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Acta Biochimica Polonica 2001One of the most unusual features of RNA viruses is their enormous genetic variability. Among the different processes contributing to the continuous generation of new... (Review)
Review
One of the most unusual features of RNA viruses is their enormous genetic variability. Among the different processes contributing to the continuous generation of new viral variants RNA recombination is of special importance. This process has been observed for human, animal, plant and bacterial viruses. The collected data reveal a great susceptibility of RNA viruses to recombination. They also indicate that genetic RNA recombination (especially the nonhomologous one) is a major factor responsible for the emergence of new viral strains or species. Although the formation and accumulation of viral recombinants was observed in numerous RNA viruses, the molecular basis of this phenomenon was studied in only a few viral species. Among them, brome mosaic virus (BMV), a model (+)RNA virus offers the best opportunities to investigate various aspects of genetic RNA recombination in vivo. Unlike any other, the BMV-based system enables homologous and nonhomologous recombination studies at both the protein and RNA levels. As a consequence, BMV is the virus for which the structural requirements for genetic RNA recombination have been most precisely established. Nevertheless, the previously proposed model of genetic recombination in BMV still had one weakness: it could not really explain the role of RNA structure in nonhomologous recombination. Recent discoveries concerning the latter problem give us a chance to fill this gap. That is why in this review we present and thoroughly discuss all results concerning nonhomologous recombination in BMV that have been obtained until now.
Topics: Animals; Base Sequence; Bromovirus; Carmovirus; Foot-and-Mouth Disease Virus; HIV Reverse Transcriptase; Humans; Models, Genetic; Molecular Sequence Data; Murine hepatitis virus; Nucleic Acid Conformation; Poliovirus; RNA Viruses; RNA, Viral; Recombination, Genetic
PubMed: 11732610
DOI: No ID Found -
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 Virology Mar 2023The three genomic and a single subgenomic RNA of (CCMV), which is pathogenic to plants, is packaged into three morphologically indistinguishable icosahedral virions...
The three genomic and a single subgenomic RNA of (CCMV), which is pathogenic to plants, is packaged into three morphologically indistinguishable icosahedral virions with T=3 symmetry. The two virion types, C1 and C2, package genomic RNAs 1 (C1) and 2 (C2), respectively. The third virion type, C3+4, copackages genomic RNA3 and its subgenomic RNA (RNA4). In this study, we sought to evaluate how the alteration of native capsid dynamics by the host and viral replicase modulate the general biology of the virus. The application of a series of biochemical, molecular, and biological assays revealed the following. (i) Proteolytic analysis of the three virion types of CCMV assembled individually revealed that, while retaining the structural integrity, C1 and C2 virions released peptide regions encompassing the N-terminal arginine-rich RNA binding motif. In contrast, a minor population of the C3+4 virion type was sensitive to trypsin-releasing peptides encompassing the entire capsid protein region. (ii) The wild-type CCMV virions purified from cowpea are highly susceptible to trypsin digestion, while those from Nicotiana benthamiana remained resistant, and (iii) finally, the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis evaluated the relative dynamics of C3+4 and B3+4 virions assembled under the control of the homologous versus heterologous replicase. The role of viral replicase in modulating the capsid dynamics was evident by the differential sensitivity to protease exhibited by B3+4 and C3+4 virions assembled under the homologous versus heterologous replicase. Our results collectively conclude that constant modulation of capsid dynamics by the host and viral replicase is obligatory for successful infection. Infectious virus particles or virions are considered static structures and undergo various conformational transitions to replicate and infect many eukaryotic cells. In viruses, conformational changes are essential for establishing infection and evolution. Although viral capsid fluctuations, referred to as dynamics or breathing, have been well studied in RNA viruses pathogenic to animals, such information is limited among plant viruses. The primary focus of this study is to address how capsid dynamics of plant-pathogenic RNA viruses, namely, (CCMV) and (BMV), are modulated by the host and viral replicase. The results presented have improved and transformed our understanding of the functional relationship between capsid dynamics and the general biology of the virus. They are likely to provide stimulus to extend similar studies to viruses pathogenic to eukaryotic organisms.
