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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 -
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 -
Journal of Controlled Release :... Mar 2021Vaccine-induced immune response can be greatly enhanced by mimicking pathogen properties. The size and the repetitive geometric shape of virus-like particles (VLPs)...
Vaccine-induced immune response can be greatly enhanced by mimicking pathogen properties. The size and the repetitive geometric shape of virus-like particles (VLPs) influence their immunogenicity by facilitating drainage to secondary lymphoid organs and enhancing interaction with and activation of B cells and innate humoral immune components. VLPs derived from the plant Bromovirus genus, specifically cowpea chlorotic mottle virus (CCMV), are T = 3 icosahedral particles. (T) is the triangulation number that refers to the number and arrangements of the subunits (pentamers and hexamers) of the VLPs. CCMV-VLPs can be easily expressed in an E. coli host system and package ssRNA during the expression process. Recently, we have engineered CCMV-VLPs by incorporating the universal tetanus toxin (TT) epitope at the N-terminus. The modified CCMV-VLPs successfully form icosahedral particles T = 3, with a diameter of ~30 nm analogous to the parental VLPs. Interestingly, incorporating TT epitope at the C-terminus of CCMV-VLPs results in the formation of Rod-shaped VLPs, ~1 μm in length and ~ 30 nm in width. In this study, we have investigated the draining kinetics and immunogenicity of both engineered forms (termed as Round-shaped CCMV-VLPs and Rod-shaped CCMV-VLPs) as potential B cell immunogens using different in vitro and in vivo assays. Our results reveal that Round-shaped CCMV-VLPs are more efficient in draining to secondary lymphoid organs to charge professional antigen-presenting cells as well as B cells. Furthermore, compared to Rod-shaped CCMV-VLPs, Round-shaped CCMV-VLPs led to more than 100-fold increased systemic IgG and IgA responses accompanied by prominent formation of splenic germinal centers. Round-shaped CCMV-VLPs could also polarize the induced T cell response toward Th1. To our knowledge, this is the first study investigating and comparing the draining kinetics and immunogenicity of one and the same VLP monomer forming nano-sized icosahedra or rods in the micrometer size.
Topics: Antibody Formation; Bromovirus; Drainage; Epitopes; Escherichia coli; Vaccines, Virus-Like Particle
PubMed: 33450322
DOI: 10.1016/j.jconrel.2021.01.012 -
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 -
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 -
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 -
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 -
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 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