Rotational and translational diffusion of biomolecules in complex liquids and HeLa cells.

Diffusive motion accompanies many physical and biological processes. The Stokes-Sutherland-Einstein relation for the translational diffusion coefficient, DT, agrees with experiments done in simple fluids but fails for complex fluids. Moreover, the interdependence between DT and rotational diffusion coefficient, DR, also deviates in complex fluids from the classical relation of DT/DR = 4r2/3 known in simple fluids. Makuch et al. Soft Matter, 2020, 16, 114-124 presented a generalization of the classical translational and rotational diffusion theory for complex fluids. In this work, we empirically verify this model based on simultaneous translational and rotational diffusion measurements. We use fluorescently stained cowpea chlorotic mottle virus (CCMV) particles as monodisperse probes and aqueous polyethylene glycol (PEG) solutions as a model complex fluid. The theory and experimental data obtained from fluorescence correlation spectroscopy (FCS) measurements agreed. Finally, we used the same model and analyzed the diffusion of Yo-Pro-1 stained large ribosomal subunits (LSU) in the cytoplasm and nucleus of living HeLa cells.

One-Step Purification Strategy for Cowpea Chlorotic Mottle Virus-Like Particles Produced by the IC-BEVS.

Virus-like particles (VLP) of the cowpea chlorotic mottle virus (CCMV), a plant virus, have been shown to be safe and noncytotoxic vehicles for delivering various cargos, including nucleic acids and peptides, and as scaffolds for presenting epitopes. Thus, CCMV-VLP have acquired increasing attention to be used in fields such as gene therapy, drug delivery, and vaccine development. Regardless of their production method, most reports purify CCMV-VLP through a series of ultracentrifugation steps using sucrose density gradient ultracentrifugation, which is a complex and time-consuming process. Here, the use of anion exchange chromatography is described as a one-step protocol for purification of CCMV-VLP produced by the insect cell-baculovirus expression vector system (IC-BEVS).

Plant Virus-Like Particles for RNA Delivery.

Plant viruses such as brome mosaic virus and cowpea chlorotic mottle virus are effectively purified through PEG precipitation and sucrose cushion ultracentrifugation. Increasing ionic strength and an alkaline pH cause the viruses to swell and disassemble into coat protein subunits. The coat proteins can be reassembled into stable virus-like particles (VLPs) that carry anionic molecules at low ionic strength and through two-step dialysis from neutral pH to acidic buffer. VLPs have been extensively studied due to their ability to protect and deliver cargo, particularly RNA, while avoiding degradation under physiological conditions. Furthermore, chemical functionalization of the surface of VLPs allows for the targeted drug delivery. VLPs derived from plants have demonstrated great potential in nanomedicine by offering a versatile platform for drug delivery, imaging, and therapeutic applications.

Microsecond time-resolved cryo-electron microscopy.

Microsecond time-resolved cryo-electron microscopy has emerged as a novel approach for directly observing protein dynamics. By providing microsecond temporal and near-atomic spatial resolution, it has the potential to elucidate a wide range of dynamics that were previously inaccessible and therefore, to significantly advance our understanding of protein function. This review summarizes the properties of the laser melting and revitrification process that underlies the technique and describes different experimental implementations. Strategies for initiating and probing dynamics are discussed. Finally, the microsecond time-resolved observation of the capsid dynamics of cowpea chlorotic mottle virus, an icosahedral plant virus, is reviewed, which illustrates important features of the technique as well as its potential.

In Vivo Delivery of Spherical and Cylindrical In Vitro Reconstituted Virus-like Particles Containing the Same Self-Amplifying mRNA.

