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Virology Oct 2003Cucumber mosaic virus (CMV, a cucumovirus) and Brome mosaic virus (BMV, a bromovirus) require the coat protein (CP) in addition to the 3a movement protein (MP) for...
Cucumber mosaic virus (CMV, a cucumovirus) and Brome mosaic virus (BMV, a bromovirus) require the coat protein (CP) in addition to the 3a movement protein (MP) for cell-to-cell movement, while Cowpea chlorotic mottle virus (CCMV, a bromovirus) does not. Using bombardment-mediated transcomplementation assays, we investigated whether the movement functions encoded by these viruses potentiate cell-to-cell movement of movement-defective Tomato mosaic virus (ToMV, a tobamovirus) and Potato virus X (PVX, a potexvirus) mutants in Nicotiana benthamiana. Coexpression of CMV 3a and CP, but neither protein alone, complemented the defective movement of ToMV and PVX. A C-terminal deletion in CMV 3a (3a Delta C33) abolished the requirement of CP in transporting the ToMV genome. The action of 3a Delta C33 was inhibited by coexpression of wild-type 3a. These findings were confirmed in tobacco with ToMV-CMV chimeric viruses. Either BMV 3a or CCMV 3a alone efficiently complemented the movement-defective phenotype of the ToMV mutant. Therefore, every 3a protein examined intrinsically possesses the activity required to act as MP. In transcomplementation of the PVX mutant, the activities of BMV 3a, CCMV 3a, and CMV 3a Delta C33 were very low. The activities of the bromovirus 3a proteins were enhanced by coexpression of the cognate CP but the activity of CMV 3a Delta C33 was not. Based on these results, possible roles of cucumo- and bromovirus CPs in cell-to-cell movement are discussed.
Topics: Base Sequence; Bromovirus; Capsid Proteins; Cucumovirus; Gene Expression Regulation, Viral; Genetic Complementation Test; Green Fluorescent Proteins; Luminescent Proteins; Solanum lycopersicum; Molecular Sequence Data; Plant Diseases; Plant Leaves; Plant Viral Movement Proteins; Potexvirus; Recombinant Fusion Proteins; Nicotiana; Tobamovirus; Viral Proteins
PubMed: 14592759
DOI: 10.1016/s0042-6822(03)00480-x -
Journal of Virology Jan 2012All positive-strand RNA viruses replicate their genomes in association with rearranged intracellular membranes such as single- or double-membrane vesicles. Brome mosaic...
All positive-strand RNA viruses replicate their genomes in association with rearranged intracellular membranes such as single- or double-membrane vesicles. Brome mosaic virus (BMV) RNA synthesis occurs in vesicular endoplasmic reticulum (ER) membrane invaginations, each induced by many copies of viral replication protein 1a, which has N-terminal RNA capping and C-terminal helicase domains. Although the capping domain is responsible for 1a membrane association and ER targeting, neither this domain nor the helicase domain was sufficient to induce replication vesicle formation. Moreover, despite their potential for mutual interaction, the capping and helicase domains showed no complementation when coexpressed in trans. Cross-linking showed that the capping and helicase domains each form trimers and larger multimers in vivo, and the capping domain formed extended, stacked, hexagonal lattices in vivo. Furthermore, coexpressing the capping domain blocked the ability of full-length 1a to form replication vesicles and replicate RNA and recruited full-length 1a into mixed hexagonal lattices with the capping domain. Thus, BMV replication vesicle formation and RNA replication depend on the direct linkage and concerted action of 1a's self-interacting capping and helicase domains. In particular, the capping domain's strong dominant-negative effects showed that the ability of full-length 1a to form replication vesicles was highly sensitive to disruption by non-productively titrating lattice-forming self-interactions of the capping domain. These and other findings shed light on the roles and interactions of 1a domains in replication compartment formation and support prior results suggesting that 1a induces replication vesicles by forming a capsid-like interior shell.
Topics: Bromovirus; Cell Nucleus; Endoplasmic Reticulum; Gene Expression Regulation, Viral; Protein Structure, Tertiary; Protein Transport; RNA Caps; RNA Helicases; RNA, Viral; Saccharomyces cerevisiae; Viral Proteins; Virus Replication
PubMed: 22090102
DOI: 10.1128/JVI.05684-11 -
PloS One 2021The vast majority of plant viruses are unenveloped, i.e., they lack a lipid bilayer that is characteristic of most animal viruses. The interactions between plant...
