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Pathogens (Basel, Switzerland) Jul 2022Broad bean mottle bromovirus infects legume plants and is transmissible by insects. Several broad bean mottle virus (BBMV) isolates have been identified, including one...
Broad bean mottle bromovirus infects legume plants and is transmissible by insects. Several broad bean mottle virus (BBMV) isolates have been identified, including one in England (isolate Ba) and five in the Mediterranean countries: Libya (LyV), Morocco (MV), Syria (SV), Sudan (TU) and Tunisia (TV). Previously, we analyzed the nucleotide sequence of the Ba RNA and here we report on and compare it with another five Mediterranean variants. The RNA segments in the latter ones were extensively homologous, with some SNPs, single nucleotide deletions and insertions, while the number of mutations was higher in isolate Ba. Both the 5' and 3' untranslated terminal regions (UTRs) among the corresponding RNAs are highly conserved, reflecting their functionality in virus replication. The AUG initiation codons are within suboptimal contexts, possibly to adjust/regulate translation. The proteins 1a, 2a, 3a and coat protein (CP) are almost identical among the five isolates, but in Ba they have more amino acid (aa) substitutions. Phylogenetic analysis revealed the isolates from Morocco and Syria clustering with the isolate from England, while the variants from Libya, Tunisia and Sudan created a different clade. The BBMV isolates encapsidate a high content of host (ribosomal and messenger) RNAs. Our studies present BBMV as a useful model for bromoviruses infecting legumes.
PubMed: 35890061
DOI: 10.3390/pathogens11070817 -
Virology Feb 2011RNA-RNA recombination salvages viral RNAs and contributes to their genomic variability. A recombinationally-active subgenomic promoter (sgp) has been mapped in Brome...
RNA-RNA recombination salvages viral RNAs and contributes to their genomic variability. A recombinationally-active subgenomic promoter (sgp) has been mapped in Brome mosaic bromovirus (BMV) RNA3 (Wierzchoslawski et al., 2004. J. Virol.78, 8552-8864) and mRNA-like 5' sgRNA3a was characterized (Wierzchoslawski et al., 2006. J. Virol. 80, 12357-12366). In this paper we describe sgRNA3a-mediated recombination in both in vitro and in vivo experiments. BMV replicase-directed co-copying of (-) RNA3 with wt sgRNA3a generated RNA3 recombinants in vitro, but it failed to when 3'-truncated sgRNA3a was substituted, demonstrating a role for the 3' polyA tail. Barley protoplast co-transfections revealed that (i) wt sgRNA3a recombines at the 3' and the internal sites; (ii) 3'-truncated sgRNA3as recombine more upstream; and (iii) 5'-truncated sgRNA3 recombine at a low rate. In planta co-inoculations confirmed the RNA3-sgRNA3a crossovers. In summary, the non-replicating sgRNA3a recombines with replicating RNA3, most likely via primer extension and/or internal template switching.
Topics: Bromovirus; Chenopodium quinoa; Gene Expression Regulation, Viral; Genome, Viral; Hordeum; Plant Diseases; RNA, Viral; Reassortant Viruses
PubMed: 21111438
DOI: 10.1016/j.virol.2010.10.037 -
Plants (Basel, Switzerland) Sep 2017The effects of 2 mM silicon (Si) and 10 mM KNO₃ (N)-prime signals for plant resistance to pathogens-were analyzed in healthy and (CCMV) or (CMMV)-infected -nodulated...
Silicon and Nitrate Differentially Modulate the Symbiotic Performances of Healthy and Virus-Infected Bradyrhizobium-nodulated Cowpea (Vigna unguiculata), Yardlong Bean (V. unguiculata subsp. sesquipedalis) and Mung Bean (V. radiata).