The dramatic effectiveness of recent mRNA (mRNA)-based COVID vaccines delivered in lipid nanoparticles has highlighted the promise of mRNA therapeutics in general. In this report, we extend our earlier work on self-amplifying mRNAs delivered in spherical in vitro reconstituted virus-like particles (VLPs), and on drug delivery using cylindrical virus particles. In particular, we carry out separate in vitro assemblies of a self-amplifying mRNA gene in two different virus-like particles: one spherical, formed with the capsid protein of cowpea chlorotic mottle virus (CCMV), and the other cylindrical, formed from the capsid protein of tobacco mosaic virus (TMV). The mRNA gene is rendered self-amplifying by genetically fusing it to the RNA-dependent RNA polymerase (RdRp) of Nodamura virus, and the relative efficacies of cell uptake and downstream protein expression resulting from their CCMV- and TMV-packaged forms are compared directly. This comparison is carried out by their transfections into cells in culture: expressions of two self-amplifying genes, enhanced yellow fluorescent protein (EYFP) and Renilla luciferase (Luc), packaged alternately in CCMV and TMV VLPs, are quantified by fluorescence and chemiluminescence levels, respectively, and relative numbers of the delivered mRNAs are measured by quantitative real-time PCR. The cellular uptake of both forms of these VLPs is further confirmed by confocal microscopy of transfected cells. Finally, VLP-mediated delivery of the self-amplifying-mRNA in mice following footpad injection is shown by in vivo fluorescence imaging to result in robust expression of EYFP in the draining lymph nodes, suggesting the potential of these plant virus-like particles as a promising mRNA gene and vaccine delivery modality. These results establish that both CCMV and TMV VLPs can deliver their in vitro packaged mRNA genes to immune cells and that their self-amplifying forms significantly enhance in situ expression. Choice of one VLP (CCMV or TMV) over the other will depend on which geometry of nucleocapsid is self-assembled more efficiently for a given length and sequence of RNA, and suggests that these plant VLP gene delivery systems will prove useful in a wide variety of medical applications, both preventive and therapeutic.

Diversity and modularity of tyrosine-accepting tRNA-like structures.

Certain positive-sense single-stranded RNA viruses contain elements at their 3' termini that structurally mimic tRNAs. These tRNA-like structures (TLSs) are classified based on which amino acid is covalently added to the 3' end by host aminoacyl-tRNA synthetase. Recently, a cryoEM reconstruction of a representative tyrosine-accepting tRNA-like structure (TLSTyr) from brome mosaic virus (BMV) revealed a unique mode of recognition of the viral anticodon-mimicking domain by tyrosyl-tRNA synthetase. Some viruses in the hordeivirus genus of Virgaviridae are also selectively aminoacylated with tyrosine, yet these TLS RNAs have a different architecture in the 5' domain that comprises the atypical anticodon loop mimic. Herein, we present bioinformatic and biochemical data supporting a distinct secondary structure for the 5' domain of the hordeivirus TLSTyr compared to those in Bromoviridae Despite forming a different secondary structure, the 5' domain is necessary to achieve robust in vitro aminoacylation. Furthermore, a chimeric RNA containing the 5' domain from the BMV TLSTyr and the 3' domain from a hordeivirus TLSTyr are aminoacylated, illustrating modularity in these structured RNA elements. We propose that the structurally distinct 5' domain of the hordeivirus TLSTyrs performs the same role in mimicking the anticodon loop as its counterpart in the BMV TLSTyr Finally, these structurally and phylogenetically divergent types of TLSTyr provide insight into the evolutionary connections between all classes of viral tRNA-like structures.

An Observation of a Very High Swelling of Bromovirus Members at Specific Ionic Strengths and pH.

Cowpea chlorotic mottle virus (CCMV) and brome mosaic virus (BMV) are naked plant viruses with similar characteristics; both form a T = 3 icosahedral protein capsid and are members of the bromoviridae 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.

Use of brome mosaic virus-like particles in feed, to deliver dsRNA targeting the white spot syndrome virus vp28 gene, reduces Penaeus vannamei mortality.

Numerous strategies have been investigated to combat viral infections in shrimp, specifically targeting the white spot syndrome virus (WSSV) that has caused outbreaks worldwide since the 1990s. One effective treatment involves intramuscular application of dsRNA-mediated interference against the viral capsid protein VP28. However, this approach presents challenges in terms of individual shrimp management, limiting its application on a large scale. To address this, our study aimed to evaluate the efficacy of oral delivery of protected dsRNA using chitosan nanoparticles or virus-like particles (VLPs) synthesized in brome mosaic virus (BMV). These delivery systems were administered before, during, and after WSSV infection to assess their therapeutic potential. Our findings indicate that BMV-derived VLPs demonstrated superior efficiency as nanocontainers for dsRNA delivery. Notably, the treatment involving vp28 dsRNA mixed in the feed and administered simultaneously to shrimp already infected with WSSV exhibited the highest survival rate (48%), while the infected group had a survival rate of zero, suggesting the potential efficacy of this prophylactic approach in commercial shrimp farms.

