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Many experimental systems in biology, especially synthetic gene networks, are amenable to perturbations that are controlled by the experimenter. We developed an optimal design algorithm that calculates optimal observation times in conjunction with optimal experimental perturbations in order to maximize the amount of information gained from longitudinal data derived from such experiments. We applied the algorithm to a validated model of a synthetic Brome Mosaic Virus (BMV) gene network and found that optimizing experimental perturbations may substantially decrease uncertainty in estimating BMV model parameters. [ABSTRACT FROM AUTHOR]

• The viral genome-linked protein, VPg, of potyviruses is involved in viral genome replication and translation. To determine host proteins that interact with Sugarcane mosaic virus (SCMV) VPg, a yeast two-hybrid screen was used and a maize (Zea mays) Elongin C (ZmElc) protein was identified. • ZmELC transcript was observed in all maize organs, but most highly in leaves and pistil extracts, and ZmElc was present in the cytoplasm and nucleus of maize cells in the presence or absence of SCMV. ZmELC expression was increased in maize tissue at 4 and 6 d post SCMV inoculation. When ZmELC was transiently overexpressed in maize protoplasts the accumulation of SCMV RNA was approximately doubled compared with the amount of virus in control protoplasts. • Silencing ZmELC expression using a Brome mosaic virus-based gene silencing vector (virusinduced gene silencing) did not influence maize plant growth and development, but did decrease RNA accumulation of two isolates of SCMV and host transcript encoding ZmeIF4E during SCMV infection. Interestingly, Maize chlorotic mottle virus, from outside the Potyviridae, was increased in accumulation after silencing ZmELC expression. • Our results describe both the location of ZmElc expression in maize and a new activity associated with an Elc: support of potyvirus accumulation. [ABSTRACT FROM AUTHOR]

In Brome mosaic virus , it was hypothesized that a physical interaction between viral replicase and capsid protein (CP) is obligatory to confer genome packaging specificity. Here we tested this hypothesis by employing Bimolecular Fluorescent Complementation (BiFC) as a tool for evaluating protein–protein interactions in living cells. The efficacy of BiFC was validated by a known interaction between replicase protein 1a (p1a) and protein 2a (p2a) at the endoplasmic reticulum (ER) site of viral replication. Additionally, co-expression in planta of a bona fide pair of interacting protein partners of p1a and p2a had resulted in the assembly of a functional replicase. Subsequent BiFC assays in conjunction with mCherry labeled ER as a fluorescent cellular marker revealed that CP physically interacts with p2a, but not p1a, and this CP:p2a interaction occurs at the cytoplasmic phase of the ER. The significance of the CP:p2a interaction in BMV replication and genome packaging is discussed. [ABSTRACT FROM AUTHOR]

Abstract: Brome mosaic virus (BMV) packages its genomic and subgenomic RNAs into three separate viral particles. BMV purified from barley, wheat, and tobacco have distinct relative abundances of the encapsidated RNAs. We seek to identify the basis for the host-dependent differences in viral RNA encapsidation. Sequencing of the viral RNAs revealed recombination events in the 3′ untranslated region of RNA1 of BMV purified from barley and wheat, but not from tobacco. However, the relative amounts of the BMV RNAs that accumulated in barley and wheat are similar and RNA accumulation is not sufficient to account for the difference in RNA encapsidation. Virions purified from barley and wheat were found to differ in their isoelectric points, resistance to proteolysis, and contacts between the capsid residues and the RNA. Mass spectrometric analyses revealed that virions from the three hosts had different post-translational modifications that should impact the physiochemical properties of the virions. Another major source of variation in RNA encapsidation was due to the purification of BMV particles to homogeneity. Highly enriched BMV present in lysates had a surprising range of sizes, buoyant densities, and distinct relative amounts of encapsidated RNAs. These results show that the encapsidated BMV RNAs reflect a combination of host effects on the physiochemical properties of the viral capsids and the enrichment of a subset of virions. The previously unexpected heterogeneity in BMV should influence the timing of the infection and also the host innate immune responses. [Copyright &y& Elsevier]

