Brome Mosaic Virus Protein Research Articles

A MATHEMATICAL MODEL OF RNA3 RECRUITMENT IN THE REPLICATION CYCLE OF BROME MOSAIC VIRUS

Positive-strand RNA viruses, such as the brome mosaic virus (BMV) and hepatitis C virus, utilize a replication cycle which involves the recruitment of RNA genomes from the cellular translation machinery to the viral replication complexes. Here, we coupled mathematical modeling with a statistical inverse problem methodology to better understand this crucial recruitment process. We developed a discrete-delay differential equation model that describes the production of BMV protein 1a and BMV RNA3, and the effect of protein 1a on RNA3 recruitment. We validated our model with experimental data generated in duplicate from a yeast strain that was engineered to express protein 1a and RNA3 under the control of inducible promoters. We used a statistical model comparison technique to test which biological assumptions in our model were correct. Our results suggest that protein 1a expression is governed by a nonlinear phenomenon and that a time delay is important for modeling RNA3 recruitment. We also performed an uncertainty analysis of two experimental designs and found that we could improve our data collection procedure in future experiments to increase the confidence in our parameter estimates.

Membrane-shaping host reticulon proteins play crucial roles in viral RNA replication compartment formation and function

Positive-strand RNA viruses replicate their genomes on membranes with virus-induced rearrangements such as single- or double-membrane vesicles, but the mechanisms of such rearrangements, including the role of host proteins, are poorly understood. Brome mosaic virus (BMV) RNA synthesis occurs in ≈70 nm, negatively curved endoplasmic reticulum (ER) membrane invaginations induced by multifunctional BMV protein 1a. We show that BMV RNA replication is inhibited 80-90% by deleting the reticulon homology proteins (RHPs), a family of membrane-shaping proteins that normally induce and stabilize positively curved peripheral ER membrane tubules. In RHP-depleted cells, 1a localized normally to perinuclear ER membranes and recruited the BMV 2a(pol) polymerase. However, 1a failed to induce ER replication compartments or to recruit viral RNA templates. Partial RHP depletion allowed formation of functional replication vesicles but reduced their diameter by 30-50%. RHPs coimmunoprecipitated with 1a and 1a expression redirected >50% of RHPs from peripheral ER tubules to the interior of BMV-induced RNA replication compartments on perinuclear ER. Moreover, RHP-GFP fusions retained 1a interaction but shifted 1a-induced membrane rearrangements from normal vesicles to double membrane layers, a phenotype also induced by excess 1a-interacting 2a(pol). Thus, RHPs interact with 1a, are incorporated into RNA replication compartments, and are required for multiple 1a functions in replication compartment formation and function. The results suggest possible RHP roles in the bodies and necks of replication vesicles.

An amphipathic alpha-helix controls multiple roles of brome mosaic virus protein 1a in RNA replication complex assembly and function.

Brome mosaic virus (BMV) protein 1a has multiple key roles in viral RNA replication. 1a localizes to perinuclear endoplasmic reticulum (ER) membranes as a peripheral membrane protein, induces ER membrane invaginations in which RNA replication complexes form, and recruits and stabilizes BMV 2a polymerase (2aPol) and RNA replication templates at these sites to establish active replication complexes. During replication, 1a provides RNA capping, NTPase and possibly RNA helicase functions. Here we identify in BMV 1a an amphipathic α-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles. We show that helix A is essential for efficient 1a–ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA replication. Strikingly, mutations in helix A give rise to two dramatically opposite 1a function phenotypes, implying that helix A acts as a molecular switch regulating the intricate balance between separable 1a functions. One class of helix A deletions and amino acid substitutions markedly inhibits 1a–membrane association and abolishes ER membrane invagination, viral RNA template recruitment, and replication, but doubles the 1a-mediated increase in 2aPol accumulation. The second class of helix A mutations not only maintains efficient 1a–membrane association but also amplifies the number of 1a-induced membrane invaginations 5- to 8-fold and enhances viral RNA template recruitment, while failing to stimulate 2aPol accumulation. The results provide new insights into the pathways of RNA replication complex assembly and show that helix A is critical for assembly and function of the viral RNA replication complex, including its central role in targeting replication components and controlling modes of 1a action.