Fast viral dynamics revealed by microsecond time-resolved cryo-EM.

Observing proteins as they perform their tasks has largely remained elusive, which has left our understanding of protein function fundamentally incomplete. To enable such observations, we have recently proposed a technique that improves the time resolution of cryo-electron microscopy (cryo-EM) to microseconds. Here, we demonstrate that microsecond time-resolved cryo-EM enables observations of fast protein dynamics. We use our approach to elucidate the mechanics of the capsid of cowpea chlorotic mottle virus (CCMV), whose large-amplitude motions play a crucial role in the viral life cycle. We observe that a pH jump causes the extended configuration of the capsid to contract on the microsecond timescale. While this is a concerted process, the motions of the capsid proteins involve different timescales, leading to a curved reaction path. It is difficult to conceive how such a detailed picture of the dynamics could have been obtained with any other method, which highlights the potential of our technique. Crucially, our experiments pave the way for microsecond time-resolved cryo-EM to be applied to a broad range of protein dynamics that previously could not have been observed. This promises to fundamentally advance our understanding of protein function.

Ecological Strategies for Resource Use by Three Bromoviruses in Anthropic and Wild Plant Communities.

Ecological strategies for resource utilisation are important features of pathogens, yet have been overshadowed by stronger interest in genetic mechanisms underlying disease emergence. The purpose of this study is to ask whether host range and transmission traits translate into ecological strategies for host-species utilisation in a heterogeneous ecosystem, and whether host utilisation corresponds to genetic differentiation among three bromoviruses. We combine high-throughput sequencing and population genomics with analyses of species co-occurrence to unravel the ecological strategies of the viruses across four habitat types. The results show that the bromoviruses that were more closely related genetically did not share similar ecological strategies, but that the more distantly related pair did. Shared strategies included a broad host range and more frequent co-occurrences, which both were habitat-dependent. Each habitat thus presents as a barrier to gene flow, and each virus has an ecological strategy to navigate limitations to colonising non-natal habitats. Variation in ecological strategies could therefore hold the key to unlocking events that lead to emergence.

Relaxational dynamics of the T-number conversion of virus capsids.

We extend a recently proposed kinetic theory of virus capsid assembly based on Model A kinetics and study the dynamics of the interconversion of virus capsids of different sizes triggered by a quench, that is, by sudden changes in the solution conditions. The work is inspired by in vitro experiments on functionalized coat proteins of the plant virus cowpea chlorotic mottle virus, which undergo a reversible transition between two different shell sizes (T = 1 and T = 3) upon changing the acidity and salinity of the solution. We find that the relaxation dynamics are governed by two time scales that, in almost all cases, can be identified as two distinct processes. Initially, the monomers and one of the two types of capsids respond to the quench. Subsequently, the monomer concentration remains essentially constant, and the conversion between the two capsid species completes. In the intermediate stages, a long-lived metastable steady state may present itself, where the thermodynamically less stable species predominate. We conclude that a Model A based relaxational model can reasonably describe the early and intermediate stages of the conversion experiments. However, it fails to provide a good representation of the time evolution of the state of assembly of the coat proteins in the very late stages of equilibration when one of the two species disappears from the solution. It appears that explicitly incorporating the nucleation barriers to assembly and disassembly is crucial for an accurate description of the experimental findings, at least under conditions where these barriers are sufficiently large.

TLR Agonists Delivered by Plant Virus and Bacteriophage Nanoparticles for Cancer Immunotherapy.