Positive-strand RNA viruses are known to rearrange the endomembrane network to make it more conducive for replication, maturation, or egress. Our previous transmission electron microscopic (TEM) analysis showed that ectopic expression of wildtype (wt) capsid protein (CP) of Brome mosaic virus (BMV) has an intrinsic property of modifying the endoplasmic reticulum (ER) to induce vesicles similar to those present in wt BMV infection. In this study, we evaluated the functional significance of CP-mediated vesicle induction to the BMV infection cycle in planta. Consequently, the cytopathologic changes induced by wt CP or its mutants defective in virion assembly due to mutations engineered in either N- or C-proximal domains were comparatively analyzed by TEM in two susceptible (Nicotiana benthamiana and Chenopodium quinoa) and one nonhost (N. clevelandii) plant species. The results showed that in susceptible hosts, CP-mediated ER-derived vesicle induction is contingent on the expression of encapsidation-competent CP. In contrast, unlike in N. benthamiana and C. quinoa, transient expression of wt CP in nonhost N. clevelandii plants eliminated vesicle induction. Additionally, comparative source-to-sink analysis of virus spread in leaves of N. benthamiana and N. clevelandii coexpressing wt BMV and Cucumber mosaic virus (CMV) showed that despite trans-encapsidation, CMV failed to complement the defective cell-to-cell movement of BMV. The significance and relation of CP-mediated vesicle induction to virus cell-to-cell movement are discussed. [ABSTRACT FROM AUTHOR]

Positive-strand RNA viruses depend on recruited host factors to control critical replication steps. Previously, it was shown that replication of evolutionarily diverse positive-strand RNA viruses, such as hepatitis C virus and brome mosaic virus, depends on host decapping activators LSm1-7, Pat1, and Dhh1 (J. Diez et al., Proc. Natl. Acad. Sci. U. S. A. 97:3913-3918, 2000; A. Mas et al., J. Virol. 80:246 -251, 2006; N. Scheller et al., Proc. Natl. Acad. Sci. U. S. A. 106:13517-13522,2009). By using a system that allows the replication of the insect Flock House virus (FHV) in yeast, here we show that LSm1-7, Pat1, and Dhh1 control the ratio of subgenomic RNA3 to genomic RNA1 production, a key feature in the FHV life cycle mediated by a long-distance base pairing within RNA1. Depletion of LSM1, PAT1, or DHH1 dramatically increased RNA3 accumulation during replication. This was not caused by differences between RNA1 and RNA3 steady-state levels in the absence of replication. Importantly, coimmunoprecipi-tation assays indicated that LSm1-7, Pat1, and Dhh1 interact with the FHV RNA genome and the viral polymerase. By using a strategy that allows dissecting different stages of the replication process, we found that LSml-7, Patl, and Dhhl did not affect the early replication steps of RNA1 recruitment to the replication complex or RNA1 synthesis. Furthermore, their function on RNA3/RNA1 ratios was independent of the membrane compartment, where replication occurs and requires ATPase activity of the Dhh1 helicase. Together, these results support that LSm1-7, Pat1, and Dhh1 control RNA3 synthesis. Their described func-tion in mediating cellular mRNP rearrangements suggests a parallel role in mediating key viral RNP transitions, such as the one required to maintain the balance between the alternative FHV RNA1 conformations that control RNA3 synthesis. [ABSTRACT FROM AUTHOR]

Abstract: The 130-kDa and 180-kDa replication proteins of Tomato mosaic virus (ToMV) covalently bind guanylate and transfer it to the 5′ end of RNA to form a cap. We found that guanylylation-competent ToMV replication proteins are in membrane-bound, disulfide-linked complexes. Guanylylation-competent replication proteins of Brome mosaic virus and Cucumber mosaic virus behaved similarly. To investigate the roles of disulfide bonding in the functioning of ToMV replication proteins, each of the 19 cysteine residues in the 130-kDa protein was replaced by a serine residue. Interestingly, three mutant proteins (C179S, C186S and C581S) failed not only to be guanylylated, but also to bind to the replication template and membranes. These mutants could trans-complement viral RNA replication. Considering that ToMV replication proteins recognize the replication templates, bind membranes, and are guanylylated in the cytoplasm that provides a reducing condition, we discuss the roles of cysteine residues and disulfide bonds in ToMV RNA replication. [Copyright &y& Elsevier]