Topics: Bromovirus; Capsid; Host Microbial Interactions; Plants; RNA, Viral; Trypsin; Viral Replicase Complex Proteins; Subgenomic RNA
PubMed: 36786601
DOI: 10.1128/jvi.01284-22 -
PLoS Pathogens Mar 2015Positive-strand RNA viruses genome replication invariably is associated with vesicles or other rearranged cellular membranes. Brome mosaic virus (BMV) RNA replication...
Positive-strand RNA viruses genome replication invariably is associated with vesicles or other rearranged cellular membranes. Brome mosaic virus (BMV) RNA replication occurs on perinuclear endoplasmic reticulum (ER) membranes in ~70 nm vesicular invaginations (spherules). BMV RNA replication vesicles show multiple parallels with membrane-enveloped, budding retrovirus virions, whose envelopment and release depend on the host ESCRT (endosomal sorting complexes required for transport) membrane-remodeling machinery. We now find that deleting components of the ESCRT pathway results in at least two distinct BMV phenotypes. One group of genes regulate RNA replication and the frequency of viral replication complex formation, but had no effect on spherule size, while a second group of genes regulate RNA replication in a way or ways independent of spherule formation. In particular, deleting SNF7 inhibits BMV RNA replication > 25-fold and abolishes detectable BMV spherule formation, even though the BMV RNA replication proteins accumulate and localize normally on perinuclear ER membranes. Moreover, BMV ESCRT recruitment and spherule assembly depend on different sets of protein-protein interactions from those used by multivesicular body vesicles, HIV-1 virion budding, or tomato bushy stunt virus (TBSV) spherule formation. These and other data demonstrate that BMV requires cellular ESCRT components for proper formation and function of its vesicular RNA replication compartments. The results highlight growing but diverse interactions of ESCRT factors with many viruses and viral processes, and potential value of the ESCRT pathway as a target for broad-spectrum antiviral resistance.
Topics: Blotting, Northern; Blotting, Western; Bromovirus; Endosomal Sorting Complexes Required for Transport; Fluorescent Antibody Technique; Host-Parasite Interactions; Immunoprecipitation; Microscopy, Confocal; Microscopy, Electron, Transmission; RNA, Viral; Virus Replication
PubMed: 25748299
DOI: 10.1371/journal.ppat.1004742 -
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 -
Pathogens (Basel, Switzerland) Jan 2024Previously, we described the RNA recombinants accumulating in tissues infected with the bromoviruses BMV (Brome mosaic virus) and CCMV (Cowpea chlorotic mottle virus)....
Previously, we described the RNA recombinants accumulating in tissues infected with the bromoviruses BMV (Brome mosaic virus) and CCMV (Cowpea chlorotic mottle virus). In this work, we characterize the recombinants encapsidated inside the purified virion particles of BMV and CCMV. By using a tool called the Viral Recombination Mapper (ViReMa) that detects recombination junctions, we analyzed a high number of high-throughput sequencing (HTS) short RNA sequence reads. Over 28% of BMV or CCMV RNA reads did not perfectly map to the viral genomes. ViReMa identified 1.40% and 1.83% of these unmapped reads as the RNA recombinants, respectively, in BMV and CCMV. Intra-segmental crosses were more frequent than the inter-segmental ones. Most intra-segmental junctions carried short insertions/deletions (indels) and caused frameshift mutations. The mutation hotspots clustered mainly within the open reading frames. Substitutions of various lengths were also identified, whereas a small fraction of crosses occurred between viral and their host RNAs. Our data reveal that the virions can package detectable amounts of multivariate recombinant RNAs, contributing to the flexible nature of the viral genomes.
PubMed: 38276169
DOI: 10.3390/pathogens13010096 -
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 -
PLoS Pathogens Apr 2018Replication of positive-strand RNA viruses [(+)RNA viruses] takes place in membrane-bound viral replication complexes (VRCs). Formation of VRCs requires virus-mediated...