Toll-like receptors (TLRs) are promising targets in cancer immunotherapy due to their role in activating the immune system; therefore, various small-molecule TLR agonists have been tested in clinical applications. However, the clinical use of TLR agonists is hindered by their non-specific side effects and poor pharmacokinetics. To overcome these limitations, we used plant virus nanoparticles (VNPs) and bacteriophage virus-like particles (VLPs) as drug delivery systems. We conjugated TLR3 or TLR7 agonists to cowpea mosaic virus (CPMV) VNPs, cowpea chlorotic mottle virus (CCMV) VNPs, and bacteriophage Qß VLPs. The conjugation of TLR7 agonist, 2-methoxyethoxy-8-oxo-9-(4-carboxybenzyl)adenine (1V209), resulted in the potent activation of immune cells and promoted the production of pro-inflammatory cytokine interleukin 6. We found that 1V209 conjugated to CPMV, CCMV, and Qß reduced tumor growth in vivo and prolonged the survival of mice compared to those treated with free 1V209 or a simple admixture of 1V209 and viral particles. Nucleic acid-based TLR3 agonist, polyinosinic acid with polycytidylic acid (poly(I:C)), was also delivered by CPMV VNPs, resulting in enhanced mice survival. All our data suggest that coupling and co-delivery are required to enhance the anti-tumor efficacy of TLR agonists and simple mixing of the VLPs with the agonists does not confer a survival benefit. The delivery of 1V209 or poly(I:C) conjugated to VNPs/VLPs probably enhances their efficacy due to the multivalent presentation, prolongation of tumor residence time, and targeting of the innate immune cells mediated by the VNP/VLP carrier.

Efficient Purification of Cowpea Chlorotic Mottle Virus by a Novel Peptide Aptamer.

The cowpea chlorotic mottle virus (CCMV) is a plant virus explored as a nanotechnological platform. The robust self-assembly mechanism of its capsid protein allows for drug encapsulation and targeted delivery. Additionally, the capsid nanoparticle can be used as a programmable platform to display different molecular moieties. In view of future applications, efficient production and purification of plant viruses are key steps. In established protocols, the need for ultracentrifugation is a significant limitation due to cost, difficult scalability, and safety issues. In addition, the purity of the final virus isolate often remains unclear. Here, an advanced protocol for the purification of the CCMV from infected plant tissue was developed, focusing on efficiency, economy, and final purity. The protocol involves precipitation with PEG 8000, followed by affinity extraction using a novel peptide aptamer. The efficiency of the protocol was validated using size exclusion chromatography, MALDI-TOF mass spectrometry, reversed-phase HPLC, and sandwich immunoassay. Furthermore, it was demonstrated that the final eluate of the affinity column is of exceptional purity (98.4%) determined by HPLC and detection at 220 nm. The scale-up of our proposed method seems to be straightforward, which opens the way to the large-scale production of such nanomaterials. This highly improved protocol may facilitate the use and implementation of plant viruses as nanotechnological platforms for in vitro and in vivo applications.

Modulation of Capsid Dynamics in Bromoviruses by the Host and Heterologous Viral Replicase.

The three genomic and a single subgenomic RNA of Cowpea chlorotic mottle virus (CCMV), which is pathogenic to plants, is packaged into three morphologically indistinguishable icosahedral virions with T=3 symmetry. The two virion types, C1V and C2V, package genomic RNAs 1 (C1) and 2 (C2), respectively. The third virion type, C3+4V, 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 in planta revealed that, while retaining the structural integrity, C1V and C2V virions released peptide regions encompassing the N-terminal arginine-rich RNA binding motif. In contrast, a minor population of the C3+4V 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+4V and B3+4V 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+4V and C3+4V 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. IMPORTANCE 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, Cowpea chlorotic mottle (CCMV) and Brome mosaic virus (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.

An Evolved 5' Untranslated Region of Alfalfa Mosaic Virus Allows the RNA Transport of Movement-Defective Variants.