The effects of 2 mM silicon (Si) and 10 mM KNO₃ (N)-prime signals for plant resistance to pathogens-were analyzed in healthy and (CCMV) or (CMMV)-infected -nodulated cowpea, yardlong bean and mung bean plants. In healthy plants of the three taxa, nodulation and growth were promoted in the order of Si + N > N > Si > controls. In the case of healthy cowpea and yardlong bean, the addition of Si and N decreased ureide and α-amino acids (AA) contents in the nodules and leaves in the order of Si + N> N > Si > controls. On the other hand, the addition of N arrested the deleterious effects of CCMV or CMMV infections on growth and nodulation in the three taxa. However, the addition of Si or Si + N hindered growth and nodulation in the CCMV- or CMMV-infected cowpea and yardlong bean, causing a massive accumulation of ureides in the leaves and nodules. Nevertheless, the AA content in leaves and nodules of CCMV- or CMMV-infected cowpea and yardlong bean was promoted by Si but reduced to minimum by Si + N. These results contrasted to the counteracting effects of Si or Si + N in the CCMV- and CMMV-infected mung bean via enhanced growth, nodulation and levels of ureide and AA in the leaves and nodules. Together, these observations suggest the fertilization with Si + N exclusively in virus-free cowpea and yardlong bean crops. However, Si + N fertilization must be encouraged in virus-endangered mung bean crops to enhance growth, nodulation and N-metabolism. It is noteworthy to see the enhanced nodulation of the three taxa in the presence of 10 mM KNO₃.
PubMed: 28914770
DOI: 10.3390/plants6030040 -
Virus Research Sep 2006Understanding the fundamental steps of virus life cycles including virus-host interactions is essential for the design of effective antiviral strategies. Such... (Review)
Review
Understanding the fundamental steps of virus life cycles including virus-host interactions is essential for the design of effective antiviral strategies. Such understanding has been deferred by the complexity of higher eukaryotic host organisms. To circumvent experimental difficulties associated with this, systems were developed to replicate viruses in the yeast Saccharomyces cerevisiae. The systems include viruses with RNA and DNA genomes that infect plants, animals and humans. By using the powerful methodologies available for yeast genetic analysis, fundamental processes occurring during virus replication have been brought to light. Here, we review the different viruses able to direct replication and gene expression in yeast and discuss their main contributions in the understanding of virus biology.
Topics: Animals; Bovine papillomavirus 1; Bromovirus; Dianthus; Fabaceae; Geminiviridae; Genome, Viral; Humans; Solanum lycopersicum; Nodaviridae; Papillomaviridae; Proteins; Saccharomyces cerevisiae; Tombusviridae; Viral Proteins; Virus Replication
PubMed: 16698107
DOI: 10.1016/j.virusres.2005.11.018 -
PLoS Pathogens Sep 2022Positive-strand RNA viruses assemble their viral replication complexes (VRCs) on specific host organelle membranes, yet it is unclear how viral replication proteins...
Positive-strand RNA viruses assemble their viral replication complexes (VRCs) on specific host organelle membranes, yet it is unclear how viral replication proteins recognize and what motifs or domains in viral replication proteins determine their destinations. We show here that an amphipathic helix, helix B in replication protein 1a of brome mosaic virus (BMV), is necessary for 1a's localization to the nuclear endoplasmic reticulum (ER) membrane where BMV assembles its VRCs. Helix B is also sufficient to target soluble proteins to the nuclear ER membrane in yeast and plant cells. We further show that an equivalent helix in several plant- and human-infecting viruses of the Alsuviricetes class targets fluorescent proteins to the organelle membranes where they form their VRCs, including ER, vacuole, and Golgi membranes. Our work reveals a conserved helix that governs the localization of VRCs among a group of viruses and points to a possible target for developing broad-spectrum antiviral strategies.
Topics: Bromovirus; Endoplasmic Reticulum; Humans; RNA, Viral; Saccharomyces cerevisiae; Viral Proteins; Virus Replication
PubMed: 36048900
DOI: 10.1371/journal.ppat.1010752 -
The Journal of Physical Chemistry. B Nov 2019A virus in its most simple form is comprised of a protein capsid that surrounds and protects the viral genome. The self-assembly of such structures, however, is a highly...