Abstract: We have previously described the efficient homologous recombination system between 5′ subgenomic RNA3a (sgRNA3a) and genomic RNA3 of Brome mosaic virus (BMV) in barley protoplasts (Sztuba-Solińska et al., 2011a). Here, we demonstrated that sequence alterations in the coat protein (CP)-binding cis-acting RNA motifs, the Bbox region (in the intercistronic RNA3 sequence) and the RNA3 packaging element (PE, in the movement protein ORF), reduced crossover frequencies in protoplasts. Additionally, the modification of Bbox-like element in the 5′ UTR region strongly debilitated crossovers. Along the lines of these observations, RNA3 mutants not expressing CP or expressing mutated CPs also reduced recombination. A series of reciprocal transfections demonstrated a functional crosstalk between the Bbox and PE elements. Altogether, our data imply the role of CP in sgRNA3a-directed recombination by either facilitating the interaction of the RNA substrates and/or by creating roadblocks for the viral replicase. [Copyright &y& Elsevier]

While most T=3 single-stranded RNA (ssRNA) viruses package in vivo about 3,000 nucleotides (nt), in vitro experiments have demonstrated that a broad range of RNA lengths can be packaged. Under the right solution conditions, for example, cowpea chlorotic mottle virus (CCMV) capsid protein (CP) has been shown to package RNA molecules whose lengths range from 100 to 10,000 nt. Furthermore, in each case it can package the RNA completely, as long as the mass ratio of CP to nucleic acid in the as-sembly mixture is 6:1 or higher. Yet the packaging efficiencies of the RNAs can differ widely, as we demonstrate by measure-ments in which two RNAs compete head-to-head for a limited amount of CP. We show that the relative efficiency depends non-monotonically on the RNA length, with 3,200 nt being optimum for packaging by the T=3 capsids preferred by CCMV CP. When two RNAs of the same length--and hence the same charge--compete for CP, differences in packaging efficiency are neces-sarily due to differences in their secondary structures and/or three-dimensional (3D) sizes. For example, the heterologous RNA1 of brome mosaic virus (BMV) is packaged three times more efficiently by CCMV CP than is RNA1 of CCMV, even though the two RNAs have virtually identical lengths. Finally, we show that in an assembly mixture at neutral pH, CP binds reversibly to the RNA and there is a reversible equilibrium between all the various RNA/CP complexes. At acidic pH, excess protein unbinds from RNA/CP complexes and nucleocapsids form irreversibly. [ABSTRACT FROM AUTHOR]

Abstract: Viral particles are biological machines that have evolved to package, protect, and deliver the viral genome into the host via regulated conformational changes of virions. We have developed a procedure to modify lysine residues with S-methylthioacetimidate across the pH range from 5.5 to 8.5. Lysine residues that are not completely modified are involved in tertiary or quaternary structural interactions, and their extent of modification can be quantified as a function of pH. This procedure was applied to the pH-dependent structural transitions of brome mosaic virus (BMV). As the reaction pH increases from 5.5 to 8.5, the average number of modified lysine residues in the BMV capsid protein increases from 6 to 12, correlating well with the known pH‐dependent swelling behavior of BMV virions. The extent of reaction of each of the capsid protein''s lysine residues has been quantified at eight pH values using coupled liquid chromatography–tandem mass spectrometry. Each lysine can be assigned to one of three structural classes identified by inspection of the BMV virion crystal structure. Several lysine residues display reactivity that indicates their involvement in dynamic interactions that are not obvious in the crystal structure. The influence of several capsid protein mutants on the pH-dependent structural transition of BMV has also been investigated. Mutant H75Q exhibits an altered swelling transition accompanying solution pH increases. The H75Q capsids show increased reactivity at lysine residues 64 and 130, residues distal from the dimer interface occupied by H75, across the entire pH range. [Copyright &y& Elsevier]