Nanoparticle-Templated Assembly of Viral Protein Cages

Self-assembly of regular protein surfaces around nanoparticle templates provides a new class of hybrid biomaterials with potential applications in medical imaging and in bioanalytical sensing. We report here the first example of efficiently self-assembled virus-like particles (VLPs) having a brome mosaic virus protein coat and a functionalized gold core. The present study indicates that functionalized gold particles can initiate VLP assembly by mimicking the electrostatic behavior of the nucleic acid component of the native virus. These VLP constructs are symmetric, with the protein stoichiometry and packaging properties indicating similarity to the icosahedral packing of the capsid. Moreover, a pH-induced swelling transition of the VLPs is observed, in direct analogy to the native virus.

Interaction between Brome Mosaic Virus Proteins and RNAs: Effects on RNA Replication, Protein Expression, and RNA Stability

Brome mosaic virus (BMV) RNA replication has been examined in a number of systems, including Saccharomyces cerevisiae. We developed an efficient T-DNA-based gene delivery system using Agrobacterium tumefaciens to transiently express BMV RNAs in Nicotiana benthamiana. The expressed RNAs can systemically infect plants and provide material to extract BMV replicase that can perform template-dependent RNA-dependent RNA synthesis in vitro. We also expressed the four BMV-encoded proteins from nonreplicating RNAs and analyzed their effects on BMV RNA accumulation. The capsid protein that coinfiltrated with constructs expressing RNA1 and RNA2 suppressed minus-strand levels but increased plus-strand RNA accumulation. The replication proteins 1a and 2a could function in trans to replicate and transcribe the BMV RNAs. None of the BMV proteins or RNA could efficiently suppress posttranscriptional silencing. However, 1a expressed in trans will suppress the production of a recombinant green fluorescent protein expressed from the nontranslated portions of BMV RNA1 and RNA2, suggesting that 1a may regulate translation from BMV RNAs. BMV replicase proteins 1a did not affect the accumulation of the BMV RNAs in the absence of RNA replication, unlike the situation reported for S. cerevisiae. This work demonstrates that the Agrobacterium-mediated gene delivery system can be used to study the cis- and trans-acting requirements for BMV RNA replication in plants and that significant differences can exist for BMV RNA replication in different hosts.

TRNA-like structure regulates translation of Brome mosaic virus RNA.

For various groups of plant viruses, the genomic RNAs end with a tRNA-like structure (TLS) instead of the 3' poly(A) tail of common mRNAs. The actual function of these TLSs has long been enigmatic. Recently, however, it became clear that for turnip yellow mosaic virus, a tymovirus, the valylated TLS(TYMV) of the single genomic RNA functions as a bait for host ribosomes and directs them to the internal initiation site of translation (with N-terminal valine) of the second open reading frame for the polyprotein. This discovery prompted us to investigate whether the much larger TLSs of a different genus of viruses have a comparable function in translation. Brome mosaic virus (BMV), a bromovirus, has a tripartite RNA genome with a subgenomic RNA4 for coat protein expression. All four RNAs carry a highly conserved and bulky 3' TLS(BMV) (about 200 nucleotides) with determinants for tyrosylation. We discovered TLS(BMV)-catalyzed self-tyrosylation of the tyrosyl-tRNA synthetase but could not clearly detect tyrosine incorporation into any virus-encoded protein. We established that BMV proteins do not need TLS(BMV) tyrosylation for their initiation. However, disruption of the TLSs strongly reduced the translation of genomic RNA1, RNA2, and less strongly, RNA3, whereas coat protein expression from RNA4 remained unaffected. This aberrant translation could be partially restored by providing the TLS(BMV) in trans. Intriguingly, a subdomain of the TLS(BMV) could even almost fully restore translation to the original pattern. We discuss here a model with a central and dominant role for the TLS(BMV) during the BMV infection cycle.

