. Through proteomic, structural, and biochemical analyses, this work shows that posttranslational modifications, specifically, phosphorylation, on the capsid protein regulate the capsid-RNA interaction and the stability of the virions and affect viral gene expression. Mutational analysis confirmed that changes in modification affected virion stability and the timing of viral infection. The mechanism for modification of the virion has striking parallels to the mechanism of regulation of chromatin packaging by nucleosomes.

Role of charge regulation and size polydispersity in nanoparticle encapsulation by viral coat proteins.

Nanoparticles can be encapsulated by virus coat proteins if their surfaces are functionalized to acquire a sufficiently large negative charge. A minimal surface charge is required to overcome (i) repulsive interactions between the positively charged RNA-binding domains on the proteins and (ii) the loss of mixing and translational entropy of RNA and capsid coat proteins. Here, we present a model describing the encapsulation of spherical particles bearing weakly acidic surface groups and investigate how charge regulation and size polydispersity impact upon the encapsulation efficiency of gold nanoparticles by model coat proteins. We show that the surface charge density of these particles cannot be assumed fixed, but that it adjusts itself to minimize electrostatic repulsion between the charges on them and maximize the attractive interaction with the RNA binding domains on the proteins. Charge regulation in combination with the natural variation of particle radii has a large effect on the encapsulation efficiency: it makes it much more gradual despite its inherently cooperative nature. Our calculations rationalize recent experimental observations on the coassembly of gold nanoparticles by brome mosaic virus coat proteins.

The Tripartite Virions of the Brome Mosaic Virus Have Distinct Physical Properties That Affect the Timing of the Infection Process

The three subsets of virions that comprise the Brome mosaic virus (BMV) were previously thought to be indistinguishable. This work tested the hypothesis that distinct capsid-RNA interactions in the BMV virions allow different rates of viral RNA release. Several results support distinct interactions between the capsid and the BMV genomic RNAs. First, the deletion of the first eight residues of the BMV coat protein (CP) resulted in the RNA1-containing particles having altered morphologies, while those containing RNA2 were unaffected. Second, subsets of the BMV particles separated by density gradients into a pool enriched for RNA1 (B1) and for RNA2 and RNA3/4 (B2.3/4) were found to have different physiochemical properties. Compared to the B2.3/4 particles, the B1 particles were more sensitive to protease digestion and had greater resistivity to nanoindentation by atomic force microscopy and increased susceptibility to nuclease digestion. Mapping studies showed that portions of the arginine-rich N-terminal tail of the CP could interact with RNA1. Mutational analysis in the putative RNA1-contacting residues severely reduced encapsidation of BMV RNA1 without affecting the encapsidation of RNA2. Finally, during infection of plants, the more easily released RNA1 accumulated to higher levels early in the infection. Viruses with genomes packaged in distinct virions could theoretically release the genomes at different times to regulate the timing of gene expression. Using an RNA virus composed of three particles, we demonstrated that the RNA in one of the virions is released more easily than the other two in vitro. The differential RNA release is due to distinct interactions between the viral capsid protein and the RNAs. The ease of RNA release is also correlated with the more rapid accumulation of that RNA in infected plants. Our study identified a novel role for capsid-RNA interactions in the regulation of a viral infection.

An Examination of the Electrostatic Interactions between the N-Terminal Tail of the Brome Mosaic Virus Coat Protein and Encapsidated RNAs

The coat protein of positive-stranded RNA viruses often contains a positively charged tail that extends toward the center of the capsid and interacts with the viral genome. Electrostatic interaction between the tail and the RNA has been postulated as a major force in virus assembly and stabilization. The goal of this work is to examine the correlation between electrostatic interaction and amount of RNA packaged in the tripartite Brome Mosaic Virus (BMV). Nanoindentation experiment using atomic force microscopy showed that the stiffness of BMV virions with different RNAs varied by a range that is 10-fold higher than that would be predicted by electrostatics. BMV mutants with decreased positive charges encapsidated lower amounts of RNA while mutants with increased positive charges packaged additional RNAs up to ∼900 nt. However, the extra RNAs included truncated BMV RNAs, an additional copy of RNA4, potential cellular RNAs, or a combination of the three, indicating that change in the charge of the capsid could result in several different outcomes in RNA encapsidation. In addition, mutant with specific arginines changed to lysines in the capsid also exhibited defects in the specific encapsidation of BMV RNA4. The experimental results indicate that electrostatics is a major component in RNA encapsidation but was unable to account for all of the observed effects on RNA encapsidation. Thermodynamic modeling incorporating the electrostatics was able to predict the approximate length of the RNA to be encapsidated for the majority of mutant virions, but not for a mutant with extreme clustered positive charges. Cryo-electron microscopy of virions that encapsidated an additional copy of RNA4 revealed that, despite the increase in RNA encapsidated, the capsid structure was minimally changed. These results experimentally demonstrated the impact of electrostatics and additional restraints in the encapsidation of BMV RNAs, which could be applicable to other viruses.