Abstract: The alphavirus-like mRNA capping enzyme of Bamboo mosaic virus (BaMV) exhibits an AdoMet-dependent guanylyltransferase activity by which the methyl group of AdoMet is transferred to GTP, leading to the formation of m7GTP, and the m7GMP moiety is next transferred to the 5′ end of ppRNA via a covalent enzyme–m7GMP intermediate. The function of the conserved H68 of the BaMV capping enzyme in the intermediate formation was analyzed by mutagenesis in this study. The nature of the bond linking the enzyme and m7GMP was changed in the H68C mutant protein, strongly suggesting that H68 covalently binds to m7GMP in the intermediate. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Positive-strand RNA virus replication involves viral proteins and cellular proteins at nearly every replication step. Brome mosaic virus (BMV) is a well-established model for dissecting virus-host interactions and is one of very few viruses whose RNA replication, gene expression and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. Previously, our laboratory identified ,100 non-essential host genes whose loss inhibited or enhanced BMV replication at least 3-fold. However, our isolation of additional BMV-modulating host genes by classical genetics and other results underscore that genes essential for cell growth also contribute to BMV RNA replication at a frequency that may be greater than that of non-essential genes. To systematically identify novel, essential host genes affecting BMV RNA replication, we tested a collection of ,900 yeast strains, each with a single essential gene promoter replaced by a doxycycline-repressible promoter, allowing repression of gene expression by adding doxycycline to the growth medium. Using this strain array of ,81% of essential yeast genes, we identified 24 essential host genes whose depleted expression reproducibly inhibited or enhanced BMV RNA replication. Relevant host genes are involved in ribosome biosynthesis, cell cycle regulation and protein homeostasis, among other cellular processes. BMV 2aPol levels were significantly increased in strains depleted for a heat shock protein (HSF1) or proteasome components (PRE1 and RPT6), suggesting these genes may affect BMV RNA replication by directly or indirectly modulating 2aPol localization, post-translational modification or interacting partners. Investigating the diverse functions of these newly identified essential host genes should advance our understanding of BMV-host interactions and normal cellular pathways, and suggest new modes of virus control. [ABSTRACT FROM AUTHOR]

Infection of maize (Zea mays) plants with the corn smut fungus Ustilago maydis leads to the formation of large tumors on the stem, leaves and inflorescences. In this biotrophic interaction, plant defense responses are actively suppressed by the pathogen, and previous transcriptome analyses of infected maize plants showed massive and stage-specific changes in host gene expression during disease progression. To identify maize genes that are functionally involved in the interaction with U. maydis, we adapted a virus-induced gene silencing (VIGS) system based on the brome mosaic virus (BMV) for maize. Conditions were established that allowed successful U. maydis infection of BMV-preinfected maize plants. This set-up enabled quantification of VIGS and its impact on U. maydis infection using a quantitative real-time PCR (qRT-PCR)-based readout. In proof-of-principle experiments, an U. maydis-induced terpene synthase was shown to negatively regulate disease development while a protein involved in cell death inhibition was required for full virulence of U. maydis. The results suggest that this system is a versatile tool for the rapid identification of maize genes that determine compatibility with U. maydis. [ABSTRACT FROM AUTHOR]

Abstract: UV spectra of viruses are complicated by overlapping protein and RNA absorbance and light scattering. We describe and validate methodology for estimating RNA and protein concentration from such spectra. Importantly, we found that encapsidation did not substantially affect RNA absorbance. Combining absorbance data with a known T number, we confirmed that brome mosaic virus packages about 3100 nucleotides/capsid, consistent with its genome. E. coli-expressed hepatitis B virus (HBV) packages host RNA based on capsid charge and volume. We examined HBV capsid protein (Cp183, +15 charge) and a less basic mutant (Cp183-EEE, +12 charge) that mimics a phosphorylated state. Cp183-EEE packaged ~3450 nucleotides per T=4 capsid and Cp183 packaged ~4800 nucleotides, correlating to the size of HBV''s RNA pre-genome and mature DNA genome, respectively. The RNA:protein charge ratio (about 1.4 phosphates per positive charge) was consistent with that of other ssRNA viruses. This spectroscopic method is generalizable to any virus-like particle. [Copyright &y& Elsevier]