Brome mosaic virus Protein 1a recruits viral RNA2 to RNA replication through a 5' proximal RNA2 signal.

Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication factors. Membrane-associated 1a protein contains a helicase-like domain and RNA capping functions. 2a, which is targeted to membranes by 1a, contains a central polymerase-like domain. In the absence of 2a and RNA replication, 1a acts through an intergenic replication signal in BMV genomic RNA3 to stabilize RNA3 and induce RNA3 to associate with cellular membrane. Multiple results imply that 1a-induced RNA3 stabilization reflects interactions involved in recruiting RNA3 templates into replication. To determine if 1a had similar effects on another BMV RNA replication template, we constructed a plasmid expressing BMV genomic RNA2 in vivo. In vivo-expressed RNA2 templates were replicated upon expression of 1a and 2a. In the absence of 2a, 1a stabilized RNA2 and induced RNA2 to associate with membrane. Deletion analysis demonstrated that 1a-induced membrane association of RNA2 was mediated by sequences in the 5'-proximal third of RNA2. The RNA2 5' untranslated region was sufficient to confer 1a-induced membrane association on a nonviral RNA. However, sequences in the N-terminal region of the 2a open reading frame enhanced 1a responsiveness of RNA2 and a chimeric RNA. A 5'-terminal RNA2 stem-loop important for RNA2 replication was essential for 1a-induced membrane association of RNA2 and, like the 1a-responsive RNA3 intergenic region, contained a required box B motif corresponding to the TPsiC stem-loop of host tRNAs. The level of 1a-induced membrane association of various RNA2 mutants correlated well with their abilities to serve as replication templates. These results support and expand the conclusion that 1a-induced BMV RNA stabilization and membrane association reflect early, 1a-mediated steps in viral RNA replication.

Helicase and capping enzyme active site mutations in brome mosaic virus protein 1a cause defects in template recruitment, negative-strand RNA synthesis, and viral RNA capping.

Brome mosaic virus (BMV) encodes two RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and 2a, which is related to polymerases. BMV 1a and 2a can direct virus-specific RNA replication in the yeast Saccharomyces cerevisiae, which reproduces the known features of BMV replication in plant cells. We constructed single amino acid point mutations at the predicted capping and helicase active sites of 1a and analyzed their effects on BMV RNA3 replication in yeast. The helicase mutants showed no function in any assays used: they were strongly defective in template recruitment for RNA replication, as measured by 1a-induced stabilization of RNA3, and they synthesized no detectable negative-strand or subgenomic RNA. Capping domain mutants divided into two groups. The first exhibited increased template recruitment but nevertheless allowed only low levels of negative-strand and subgenomic mRNA synthesis. The second was strongly defective in template recruitment, made very low levels of negative strands, and made no detectable subgenomes. To distinguish between RNA synthesis and capping defects, we deleted chromosomal gene XRN1, encoding the major exonuclease that degrades uncapped mRNAs. XRN1 deletion suppressed the second but not the first group of capping mutants, allowing synthesis and accumulation of large amounts of uncapped subgenomic mRNAs, thus providing direct evidence for the importance of the viral RNA capping function. The helicase and capping enzyme mutants showed no complementation. Instead, at high levels of expression, a helicase mutant dominantly interfered with the function of the wild-type protein. These results are discussed in relation to the interconnected functions required for different steps of positive-strand RNA virus replication.