A virus in its most simple form is comprised of a protein capsid that surrounds and protects the viral genome. The self-assembly of such structures, however, is a highly complex, multiprotein, multiinteraction process and has been a topic of study for a number of years. This self-assembly process is driven by the (mainly electrostatic) interaction between the capsid proteins (CPs) and the genome as well as by the protein-protein interactions, which primarily rely on hydrophobic interactions. Insight in the thermodynamics that is involved in virus and virus-like particle (VLP) formation is crucial in the detailed understanding of this complex assembly process. Therefore, we studied the assembly of CPs of the cowpea chlorotic mottle virus (CCMV) templated by polyanionic species (cargo), that is, single-stranded DNA (ssDNA), and polystyrene sulfonate (PSS) using isothermal titration calorimetry. By separating the electrostatic CP-cargo interaction from the full assembly interaction, we conclude that CP-CP interactions cause an enthalpy change of -3 to -4 kcal mol CP. Furthermore, we quantify that upon reducing the CP-CP interaction, in the case of CCMV by increasing the pH to 7, the CP-cargo starts to dominate VLP formation. This is highlighted by the three times higher affinity between CP and PSS compared to CP and ssDNA, resulting in the disassembly of CCMV at neutral pH in the presence of PSS to yield PSS-filled VLPs.
Topics: Bromovirus; Capsid Proteins; DNA, Single-Stranded; Hydrogen-Ion Concentration; Polyelectrolytes; Polymers; Polystyrenes; Static Electricity; Temperature; Thermodynamics; Virus Assembly
PubMed: 31661278
DOI: 10.1021/acs.jpcb.9b06258 -
Journal of Virology Jul 1991Brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are related positive-strand RNA viruses with genomes divided among RNAs 1, 2, and 3. RNAs 1 and 2...
Brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are related positive-strand RNA viruses with genomes divided among RNAs 1, 2, and 3. RNAs 1 and 2 encode the viral RNA replication factors, which share extensive conservation with proteins encoded by the animal alphaviruses and diverse plant viruses. In barley protoplasts, CCMV RNAs 1 and 2 support high but distinguishable amplification of either BMV RNA3 (B3) or CCMV RNA3 (C3), while BMV RNAs 1 and 2 show even greater discrimination, amplifying C3 poorly relative to B3. To identify the cis-acting determinants of these template-specific and virus-specific differences in RNA3 accumulation, we constructed and tested a series of B3/C3 hybrids that exchange in turn the 5',3', and intercistronic noncoding regions, which contain all sequences required in cis for efficient B3 and C3 amplification. Despite suggestive prior in vitro results, the 3' noncoding regions were not the major determinant of the differences in amplification of B3 and C3 in vivo. Rather, 3' exchanges had relatively modest effects and did not transfer the distinctive asymmetry of amplification between B3 and C3. Intercistronic exchanges produced larger effects on RNA3 accumulation and transferred some of the polarized characteristics of the wild-type B3 and C3 behaviors. 5' exchanges revealed context-specific effects showing that the contribution of the B3 5' region to RNA3 amplification is dependent on some other B3 segment or segments. Together with previous results implicating the BMV and CCMV 1a genes in trans-acting discrimination between B3 and C3 (P. Traynor and P. Ahlquist, J. Virol. 64:69-77, 1990), these observations should help to guide studies of protein-RNA interactions governing template specificity in bromovirus RNA replication.
Topics: Cloning, Molecular; Gene Amplification; Gene Expression Regulation, Viral; Genes, Viral; Plant Viruses; RNA, Viral; Templates, Genetic
PubMed: 2041089
DOI: 10.1128/JVI.65.7.3693-3703.1991 -
Journal of Virology Mar 2020Viral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replication-derived four-molecule...