Abstract: Viral capsid proteins (CPs) can regulate gene expression and encapsulate viral RNAs. Low-level expression of the brome mosaic virus (BMV) CP was found to stimulate viral RNA accumulation, while higher levels inhibited translation and BMV RNA replication. Regulation of translation acts through an RNA element named the B box, which is also critical for the replicase assembly. The BMV CP has also been shown to preferentially bind to an RNA element named SLC that contains the core promoter for genomic minus-strand RNA synthesis. To further elucidate CP interaction with RNA, we used a reversible cross-linking–peptide fingerprinting assay to identify peptides in the capsid that contact the SLC, the B-box RNA, and the encapsidated RNA. Transient expression of three mutations made in residues within or close by the cross-linked peptides partially released the normal inhibition of viral RNA accumulation in agroinfiltrated Nicotiana benthamiana. Interestingly, two of the mutants, R142A and D148A, were found to retain the ability to down-regulate reporter RNA translation. These two mutants formed viral particles in inoculated leaves, but only R142A was able to move systemically in the inoculated plant. The R142A CP was found to have higher affinities for SLC and the B box compared with those of wild-type CP and to alter contacts to the RNA in the virion. These results better define how the BMV CP can interact with RNA and regulate different viral processes. [Copyright &y& Elsevier]

Abstract: Brome mosaic virus (BMV) and Spring beauty latent virus (SBLV) are closely related, tripartite RNA plant viruses. In Arabidopsis thaliana, BMV shows limited multiplication whereas SBLV efficiently multiplies. Such distinct multiplication abilities have been observed commonly in all Arabidopsis accessions tested. We used this model system to analyze the molecular mechanism of viral resistance in plants at the species level. Unlike SBLV, BMV multiplication was limited even in protoplasts and a reassortment assay indicated that at least viral RNA1 and/or RNA2 determine such distinct infectivities. By screening Arabidopsis mutants with altered defense responses, we found that BMV multiplies efficiently in cpr5-2 mutant plants. This mutation specifically enhanced BMV multiplication in protoplasts, which depended on the functions of RNA1 and RNA2. In the experiment using DNA vectors to express BMV replication proteins encoded by RNA1 and RNA2, BMV RNA3 accumulation in cpr5-2 protoplasts was similar to that in wild-type Col-0 protoplasts, despite significant reduction of accumulation levels of replication proteins, suggesting that cpr5-2 mutation could enhance BMV multiplication independently of increased accumulation, therefore enhanced translation and stabilization, of the replication proteins. [Copyright &y& Elsevier]

The yeast Saccharomyces cerevisiae is invaluable for understanding fundamental cellular processes and disease states of relevance to higher eukaryotes. Plant viruses are intracellular parasites that take advantage of resources of the host cell, and a simple eukaryotic cell, such as yeast, can provide all or most of the functions for successful plant virus replication. Thus, yeast has been used as a model to unravel the interactions of plant viruses with their hosts. Indeed, genome-wide and proteomics studies using yeast as a model host with bromoviruses and tombusviruses have facilitated the identification of replication-associated factors that affect host-virus interactions, virus pathology, virus evolution, and host range. Many of the host genes that affect the replication of the two viruses, which belong to two dissimilar virus families, are distinct, suggesting that plant viruses have developed different ways to utilize the resources of host cells. In addition, a surprisingly large number of yeast genes have been shown to affect RNA-RNA recombination in tombusviruses; this opens an opportunity to study the role of the host in virus evolution. The knowledge gained about host-virus interactions likely will lead to the development of new antiviral methods and applications in biotechnology and nanotechnology, as well as new insights into cellular functions of individual genes and the basic biology of the host cell. [ABSTRACT FROM AUTHOR]

Abstract: The interaction between brome mosaic virus (BMV) coat protein (CP) and viral RNA is a carefully orchestrated process resulting in the formation of homogeneous population of infectious virions with T =3 symmetry. Expression in vivo of either wild type or mutant BMV CP through homologous replication never results in the assembly of aberrant particles. In this study, we report that deletion of amino acid residues 41–47 from the N-proximal region of BMV CP resulted in the assembly of polymorphic virions in vivo. Purified virions from symptomatic leaves remain non-infectious and Northern blot analysis of virion RNA displayed packaging defects. Biochemical characterization of variant CP by circular dichroism and MALDI-TOF, respectively, revealed that the engineered deletion affected the protein structure and capsid dynamics. Most significantly, CP subunits dissociated from polymorphic virions are incompetent for in vitro reassembly. Based on these observations, we propose a chaperon-mediated mechanism for the assembly of variant CP in vivo and also hypothesize that 41KAIKAIA47 N-proximal peptide functions as a molecular switch in regulating T =3 virion symmetry. [Copyright &y& Elsevier]