The Mode of Action of Hirsutellin A on Eukaryotic Cells

A 16-kDa protein toxin was purified fromHirsutella thompsoniivarthompsoniiand named hirsutellin A (HtA). At 0.5 and 5.0 μmconcentrations, HtA caused detectable cytopathic effects onSpodoptera frugiperdacells (Sf-9) within 2–4 hr and completely inhibited Sf-9 cell growth at 4 days posttreatment. Electron microscope data showed that the HtA treated Sf-9 cells became hypotrophied and internal organelles and cell membranes were disrupted. At the same concentration, HtA effectively inhibited Brome mosaic virus protein synthesis of both rabbit reticulocyte and wheat germin vitrotranslation systems. The ribosomal RNA extracted from HtA treated Sf-9 cells produced a smaller RNA (approximately 528 bases) than untreated Sf-9 cells. In summary, HtA is the first mycotoxin of a invertebrate mycopathogen determined to possess ribosomal inhibiting activity and appears to possess some specificity to invertebrate cells.

Targeting of the site of nonhomologous genetic recombination in brome mosaic virus.

The genome of brome mosaic virus (BMV) consists of three positive strand RNA segments that share a high degree of sequence homology in the 3' noncoding region. The phenomenon of both homologous and nonhomologous intersegment RNA-RNA recombinant has been demonstrated within the 3' noncoding region of BMV RNAs. It has been postulated that nonhomologous crossovers occur at local heteroduplexes formed between the recombining BMV RNA substrates of the same polarity and that the formation of double-stranded regions facilitates strand switching by the replicase. To test the hypothesis of hybridization-mediated recombination in BMV, RNA-3 constructs carrying short antisense RNA1-derived sequences have been used to induce nonhomologous recombination events between RNA-1 and RNA-3 at or near the site of hybridization. We find that both the incidence of recombination and the location of recombinant junctions depends on the structure and the stability of heteroduplexes. Furthermore, our preliminary results demonstrate that mutations in the helicase-like domain of BMV protein 1a affect the location of recombinant junctions. This provides experimental evidence that BMV replicase protein 1a participates in recombination.

Involvement of a nonstructural protein in the RNA synthesis of brome mosaic virus

RNA-dependent RNA polymerase (RdRp) was prepared from brome mosaic virus (BMV)-infected barley by a procedure including Nonidet-P40 treatment. The enzyme proved to be highly active, specific, and almost completely template dependent without the need for nuclease treatment [ W. A. Miller, and T. C. Hall (1983) Virology 125, 236–241] or DEAE ion exchange chromatography [ K. Maekawa and I. Furusawa (1984) Ann. Phytopathol. Soc. Japan 50, 491–499]. Two C-terminal peptides P1C and P2C derived from the nonstructural BMV proteins P1 and P2, respectively, were synthesized. Antibodies raised against these peptides were able to recognize the corresponding native proteins present in RdRp preparations. Antibodies directed against P1 C were capable of completely blocking the transcription of BMV RNA in vitro. This is the first experimental evidence that a nonstructural viral protein is present in an enzyme complex involved in tricornaviral RNA synthesis.

Self-assembly of brome mosaic virus protein into capsids: Initial and final states of aggregation

The pH and ionic strength dependence of the states of aggregation of brome mosaic virus protein has been investigated by small angle neutron scattering, quasielastic light-scattering, analytical centrifugation and electron microscopy. At pH above neutrality, protein oligomers are found in dynamical equilibrium, comprising monomers, dimers and aggregates of higher molecular weight. By lowering the pH. capsids assemble spontaneously with dimensions in solution which depend on ionic strength. If formed by dialysis, they contain 180 monomers, but are 30 Å larger in diameter than the native virus. If formed by pH-jump, they contain less monomers; the deficiency decreases with decreasing the final pH and the initial protein concentration. Upon dehydration for electron microscopy, capsids contract by 10%.