The coat protein leads the way: an update on basic and applied studies with the Brome mosaic virus coat protein.

The Brome mosaic virus (BMV) coat protein (CP) accompanies the three BMV genomic RNAs and the subgenomic RNA into and out of cells in an infection cycle. In addition to serving as a protective shell for all of the BMV RNAs, CP plays regulatory roles during the infection process that are mediated through specific binding of RNA elements in the BMV genome. One regulatory RNA element is the B box present in the 5' untranslated region (UTR) of BMV RNA1 and RNA2 that play important roles in the formation of the BMV replication factory, as well as the regulation of translation. A second element is within the tRNA-like 3' UTR of all BMV RNAs that is required for efficient RNA replication. The BMV CP can also encapsidate ligand-coated metal nanoparticles to form virus-like particles (VLPs). This update summarizes the interaction between the BMV CP and RNAs that can regulate RNA synthesis, translation and RNA encapsidation, as well as the formation of VLPs.

Functional analysis of brome mosaic virus coat protein RNA-interacting domains

The coat proteins (CP) of cowpea chlorotic mottle (CCMV) and brome mosaic virus (BMV), two members of the genus Bromovirus, share 70% identity at the amino acid (aa) level and contain four highly conserved regions, identified as putative RNA-interacting domains (RIDs). To assess the contribution of the conserved aa sequence within each RID and the structural features contained therein toward virion assembly and RNA packaging, we engineered a set of fourteen independent mutations (deletions and substitutions) encompassing all four RIDs. The effect of each mutation on viral biology, pathogenesis, and RNA packaging was analyzed in whole-plant infection assays. Among the four RIDs, two mutations engineered into the N-proximal domain (RID I) and two of the four mutations engineered into the C-proximal domain (RID IV) proved to be more debilitating (compared to wild-type) while only selected regions in the central domains (RID II or III) showed a detectable effect. Neutral effects were observed when aa residues that are predicted to affect calcium binding were mutated. To further analyze the importance of N and C terminal interactions leading to virus assembly and RNA packaging, four CP hybrids were constructed by precisely exchanging either the N-terminal 77 or the C-terminal 113/112aa between BMV and CCMV. Despite the fact that the CP composition of the hybrid viruses is distinct from either of the parents, the symptom phenotype in Chenopodium quinoa, migration pattern of CP in Western blots and virion mobility in agarose gels was indistinguishable from the respective parent providing the genetic background. Collectively, the data provide insight for assessing the relative importance of each RID during genome packaging and in molecular processes regulating the overall architecture of the assembled virions.

Characterization of a novel barley protein, HCP1, that interacts with the Brome mosaic virus coat protein.

Brome mosaic virus (BMV) requires the coat protein (CP) not only for encapsidation but also for viral cell-to-cell and long-distance movement in barley plants. This suggests that BMV infection is controlled by interactions of CP with putative host factors as well as with viral components. To identify the host factors that interact with BMV CP, we screened a barley cDNA library containing 2.4 x 10(6) independent clones, using a yeast two-hybrid system. Using full-length and truncated BMV CPs as baits, four candidate cDNA clones were isolated. One of the candidate cDNAs encodes a unique oxidoreductase enzyme, designated HCP1. HCP1 was found predominantly in the soluble fractions after differential centrifugation of BMV-infected and mock-inoculated barley tissues. A two-hybrid binding assay using a series of truncated BMV CPs demonstrated that a C-terminal portion of CP is essential for its interaction with HCP1. Interestingly, experiments with CP mutants bearing single amino acid substitutions at the C-terminus revealed that the capacity for mutant CP-HCP1 binding correlates well with the infectivity of the corresponding mutant viruses in barley. These results indicate that CP-HCP1 binding controls BMV infection of barley, interacting directly with CP, probably in the cell cytoplasm.