Arrays of >5,000 Saccharomyces cerevisiae proteins were screened to identify proteins that can preferentially bind a small RNA hairpin that contains a clamped adenine motif (CAM). A CAM is required for the replication of Brome Mosaic Virus (BMV), a plant-infecting RNA virus that can replicate in S. cerevisiae. Several hits were selected for further characterization in Nicotiana benthamiana. Pseudouridine Synthase 4 (Pus4) and the Actin Patch Protein 1 (App1) modestly reduced BMV genomic plus-strand RNA accumulation, but dramatically inhibited BMV systemic spread in plants. Pus4 also prevented the encapsidation of a BMV RNA in plants and the reassembly of BMV virions in vitro. These results demonstrate the feasibility of using proteome arrays to identify specific RNA-binding proteins for antiviral activities. Furthermore, the effects of Pus4 suggest that the CAM-containing RNA motif provides a regulatory link between RNA replication and encapsidation. [ABSTRACT FROM AUTHOR]

Ecotypes of Arabidopsis thaliana supported the replication and systemic spread of Brome mosaic virus (BMV) RNAs. Infection was induced either by manual inoculation with viral RNA or by BMV virions, demonstrating that virus disassembly did not prevent infection. When in vitro-transcribed BMV RNAs 1–3 were used, production of subgenomic RNA4 was observed, showing that BMV RNA replication and transcription had occurred. Furthermore, inoculations of the transgenic Arabidopsis line that expressed a suppressor of RNA interference (RNAi) pathway markedly increased the BMV RNA concentrations. Inoculations with designed BMV RNA3 recombination vectors generated both homologous and nonhomologous BMV RNA–RNA recombinants. Thus, all cellular factors essential for BMV RNA replication, transcription, and RNA recombination were shown to be present in Arabidopsis. The current scope of understanding of the model Arabidopsis plant system should facilitate the identification of these factors governing the BMV life cycle. [Copyright &y& Elsevier]

Brome mosaic virus (BMV), a tripartite RNA plant virus, accumulates RNA3-derived defective RNAs (D-RNAs) in which 477–500 nucleotides (nt) are deleted in the central region of the 3a protein open reading frame (ORF), after prolonged infection in barley. In the present study, six artificial D-RNAs (AD-RNAs), having deletions of the same size as the naturally occurring D-RNA but at different positions in the 3a ORF, were constructed and tested for their amplification and encapsidation in barley protoplasts by coinoculation with BMV RNA1 and 2, or RNA1, 2, and 3. Northern blot analysis of RNA accumulation in total and virion fractions showed that deletions of 492 nt in the 3′-proximal and the 5′-proximal regions of the 3a ORF decreased encapsidation efficiency of the AD-RNAs compared with that of RNA3, whereas deletions in the central region enhanced encapsidation efficiency. The present results also show that deletion positions affect competition with RNA3 in the amplification and encapsidation of AD-RNAs. [Copyright &y& Elsevier]

Summary We have constructed a new system for inducible high-level expression of mRNA for foreign genes in transgenic plants by introducing a glucocorticoid-inducible transcription system into the previously developed ‘mRNA amplification system’ where target mRNA can be amplified as a subgenomic RNA by the replicase of a plant tripartite RNA virus, Brome mosaic virus (BMV). In the new amplification system, the amplification of mRNA is tightly regulated by the expression of a subunit of the BMV replicase. Transgenic Nicotiana benthamiana plants (designated GVG1 × 2FR) were produced that contained cDNA of BMV RNA1 coding a subunit of replicase under the control of a tightly regulated, glucocorticoid-inducible promoter. In addition GVG1 × 2FR plants contain cDNAs of BMV RNA2 coding another subunit of the replicase, and a replicable engineered BMV RNA3 derivative (FCP2IFN) carrying the human gamma interferon (IFN) gene under the control of the Cauliflower mosaic virus 35S promoter. When transgenic plants were treated with dexamethasone (DEX), a strong synthetic glucocorticoid, induction of replication and amplification of the 35S-driven FCP2IFN and synthesis of subgenomic mRNA for IFN were observed. Accumulation levels of amplified FCP2IFN were over 300 times higher than those of the 35S-driven FCP2IFN in the GVG1 × 2FR plant without the treatment and those of the mRNA for IFN were 30–230 times higher than in the previous, non-inducible mRNA amplification system. Without DEX treatment, no subgenomic mRNA for IFN was detected in the GVG1 × 2FR plant. The advantages and potential uses of this system are also discussed. [ABSTRACT FROM AUTHOR]