The specific involvement of coat protein in tobacco mosaic virus cross protection

Nicotiana sylvestris infected by strains of tobacco mosaic virus (TMV) causing mosaic can be superinfected in the dark green leaf tissue, but not light green tissue, by necrotizing strains of TMV. The dark green tissue, however, is much less susceptible than healthy tissue, to some extent, even to unrelated viruses. The RNA of necrotizing strains of TMV was relatively more infectious than intact virus on mosaic than on healthy leaves and caused lesions in both light and dark green tissues. The same relationship was found in Nicotiana longiflora and, when the protecting strain in N. sylvestris could be used as a challenge, in Capsicum baccatum. The efficiency of superinfection by RNA was not found with viruses unrelated to TMV. When bentonite at 1 mg/ml, which is known to strip protein from TMV, was included in the inoculum of intact TMV it superinfected in the same manner as RNA. RNA of a necrotizing strain of TMV, encapsidated in brome mosaic virus protein and used as a challenge, superinfected in the same manner as RNA. When encapsidated in common TMV protein, however, it behaved as native virus. Cross protection apparently results from the prevention of uncoating of related challenge virus in light green tissue of N. sylvestris. Locally inoculated N. sylvestris leaves were insusceptible to challenge RNA or intact virus when the protecting virus was increasing. After increase ceased, RNA was more infectious than intact virus.

Regulation of Brome Mosaic Virus Gene Expression by Restriction of Initiation of Protein Synthesis

The translation of total and individual brome mosaic virus (BMV) RNAs was examined in a wheat germ cell-free system in the presence of various inhibitors. Inhibitors of the initiation of polypeptide synthesis, e.g., potassium ions, 7-methylguanosine 5' -monophosphate, and aurintricarboxylic acid, were shown not only to inhibit overall BMV protein synthesis but also to change the ratio of BMV polypeptides synthesized. Under conditions restrictive for initiation, the translation of nonstructural BMV genes was suppressed, but coat protein synthesis proceeded at a high rate. A similar discrimination among BMV messengers was exerted by a regulatory protein kinase isolated from wheat germ. These results suggest that the regulation of the expression of BMV genes is based on a difference in the mechanism of formation of initiation complexes for individual BMV messages.

In Vitro encapsidation of the four RNA species of brome mosaic virus

The products of in vitro reconstitution of brome mosaic virus (BMV) protein and RNA were studied using analytical sedimentation and polyacrylamide gel electrophoresis. The product formed depended on the protein concentration, even though the ratio of RNA to protein was constant (1:4). At a low ionic strength with protein at 0.2 g/liter, mostly 78 S particles (like native BMV) were made, whereas at 2 g/liter of protein, reconstitution proceeded erratically. When the ionic strength was decreased to various levels by dialysis after mixing RNA and protein at I = 1.0, the four RNAs were encapsidated at different rates according to species, with RNAs 4 and 3 at the highest rates. The same sequential encapsidation in order of increasing molecular weight occurred as the ratio of protein to RNA was increased. Some particles containing three copies of the smallest RNA were found. These observations show that BMV protein displays in vitro a differential affinity for the various BMV RNA species.

Some tryptic peptides of bromovirus proteins.

Abstract The action of trypsin on brome mosaic virus (BMV) at pH 8 resulted in the dissociation of the virus particles into an 8 S component which contained two proteins, I and F. Protein I was capable of reassembly at pH 8 or 5 into 16-nm spherical particles; protein F formed these particles only at pH 5. SDS-polyacrylamide-gel electrophoresis and amino acid analysis indicated that protein F was about 23 and protein I about 18 amino acid residues smaller than native BMV protein. Six peptides, a cleavage product of the acetylated N-terminal peptide, free arginine, and eight other peptides containing more than one basic amino acid were isolated and their amino acid compositions and N-terminal residues were determined. The BMV peptides were similar or identical to peptides isolated after the limited proteolysis of cowpea chlorotic mottle virus protein at the N-terminus. The sequences of these portions of both viral proteins are probably very similar and probably function as sites of RNA-protein interaction. Peptides isolated from a total tryptic digestion of broad bean mottle virus protein were unlike peptides cleaved in limited proteolysis of BMV and cowpea chlorotic mottle virus.