The vast majority of plant viruses are unenveloped, i.e., they lack a lipid bilayer that is characteristic of most animal viruses. The interactions between plant viruses, and between viruses and surfaces, properties that are essential for understanding their infectivity and to their use as bionanomaterials, are largely controlled by their surface charge, which depends on pH and ionic strength. They may also depend on the charge of their contents, i.e., of their genes or-in the instance of virus-like particles-encapsidated cargo such as nucleic acid molecules, nanoparticles or drugs. In the case of enveloped viruses, the surface charge of the capsid is equally important for controlling its interaction with the lipid bilayer that it acquires and loses upon leaving and entering host cells. We have previously investigated the charge on the unenveloped plant virus Cowpea Chlorotic Mottle Virus (CCMV) by measurements of its electrophoretic mobility. Here we examine the electrophoretic properties of a structurally and genetically closely related bromovirus, Brome Mosaic Virus (BMV), of its capsid protein, and of its empty viral shells, as functions of pH and ionic strength, and compare them with those of CCMV. From measurements of both solution and gel electrophoretic mobilities (EMs) we find that the isoelectric point (pI) of BMV (5.2) is significantly higher than that of CCMV (3.7), that virion EMs are essentially the same as those of the corresponding empty capsids, and that the same is true for the pIs of the virions and of their cleaved protein subunits. We discuss these results in terms of current theories of charged colloidal particles and relate them to biological processes and the role of surface charge in the design of new classes of drug and gene delivery systems.
Topics: Bromovirus; Capsid Proteins; Hordeum; Osmolar Concentration; Plant Leaves; RNA, Viral; Virus Assembly; Virus Replication
PubMed: 34506491
DOI: 10.1371/journal.pone.0255820 -
Virology Nov 1998Brome mosaic bromovirus (BMV) and cucumber mosaic cucumovirus (CMV) are structurally and genetically very similar. The specificity of the BMV and CMV coat proteins (CPs)...
Brome mosaic bromovirus (BMV) and cucumber mosaic cucumovirus (CMV) are structurally and genetically very similar. The specificity of the BMV and CMV coat proteins (CPs) during in vivo encapsidation was studied using two RNA3 chimera in which the respective CP genes were exchanged. The replicative competence of each chimera was analyzed in Nicotiana benthamiana protoplasts, and their ability to cause infections was examined in two common permissive hosts, Chenopodium quinoa and N. benthamiana. Each RNA3 chimera replicated to near wild-type (wt) levels and synthesized CPs of expected parental origin when co-inoculated with their respective genomic wt RNAs 1 and 2. However, inoculum containing each chimera was noninfectious in the common permissive hosts tested. Encapsidation assays in N. benthamiana protoplasts revealed that CMV CP expressed from chimeric BMV RNA3 was capable of packaging heterologous BMV RNA, however, at a lower efficiency than parental BMV CP. By contrast, BMV CP expressed from chimeric CMV RNA3 was unable to package heterologous CMV RNA. These observations demonstrate that BMV CP, but not CMV CP, exhibits a high degree of specificity during in vivo packaging. The reasons for the noninfectious nature of each chimera in the host plants tested and factors likely to affect encapsidation in vivo are discussed.
Topics: Bromovirus; Capsid; Cucumovirus; DNA Mutational Analysis; DNA, Viral; Edible Grain; Genome, Viral; Plants, Toxic; RNA, Viral; Nicotiana; Transcription, Genetic; Transfection; Virus Assembly
PubMed: 9837807
DOI: 10.1006/viro.1998.9421 -
Journal of the American Chemical Society Jul 2022Cowpea chlorotic mottle virus (CCMV) is a widely used model for virus replication studies. A major challenge lies in distinguishing between the roles of the interaction...