Amide hydrogen exchange and mass spectrometry have been used to study the pH-induced structural changes in the capsid of brome mosaic virus (BMV). Capsid protein was labeled in a structurally sensitive way by incubating intact viral particles in D2O at pH 5.4 and 7.3. Deuterium levels in the intact coat protein and its proteolytic fragments were determined by mass spectrometry. The largest deuterium increases induced by structural alteration occurred in the regions around the quasi-threefold axes, which are located at the center of the asymmetric unit. The increased levels of deuterium indicate loosening of structure in these regions. This observation confirms the previously proposed swelling model for BMV and cowpea chlorotic mottle virus (CCMV) and is consistent with the structure of swollen CCMV recently determined by cryo-electron microscopy and image reconstruction. Structural changes in the extended N- and C-terminal arms were also detected and compared with the results obtained with other swollen plant viruses. This study demonstrates that protein fragmentation/amide hydrogen exchange is a useful tool for probing structural changes in viral capsids. [ABSTRACT FROM AUTHOR]

Previous studies on the occurrence of "barley yellow dwarf virus (BYDV) disease" in South Africa have led to the conclusion that, although this virus is present, the main causative agent of "yellow dwarf" disease in cereals appears to be the unrelated brome mosaic virus (BMV). In this study, material from South Africa, Britain and Australia that had been identified symptomatically as being infected with BYDV, was found by serological testing to contain BMV. No BYDV could be detected in the same samples. This report discusses the hazards of relying on symptom expression for the diagnosis of a common world-wide disease problem. [ABSTRACT FROM AUTHOR]

The extent of contamination of wheat seedlots by brome mosaic virus (BMV), and the possible transmission of the virus through seed, were investigated by radial immunodiffusion (RID), enzyme linked immunosorbent assay (ELISA), and enzyme-assisted immunoelectroblotting (IEB) tests. BMV was purified from seed washings, and from plants grown from contaminated seed. All three serological tests proved of value in detecting contaminated seeds, while ELISA and IEB were especially useful in detecting virus in infected plants grown from such seed. Rhopalosiphum padi aphids were shown to increase the incidence of BMV infection in seedling batches containing a few seed-infected plants. The implications of these findings for wheat breeding schemes are discussed. [ABSTRACT FROM AUTHOR]

The toxicity of the a, β unsaturated carbonyl compounds (a, β UCCs) (patulin, penicillic acid, parasorbic acid, tulipalin and plumbagin) towards Pythium sp. group F (Van Der PLAAT-NlTERINKS 1981) was neutralized by the addition of an excess of cysteine. This suggests that the mode of action of these compounds could be due to a binding of the a, β UCCs to sulphydryl groups in enzymes or other macromolecules. Alcohol dehydrogenase (ADH), an enzyme with a sulphydryl group at the active site, was assayed spectrophotometrically and all the a, β UCCs inhibited ADH. [ABSTRACT FROM AUTHOR]

Back transmission trials on young forest plants with isolates of purified viruses from the same tree species were performed using different inoculation techniques. Spruce seedlings and willow plants were successfully infected with tobacco necrosis virus (TNV) by the conventional method of mechanical inoculation of virus suspension mixed with celite as abrasive. Cherry leaf roll virus (CLRV) was transmitted to birches only after adding poIy-L-ornithine (PLO) to the inoculum. The same method was successful with brome mosaic virus (BMV) on beech seedlings. PLO also improved the rate of infection on TNV in willows. In only one case, was CLRV transmitted conventionally to a white ash seedling. The infection of white ash was increased when frozen powders of infected ash leaves were directly rubbed onto leaves. BMV could not be transmitted to beech seedlings by carborundum pressure-inoculation. Stem slashing-inoculation of BMV and CLRV was successful with CLRV in one beech out of 60 seedlings. [ABSTRACT FROM AUTHOR]