Replication of positive-strand RNA viruses [(+)RNA viruses] takes place in membrane-bound viral replication complexes (VRCs). Formation of VRCs requires virus-mediated manipulation of cellular lipid synthesis. Here, we report significantly enhanced brome mosaic virus (BMV) replication and much improved cell growth in yeast cells lacking PAH1 (pah1Δ), the sole yeast ortholog of human LIPIN genes. PAH1 encodes Pah1p (phosphatidic acid phosphohydrolase), which converts phosphatidate (PA) to diacylglycerol that is subsequently used for the synthesis of the storage lipid triacylglycerol. Inactivation of Pah1p leads to altered lipid composition, including high levels of PA, total phospholipids, ergosterol ester, and free fatty acids, as well as expansion of the nuclear membrane. In pah1Δ cells, BMV replication protein 1a and double-stranded RNA localized to the extended nuclear membrane, there was a significant increase in the number of VRCs formed, and BMV genomic replication increased by 2-fold compared to wild-type cells. In another yeast mutant that lacks both PAH1 and DGK1 (encodes diacylglycerol kinase converting diacylglycerol to PA), which has a normal nuclear membrane but maintains similar lipid compositional changes as in pah1Δ cells, BMV replicated as efficiently as in pah1Δ cells, suggesting that the altered lipid composition was responsible for the enhanced BMV replication. We further showed that increased levels of total phospholipids play an important role because the enhanced BMV replication required active synthesis of phosphatidylcholine, the major membrane phospholipid. Moreover, overexpression of a phosphatidylcholine synthesis gene (CHO2) promoted BMV replication. Conversely, overexpression of PAH1 or plant PAH1 orthologs inhibited BMV replication in yeast or Nicotiana benthamiana plants. Competing with its host for limited resources, BMV inhibited host growth, which was markedly alleviated in pah1Δ cells. Our work suggests that Pah1p promotes storage lipid synthesis and thus represses phospholipid synthesis, which in turn restricts both viral replication and cell growth during viral infection.
Topics: Bromovirus; Gene Expression Regulation, Fungal; Gene Expression Regulation, Plant; Genome, Viral; Nuclear Envelope; Phosphatidate Phosphatase; Phospholipids; Saccharomyces cerevisiae; Nicotiana; Virus Replication
PubMed: 29649282
DOI: 10.1371/journal.ppat.1006988 -
Journal of Visualized Experiments : JoVE Nov 2012The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for...
The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. (1) and (2) for further information on VNPs). From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies. In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.
Topics: Animals; Bromovirus; Colonic Neoplasms; Comovirus; Fabaceae; HT29 Cells; Humans; Mice; Mice, Nude; Microscopy, Electron, Transmission; Nanoparticles; Plant Viruses; Potexvirus; Spectrophotometry, Ultraviolet; Nicotiana; Tobacco Mosaic Virus
PubMed: 23183850
DOI: 10.3791/4352 -
Virology May 2000Specific interactions are likely to occur between the highly conserved N-proximal arginine-rich motif (ARM) of Brome mosaic virus (BMV) coat protein (CP) and each of...
Specific interactions are likely to occur between the highly conserved N-proximal arginine-rich motif (ARM) of Brome mosaic virus (BMV) coat protein (CP) and each of three genomic RNAs and a single subgenomic RNA during in vivo encapsidation. To characterize these interactions, three independent deletions were engineered into a biologically active clone of BMV RNA3 (B3) such that the matured CP of each B3 variant precisely lacks either the entire ARM (B3/Delta919) or two consecutive arginine residues (B3/13DeltaDelta14 and B3/18DeltaDelta19) within the ARM. Analysis of virion RNA for each B3 variant recovered from symptomatic leaves of Chenopodium quinoa revealed that the interactions between the N-terminal ARM of BMV CP and each of three genomic RNAs is distinct. Northern blot hybridization of B3Delta919 virion RNA revealed that the deleted ARM region specifically affected the stability of virions containing RNA1. An abundant truncated RNA species recurrently found in the virions of B3Delta919 was identified to be a derivative of genomic RNA1, lacking the 5' 943 nucleotides. Additional Northern blot analysis of virion RNAs from B3/Delta919, B3/13DeltaDelta14, and B3/18DeltaDelta19, and in vitro reassembly assays revealed that the N-terminal ARM region contains crucial amino acids required for RNA4 packaging, independent of genomic RNA3. The significance of these observations in relation to Bromovirus CP-RNA interactions during virion assembly is discussed.
Topics: Amino Acid Sequence; Arginine; Bromovirus; Capsid; Molecular Sequence Data; Nucleic Acid Conformation; Protein Conformation; RNA, Viral; Virion; Virus Replication
PubMed: 10792997
DOI: 10.1006/viro.2000.0312