TRNA elements mediate the assembly of an icosahedral RNA virus.

tRNAs, the adapter molecules in protein synthesis, also serve as metabolic cofactors and as primers for viral RNA-directed DNA synthesis. The genomic and subgenomic RNAs of some plant viruses have a 3'-terminal tRNA-like structure (TLS) that can accept a specific amino acid and serve as a site for initiation of replication and as a simple telomere. We report a previously undescribed role for the TLS of brome mosaic virus (BMV), and potentially for cellular tRNA, in mediating the assembly of its icosahedral virions. BMV genomic RNAs and subgenomic RNA lacking the TLS failed to assemble into virions when incubated with purified BMV coat protein. Assembly was restored by addition of a 201-nt RNA containing the BMV TLS. TLSs from two other plant viruses as well as tRNAs from wheat germ and yeast were similarly active in the BMV virion assembly reaction, but ribosomal RNA and polyadenylate did not facilitate assembly. Surprisingly, virions assembled from TLS-less BMV RNA in the presence of tRNAs or TLS-containing short RNA did not incorporate the latter molecules. Consistent with a critical role for the BMV TLS in virion assembly, mutations in the BMV genomic RNAs that were designed to disrupt the folding of the TLS also abolished virion assembly. We discuss the likely roles of the TLS in early stages of virion assembly.

Molecular Studies on Bromovirus Capsid Protein: VII. Selective Packaging of BMV RNA4 by Specific N-Terminal Arginine Residues

An arginine-rich RNA-binding motif (ARM) found at the N-proximal region of Brome mosaic virus (BMV) coat protein (CP) adopts α-helical conformation and shares homology with CPs of plant and insect RNA viruses, HIV-Rev and Tat proteins, bacterial antiterminators, and ribosomal splicing factors. The ARM of BMV CP, consisting of amino acids 9 through 21 with six arginine residues, is essential for RNA binding and subsequent packaging. In this study analysis of the α-helical contents of wild-type and mutant peptides by circular dichroism spectra identified protein determinants required for such conformation. Electrophoretic mobility-shift assays between viral RNA and BMV CP peptides with either proline or alanine substitutions revealed that the interaction is nonspecific. Expression in vivo of mature full-length BMV CP subunits, having the same substitutions for each arginine within the ARM, derived from biologically active clones was found to be competent to assemble into infectious virions and cause visible symptom phenotypes in whole plants. However, analysis of virion progeny RNA profiles of CP variants and subsequent in vitro reassembly assays between mutant CP and four BMV RNAs unveiled the ability of arginine residues at positions 10, 13, or 14 of the ARM to confer selective packaging of BMV RNA4. Thus, BMV CP contains determinants that specifically interact with RNA4 to ensure selective packaging.

Packaging of Tobacco Mosaic Virus Subgenomic RNAs by Brome Mosaic Virus Coat Protein Exhibits RNA Controlled Polymorphism

The coat protein (CP) of icosahedral Brome mosaic virus (BMV) was expressed from a genetically engineered rod-shape Tobacco mosaic virus. Molecular characterization of the progeny recovered from symptomatic plants revealed that BMV CP selectively packaged the three subgenomic RNAs of the hybrid virus into two differently sized icosahedral virus-like particles (VLPs). The smaller VLPs packaged only the two smaller subgenomic RNAs. Additional in vitro reassembly assays with BMV CP subunits and transcripts of hybrid subgenomic RNAs further demonstrated that the ability of BMV capsids to display polymorphism is not dependent on the RNA size alone and appears to be controlled by some other feature(s) of the genetically engineered RNA.

Molecular Studies on Bromovirus Capsid Protein: VI. Contributions of the N-Terminal Arginine-Rich Motif of BMV Capsid Protein to Virion Stability and RNA Packaging

Specific interactions are likely to occur between the highly conserved N-proximal arginine-rich motif (ARM) of Brome mosaic virus (BMV) coat protein (CP) and each of three genomic RNAs and a single subgenomic RNA during in vivo encapsidation. To characterize these interactions, three independent deletions were engineered into a biologically active clone of BMV RNA3 (B3) such that the matured CP of each B3 variant precisely lacks either the entire ARM (B3/Δ919) or two consecutive arginine residues (B3/13ΔΔ14 and B3/18ΔΔ19) within the ARM. Analysis of virion RNA for each B3 variant recovered from symptomatic leaves of Chenopodium quinoa revealed that the interactions between the N-terminal ARM of BMV CP and each of three genomic RNAs is distinct. Northern blot hybridization of B3Δ919 virion RNA revealed that the deleted ARM region specifically affected the stability of virions containing RNA1. An abundant truncated RNA species recurrently found in the virions of B3Δ919 was identified to be a derivative of genomic RNA1, lacking the 5′ 943 nucleotides. Additional Northern blot analysis of virion RNAs from B3/Δ919, B3/13ΔΔ14, and B3/18ΔΔ19, and in vitro reassembly assays revealed that the N-terminal ARM region contains crucial amino acids required for RNA4 packaging, independent of genomic RNA3. The significance of these observations in relation to Bromovirus CP-RNA interactions during virion assembly is discussed.