Viral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replication-derived four-molecule RNA progeny of (BMV) is packaged by a single capsid protein (CP) into three types of morphologically indistinguishable icosahedral virions with T=3 quasisymmetry. Type 1 (B1) and type 2 (B2) virions package genomic RNA1 and RNA2, respectively, while type 3 (B3+4) virions copackage genomic RNA3 (B3) and its subgenomic RNA4 (sgB4). In this study, the application of a robust -mediated transient expression system allowed us to assemble each virion type separately Experimental approaches analyzing the morphology, size, and electrophoretic mobility failed to distinguish between the virion types. Thermal denaturation analysis and protease-based peptide mass mapping experiments were used to analyze stability and the conformational dynamics of the individual virions, respectively. The crystallographic structure of the BMV capsid shows four trypsin cleavage sites (K, R, K, and K on the CP subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1 and B2 released a single peptide encompassing amino acids 2 to 8 of the N-proximal arginine-rich RNA binding motif. In contrast, B3+4 capsids were unstable with trypsin, releasing several peptides in addition to the peptides encompassing four predicted sites exposed on the capsid exterior. These results, demonstrating qualitatively different dynamics for the three types of BMV virions, suggest that the different RNA genes they contain may have different translational timing and efficiency and may even impart different structures to their capsids. The majority of viruses contain RNA genomes protected by a shell of capsid proteins. Although crystallographic studies show that viral capsids are static structures, accumulating evidence suggests that, in solution, virions are highly dynamic assemblies. The three genomic RNAs (RNA1, -2, and -3) and a single subgenomic RNA (RNA4) of Brome mosaic virus (BMV), an RNA virus pathogenic to plants, are distributed among three physically homogeneous virions. This study examines the thermal stability by differential scanning fluorimetry (DSF) and capsid dynamics by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analyses following trypsin digestion of the three virions assembled separately using the -mediated transient expression approach. The results provide compelling evidence that virions packaging genomic RNA1 and -2 are distinct from those copackaging RNA3 and -4 in their stability and dynamics, suggesting that RNA-dependent capsid dynamics play an important biological role in the viral life cycle.
Topics: Agrobacterium; Bromovirus; Capsid; Capsid Proteins; Genome, Viral; Peptide Mapping; RNA, Bacterial; RNA, Viral; Virion; Virus Assembly; Virus Replication
PubMed: 31996436
DOI: 10.1128/JVI.01794-19 -
Molecular Plant Pathology May 2011The 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... (Review)
Review
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.
Topics: Bromovirus; Capsid Proteins; Gene Expression Regulation, Viral; Protein Structure, Secondary; RNA, Viral
PubMed: 21453435
DOI: 10.1111/j.1364-3703.2010.00678.x -
Journal of Materials Chemistry. B Jul 2021Long-term tracking of nanoparticles to resolve intracellular structures and motions is essential to elucidate fundamental parameters as well as transport processes...
Long-term tracking of nanoparticles to resolve intracellular structures and motions is essential to elucidate fundamental parameters as well as transport processes within living cells. Fluorescent nanodiamond (ND) emitters provide cell compatibility and very high photostability. However, high stability, biocompatibility, and cellular uptake of these fluorescent NDs under physiological conditions are required for intracellular applications. Herein, highly stable NDs encapsulated with Cowpea chlorotic mottle virus capsid proteins (ND-CP) are prepared. A thin capsid protein layer is obtained around the NDs, which imparts reactive groups and high colloidal stability, while retaining the opto-magnetic properties of the coated NDs as well as the secondary structure of CPs adsorbed on the surface of NDs. In addition, the ND-CP shows excellent biocompatibility both in vitro and in vivo. Long-term 3D trajectories of the ND-CP with fine spatiotemporal resolutions are recorded; their intracellular motions are analyzed by different models, and the diffusion coefficients are calculated. The ND-CP with its brilliant optical properties and stability under physiological conditions provides us with a new tool to advance the understanding of cell biology, e.g., endocytosis, exocytosis, and active transport processes in living cells as well as intracellular dynamic parameters.
Topics: Biocompatible Materials; Bromovirus; Capsid Proteins; Capsules; Fluorescence; Nanodiamonds; Particle Size
PubMed: 34184014
DOI: 10.1039/d1tb00890k