Natural fluorescence properties of brome mosaic virus protein

Some of the fluorescence properties of brome mosaic virus protein in different states of aggregation (dimer, capsid) have been studied, in particular the emission spectra, fluorescence quantum yields and lifetimes, as well as the effect of external quenchers and of temperature on the fluorescence. Brome mosaic virus protein, which contains two tryptophan and five tyrosine residues per monomer, displayed an unusual fluorescence spectrum maximum of 308 nm at pH 7.4 when excited at 280 nm. The emission maximum was shifted to 327 nm when excited at 295 nm. Analysis of the results showed that the tyrosine emission is characterized by a high value for the quantum yield ( ϕ = 0.07), which is consistent with a location of most of these residues in helical regions of the protein, while the tryptophan emission is strongly quenched ( ϕ = 0.035). The effects of external quenchers suggested that two kinds of tryptophan residues might exist, one buried ( ϕ = 0.014). The tryptophan fluorescence quenching is partially removed at pH 8.4 and totally eliminated after chemical (guanidinium chloride) or thermal denaturation of the protein. Formation of capsid induced an additional quenching of the exposed tryptophan residue while interaction with the RNA in the virus did not modify the emission parameters of the protein.

Aggregation states of Brome mosaic virus protein

The self-assembly of Brome mosaic virus protein has been studied as a function of pH and ionic strength at 22°. At ionic strengths above 0.25, the dissociated protein (dimers of the subunit) polymerized quantitatively below pH 5 into empty capsids or PTC (pseudo top component) in a fully reversible manner. The threshold of reassociation was displaced to higher pH values with increasing ionic strength. Once formed, PTC particles could as well be dissociated into ribbons of protein at low pH, but at low ionic strength only; in the latter configuration, BMV protein was unable to form shells upon reverting to initial conditions. Filamentous structures also formed from dissociated protein when reassociation was performed at low ionic strength; these ribbons too could hardly, if ever, give rise to PTC particles upon shifting to conditions promoting shell formation. Here again, an increase in ionic strength at constant pH resulted in a higher degree of polymerization, suggesting a major role for hydrophobic forces in the formation of organized structures from BMV protein subunits.

Genomic masking in mixed infections with brome mosaic and barley stripe mosaic viruses

Barley plants infected with both barley stripe mosaic virus (BSMV) and brome mosaic virus (BMV) had more severe symptoms than plants infected with either virus alone, although the viruses were present in lower concentrations than in singly infected plants. To determine whether BMV protein encapsidated BSMV RNA in vivo, assay plants were inoculated with BMV virions purified from doubly infected plants. These assay plants later contained a small number of BSMV virions in addition to a high concentration of BMV virions. The BSMV virions presumably resulted from infection by BSMV RNA encapsidated by BMV protein.

Structures derived from cowpea chlorotic mottle and brome mosaic virus protein

The behavior at different salt and acidic pH levels of protein isolated from three spherical plant viruses was investigated. Broad bean mottle virus protein did not aggregate into recognizable organized structures. Protein from brome mosaic virus formed capsids similar to those of the virus at a pH and salt-dependent efficiency with best yields at pH 5.0 in 0.2 M NaCl. Best yields for cowpea chlorotic mottle virus protein capsids occurred at pH 5.0 in 0.1 M NaCl or from pH 3.5 to 5.0 in 0.2 M NaCl. Protein from the latter virus also formed double-shelled and rosettelike particles in 0.2 M NaCl from pH 5.3 to 5.7. Narrow tubes were made at pH levels of 6.0 and higher, and the tubes were accompanied by T = 1 and T = 3 particles if ribonu-clease-digested RNA was present. In the absence of NaCl in 0.01 M acetate buffer at pH 4.0 and 4.5, laminar and platelike aggregates were found with CCMV protein. The production and some of the properties of the various forms are described.