Characterization of the Brome Mosaic Virus Movement Protein Expressed inE. coli

The biochemical and functional properties of the movement protein (MP) of brome mosaic virus (BMV) were investigated. Expression and purification of the BMV MP fromEscherichia coliresulted in a pure and soluble protein preparation. Sucrose gradient centrifugation revealed that BMV MP forms oligomers consisting of two or more copies but no higher order multimers even when different ionic strengths and pHs were applied. Nitro-cellulose filter binding and gel retardation studies showed thatin vitrothe BMV MP preferentially bound to ss nucleic acids (RNA and DNA); the affinity to ssRNA was lower compared to BMV coat protein. The binding to ss nucleic acid was cooperative and not sequence specific and the hypothetical binding site was calculated to be around three to six nucleotides per MP monomer. The nucleic acid binding properties of the BMV MP are discussed in relation to the recent finding that this protein is also able to form tubular structures in infected protoplasts.

Molecular Studies on Bromovirus Capsid Protein

Two members of the bromovirus group, brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV), selectively infect barley and cowpea, respectively, and also differ in their ability to systemically infect a common permissive host,Chenopodium quinoa.CCMV is confined to inoculated leaves ofC. quinoa,whereas BMV causes rapid systemic mottling. To examine whether host-specific determinants for systemic movement of BMV and CCMV in each of these hosts are localized in the coat protein (CP), sequences encoding this gene were exchanged between biologically active clones of BMV RNA3 (B3) and CCMV RNA3 (C3) to create chimera expressing heterologous CP genes (B3/CCP and C3/BCP). Inoculation of each chimera with its respective wild-type (wt) RNAs 1 and 2 to barley or cowpea orC. quinoaplants resulted in symptom phenotype and long distance movement characteristics similar to those of the parental virus donating RNAs 1 and 2. These observations suggest that neither BMV CP nor CCMV CP has host-specific determinants for long distance movement. Inoculation of additional recombinant viruses, constructed by reassorting wt genomic RNAs 1 and 2 of BMV and CCMV with either heterologous wt RNA3 (i.e., B1+B2+C3 and C1+C2+B3) or heterologous chimeric RNA3 (i.e., B1+B2+C3/BCP and C1+C2+B3/CCP), to susceptible hosts resulted only in localized infections. The significance of these observations in relation to bromovirus movement is discussed.

Biological Significance of the Seven Amino-Terminal Basic Residues of Brome Mosaic Virus Coat Protein

Inoculation of six brome mosaic virus (BMV) RNA3 transcripts with defined deletions in the coat protein (CP) gene to three Chenopodium spp demonstrated that synthesis of a functional, encapsidation-competent CP is required for the induction of local lesions. The BMV CP open reading frame contains two in-frame AUG codons separated by seven amino acids, resulting in the synthesis of two CPs (CP1 and CP2). To elucidate the biological significance of the N-terminal basic region of BMV CP, RNA3 variants capable of producing either CP1 or CP2 but not both were constructed. Infection phenotypes elicited on three Chenopodium spp by each RNA3 variant revealed that amino-terminal residues 1 to 7 are required to establish chlorotic local lesions and systemic infection in Chenopodium quinoa. Deletion of this region has no effect on infection in barley plants but resulted in the induction of the hypersensitive response on the inoculated leaves of C. quinoa and blocked systemic spread. Analysis of seven additional RNA3 variant transcripts, each having a six base deletion (two amino acids) in the sequence encoding the N-terminal seven residues, indicated that variants that share a common deletion of positively charged lysine rendered the CP encapsidation-incompetent and failed to establish infection. Taken together, these results suggest that residues 1 to 7 of the BMV CP play an important role in virus host interactions and contribute differently to the virulence phenotype in different host plants.