Cowpea chlorotic mottle virus (CCMV) is a widely used model for virus replication studies. A major challenge lies in distinguishing between the roles of the interaction between coat proteins and that between the coat proteins and the viral RNA in assembly and disassembly processes. Here, we report on the spontaneous and reversible size conversion of the empty capsids of a CCMV capsid protein functionalized with a hydrophobic elastin-like polypeptide which occurs following a pH jump. We monitor the concentrations of = 3 and = 1 capsids as a function of time and show that the time evolution of the conversion from one number to another is not symmetric: The conversion from = 1 to = 3 is a factor of 10 slower than that of = 3 to = 1. We explain our experimental findings using a simple model based on classical nucleation theory applied to virus capsids, in which we account for the change in the free protein concentration, as the different types of shells assemble and disassemble by shedding or absorbing single protein subunits. As far as we are aware, this is the first study confirming that both the assembly and disassembly of viruslike shells can be explained through classical nucleation theory, reproducing quantitatively results from time-resolved experiments.
Topics: Bromovirus; Capsid; Capsid Proteins; RNA, Viral; Virion; Virus Assembly
PubMed: 35792573
DOI: 10.1021/jacs.2c04074 -
Virology Dec 1996The N-terminal region of the brome mosaic bromovirus (BMV) coat protein (CP) contains an arginine-rich motif that is conserved among plant and nonplant viruses and...
The N-terminal region of the brome mosaic bromovirus (BMV) coat protein (CP) contains an arginine-rich motif that is conserved among plant and nonplant viruses and implicated in binding the RNA during encapsidation. To elucidate the functional significance of this conserved motif in the BMV CP, a series of deletions encompassing the arginine-rich motif was introduced into a biologically active clone of BMV RNA3, and their effect on replication, encapsidation, and infection in plants was examined. Analysis of infection phenotypes elicited on Chenopodium quinoa revealed the importance of the first 19 N-proximal amino acids of BMV CP in encapsidation and pathogenicity. Inoculation of C. quinoa with three viable variants of BMV RNA3 lacking the first 11, 14, and 18 N-terminal amino acids of the CP resulted in the development of necrotic local lesions and restricted the spread of infection to inoculated leaves. Progeny analysis from symptomatic leaves revealed that, in each case, virus accumulation was severely affected by the introduced mutations and each truncated CP differed in its ability to package genomic RNA. In contrast to these observations in C. quinoa, none of the CP variants was able to establish either local or systemic infections in barley plants. The intrinsic role played by the N-terminal arginine-rich motif of BMV CP in packaging viral RNAs and the interactions between the host and the truncated CPs in modulating symptom expression and movement are discussed.
Topics: Amino Acid Sequence; Arginine; Binding Sites; Blotting, Western; Bromovirus; Capsid; Edible Grain; Hordeum; Molecular Sequence Data; Plant Diseases; Protoplasts; RNA, Viral; Sequence Deletion; Virus Assembly
PubMed: 8955049
DOI: 10.1006/viro.1996.0657 -
Virology Jan 1995The nonstructural 3a protein of the positive-strand RNA bromoviruses is required for infection spread in plants and is a crucial determinant of host specificity in...
The nonstructural 3a protein of the positive-strand RNA bromoviruses is required for infection spread in plants and is a crucial determinant of host specificity in systemic infection. To determine the paths of wild-type (wt) bromovirus infection spread, the step at which 3a mutants are arrested, and the nature of the host specificity associated with the 3a gene, we used in situ hybridization to examine infection spread by cowpea chlorotic mottle bromovirus (CCMV) and its derivatives at the level of individual cells in cowpea leaf epidermis. From 1 to 3 days post inoculation (dpi), wt CCMV spread from initially infected cells to adjacent cells, creating expanding infection foci whose radii grew by one additional epidermal cell diameter every 5 hr. By 3 to 4 dpi, vascular elements contacting such foci acted as conduits for further infection spread. By contrast, a 3a frameshift derivative multiplied in initially infected epidermal cells but failed to move into neighboring cells even by 4 dpi, showing that the 3a gene is essential for cell-to-cell spread. Most interestingly, a CCMV derivative with the 3a gene replaced by that of a bromovirus not adapted to cowpea, brome mosaic virus (BMV), initially spread from cell to cell in cowpea plants, but stopped spreading between 1 and 2 dpi, when most infection foci encompassed 40-80 epidermal cells. Thus, the host-specificity restriction imposed by BMV 3a protein did not result from an inability to direct the spread of infection out of initially infected cowpea cells, but from a much later block. The apparent absence of any preexisting anatomical boundary at the limit of infection spread and localized tissue changes at the infection foci suggested that induced host responses might have contributed to this block.