Foreign Complementary Sequences Facilitate Genetic RNA Recombination in Brome Mosaic Virus

We have demonstrated that local antisense sequences can mediate genetic recombination within the 3′ noncoding region among brome mosaic virus (BMV) RNAs (P. Nagy and J. J. Bujarski, 1993, Proc, Natl. Acad. Sci. USA 90, 6390-6394). Here we show that foreign complementary inserts can direct crossovers between BMV RNA3 components within an internal region. A 170-nt polynucleotide derived from the cowpea chlorotic mottle virus (CCMV) RNA3 was inserted just upstream of the initiation cordon of the BMV coat protein open reading frame in either sense or antisense orientations. The resulting respective mutants, BCC+ and BCC-, maintained unchanged CCMV inserts when inoculated separately on leaves of a local lesion host for BMV. In contrast, when a mixture containing both mutated RNAs3 was inoculated, a significant fraction of lesions accumulated the BMV RNA3 lacking the CCMV insert. The presence of a 3′ marker mutation confirmed that the BMV RNA3 progeny arose due to crossovers between BCC+ and BCC- within the complementary sequences. The highest frequency of recombinant appearance was observed when the RNA mixtures were annealed prior to inoculation on the host plants. Our results confirm a concept predicting the general nature of the heteroduplex-mediated recombination functioning in RNA viruses. Examples of possible applications of this approach in recombinant RNA technology are discussed.

The 3′-untranslated region of brome mosaic virus RNA does not enhancetranslation of capped mRNAs in vitro

The translation enhancing ability of cis-acting 3′-terminal untranslated region (3′-UTR) of brome mosaic virus (BMV) was examined. Two chimeric mRNA constructs translated in rabbit reticulocyte lysates contained the BMV coat protein (CP) gene and NPTI gene, respectively. It was shown that the 3′-UTR of BMV RNA enhanced the translational efficiency of uncapped but not capped messages.

Effects of deletions in the N-terminal basic arm of brome mosaic virus coat protein on RNA packaging and systemic infection.

The first 25 amino acids of brome mosaic virus (BMV) coat protein include 8 basic and no acidic residues and are implicated in binding the encapsidated RNA. Using infectious transcripts from BMV RNA3 cDNA clones, we modified this region of the coat gene. A coat protein mutant with the first 25 amino acids deleted failed to direct either packaging of viral RNA in protoplasts or systemic infection of whole barley plants. Neither symptoms, virions, nor viral RNA was detectable in plants inoculated with this mutant or a mutant with a frameshift mutation in the coat gene. Mutants with the normal start codon changed to AAG or with the first eight codons deleted allowed translation to start at a downstream AUG, resulting in a deletion of the first 7 amino acids of the mature wild-type coat protein. These mutants not only packaged viral RNA in protoplasts but directed symptomatic, systemic infections that developed with normal speed and degree of spread within the host. The AUG-to-AAG point substitution did not revert to the wild type after long-term culture in planta. Wild-type BMV virions were also found to contain small amounts of a protein that coelectrophoresed with the truncated coat protein produced by the viable AAG and eight-codon-deletion mutants. This minor coat protein species presumably arose by infrequent translation initiation at the second AUG in the wild-type coat protein gene. Absence of encapsidation-competent coat protein appeared to stimulate production of nonstructural proteins in protoplast infections.

Hybrid brome mosaic virus RNAs express and are packaged in tobacco mosaic virus coat protein in vivo

Brome mosaic virus (BMV) is an icosahedral virus with a tripartite RNA genome which infects monocotyledonous plants, while the cowpea or legume strain of tobacco mosaic virus (CcTMV) is a rod-shaped virus with a single component RNA genome which infects dicotyledonous plants. To examine the potential for exchanging entire genes between RNA viruses, biologically active cDNA clones were used to replace the natural coat gene of BMV RNA3 with the coat gene and encapsidation origin of CcTMV. In protoplasts coinoculated with BMV RNAs 1 and 2, the resulting hybrid RNA3 was replicated by BMV trans-acting factors but was packaged in TMV coat protein to give rod-shaped particles rather than the usual BMV icosahedra. When the CcTMV encapsidation origin was suitably inserted in derivatives of BMV RNAs 1 and 2, these RNAs were also packaged in a ribonuclease-resistant form in protoplasts coinoculated with the hybrid RNA3 expressing TMV rather than BMV coat protein. Thus, despite the markedly divergent nature of BMV and TMV, replicating hybrids bearing characters derived from both parent viruses were produced. Such hybrid viruses could be of considerable value for studying specific steps in infection and for assigning functions to particular virus genes.

Further studies on inhibitory effect of brome mosaic virus coat protein on the viral RNA synthesis in vitro.

Further studies on inhibitory effect of brome mosaic virus coat protein on the viral RNA synthesis in vitro.