Topics: Bromovirus; Fabaceae; Gene Expression; In Situ Hybridization; Plants, Medicinal; Protoplasts; RNA, Viral; Viral Nonstructural Proteins
PubMed: 7831782
DOI: 10.1016/s0042-6822(95)80043-3 -
Virology Nov 1997Two members of the bromovirus group, brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV), selectively infect barley and cowpea, respectively, and also...
Molecular studies on bromovirus capsid protein. IV. Coat protein exchanges between brome mosaic and cowpea chlorotic mottle viruses exhibit neutral effects in heterologous hosts.
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 of C. 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 or C. quinoa plants 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.
Topics: Bromovirus; Capsid; Hordeum; Pisum sativum; RNA, Viral; Reassortant Viruses; Virus Replication
PubMed: 9400617
DOI: 10.1006/viro.1997.8849 -
Viruses Oct 2023(CCMV) and (BMV) are naked plant viruses with similar characteristics; both form a T = 3 icosahedral protein capsid and are members of the family. It is well known...
(CCMV) and (BMV) are naked plant viruses with similar characteristics; both form a T = 3 icosahedral protein capsid and are members of the family. It is well known that these viruses completely disassemble and liberate their genome at a pH around 7.2 and 1 M ionic strength. However, the 1 M ionic strength condition is not present inside cells, so an important question is how these viruses deliver their genome inside cells for their viral replication. There are some studies reporting the swelling of the CCMV virus using different techniques. For example, it is reported that at a pH~7.2 and low ionic strength, the swelling observed is about 10% of the initial diameter of the virus. Furthermore, different regions within the cell are known to have different pH levels and ionic strengths. In this work, we performed several experiments at low ionic strengths of 0.1, 0.2, and 0.3 and systematically increased the pH in 0.2 increments from 4.6 to 7.4. To determine the change in virus size at the different pHs and ionic strengths, we first used dynamic light scattering (DLS). Most of the experiments agree with a 10% capsid swelling under the conditions reported in previous works, but surprisingly, we found that at some particular conditions, the virus capsid swelling could be as big as 20 to 35% of the original size. These measurements were corroborated by atomic force microscopy (AFM) and transmission electron microscopy (TEM) around the conditions where the big swelling was determined by DLS. Therefore, this big swelling could be an easier mechanism that viruses use inside the cell to deliver their genome to the cell machinery for viral replication.
Topics: Bromovirus; Plant Viruses; Capsid Proteins; Capsid; Osmolar Concentration
PubMed: 37896823
DOI: 10.3390/v15102046 -
Advances in Virus Research 1994It is well known that DNA-based organisms rearrange and repair their genomic DNA through recombination processes, and that these rearrangements serve as a powerful... (Review)
Review
It is well known that DNA-based organisms rearrange and repair their genomic DNA through recombination processes, and that these rearrangements serve as a powerful source of variability and adaptation for these organisms. In RNA viruses' genetic recombination is defined as any process leading to the exchange of information between viral RNAs. There are two types of recombination events: legitimate and illegitimate. While legitimate (homologous) recombination occurs between closely related sequences at corresponding positions, illegitimate (nonhomologous) recombination could happen at any position among the unrelated RNA molecules. In order to differentiate between the symmetrical and asymmetrical homologous crosses, Lai defined the former as homologous recombination and the latter as aberrant homologous recombination. This chapter uses brome mosaic virus (BMV), a multicomponent plant RNA virus, as an example to discuss the progress in studying the mechanism of genetic recombination in positive-stranded RNA viruses. Studies described in this chapter summarize the molecular approaches used to increase the frequency of recombination among BMV RNA segments and, more importantly, to target the sites of crossovers to specific BMV RNA regions. It demonstrates that the latter can be accomplished by introducing local complementarities to the recombining substrates.
Topics: Animals; Base Sequence; Bromovirus; Models, Genetic; Molecular Sequence Data; RNA, Viral; Recombination, Genetic; Viruses
PubMed: 8191956
DOI: 10.1016/s0065-3527(08)60051-2