Although the coat protein (CP) has a relevant role in the long-distance movement of alfalfa mosaic virus (AMV) and brome mosaic virus (BMV), its precise function is not fully understood. Previous results showed that a specific interaction between the C termini of the movement protein (MP) and the cognate CP is required for systemic transport. Thus, we have performed a compensatory evolution experiment using an AMV RNA3 derivative defective in long-distance transport that carries a BMV MP lacking the C-terminal 48 residues and unable to interact with the AMV CP. After several passages, five independent evolution lineages were able to move long distance. The analysis of the viral RNA of these lineages showed the presence of three different modifications located exclusively at the 5' untranslated region (5' UTR). The three evolved 5' UTR variants accumulated comparable levels of viral RNA and CP but reduced the accumulation of virus particles and the affinity between the 5' UTR and the AMV CP. In addition, the evolved 5' UTR increased cell-to-cell transport for both the AMV RNA3 carrying the BMV MP and that carrying the AMV MP. Finally, the evolved 5' UTRs allowed the systemic transport of an AMV RNA3 carrying a CP mutant defective in virus particles and increased the systemic transport of several AMV RNA3 derivatives carrying different viral MPs associated with the 30K superfamily. Altogether, our findings indicate that virus particles are not required for the systemic transport of AMV but also that BMV MP is competent for the short- and long-distance transport without the interaction with the CP. IMPORTANCE The results obtained in the present work could challenge the view of the role of the virus particle in the systemic transport of plant viruses. In this sense, we show that two different MPs are competent to systemically transport the AMV genome without the requirement of the virus particles, as reported for viruses lacking a CP (e.g., Umbravirus). The incapability of the viral MP to interact with the CP triggered virus variants that evolved to reduce the formation of virus particles, probably to increase the accessibility of the MP to the viral progeny. Our results point to the idea that virus particles would not be necessary for the viral systemic transport but would be necessary for vector virus transmission. This idea is reinforced by the observation that heterologous MPs also increased the systemic transport of the AMV constructs that have reduced encapsidation capabilities.

Single-particle studies of the effects of RNA-protein interactions on the self-assembly of RNA virus particles.

Understanding the pathways by which simple RNA viruses self-assemble from their coat proteins and RNA is of practical and fundamental interest. Although RNA-protein interactions are thought to play a critical role in the assembly, our understanding of their effects is limited because the assembly process is difficult to observe directly. We address this problem by using interferometric scattering microscopy, a sensitive optical technique with high dynamic range, to follow the in vitro assembly kinetics of more than 500 individual particles of brome mosaic virus (BMV)-for which RNA-protein interactions can be controlled by varying the ionic strength of the buffer. We find that when RNA-protein interactions are weak, BMV assembles by a nucleation-and-growth pathway in which a small cluster of RNA-bound proteins must exceed a critical size before additional proteins can bind. As the strength of RNA-protein interactions increases, the nucleation time becomes shorter and more narrowly distributed, but the time to grow a capsid after nucleation is largely unaffected. These results suggest that the nucleation rate is controlled by RNA-protein interactions, while the growth process is driven less by RNA-protein interactions and more by protein-protein interactions and intraprotein forces. The nucleated pathway observed with the plant virus BMV is strikingly similar to that previously observed with bacteriophage MS2, a phylogenetically distinct virus with a different host kingdom. These results raise the possibility that nucleated assembly pathways might be common to other RNA viruses.

A conserved viral amphipathic helix governs the replication site-specific membrane association.

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.

Dynamic stability of salt stable cowpea chlorotic mottle virus capsid protein dimers and pentamers of dimers.

Intermediates of the self-assembly process of the salt stable cowpea chlorotic mottle virus (ss-CCMV) capsid can be modelled atomistically on realistic computational timescales either by studying oligomers in equilibrium or by focusing on their dissociation instead of their association. Our previous studies showed that among the three possible dimer interfaces in the icosahedral capsid, two are thermodynamically relevant for capsid formation. The aim of the current study is to evaluate the relative structural stabilities of the three different ss-CCMV dimers and to find and understand the conditions that lead to their dissociation. Long timescale molecular dynamics simulations at 300 K of the various dimers and of the pentamer of dimers underscore the importance of large contact surfaces on stabilizing the capsid subunits within an oligomer. Simulations in implicit solvent show that at higher temperature (350 K), the N-terminal tails of the protein units act as tethers, delaying dissociation for all but the most stable interface. The pentamer of dimers is also found to be stable on long timescales at 300 K, with an inherent flexibility of the outer protein chains.

The Dynamics of Viruslike Capsid Assembly and Disassembly.

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 T = 3 and T = 1 capsids as a function of time and show that the time evolution of the conversion from one T number to another is not symmetric: The conversion from T = 1 to T = 3 is a factor of 10 slower than that of T = 3 to T = 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.

Production and characterization of chimeric SARS-CoV-2 antigens based on the capsid protein of cowpea chlorotic mottle virus.

The COVID-19 pandemic has highlighted the need for new vaccine platforms to rapidly develop solutions against emerging pathogens. In particular, some plant viruses offer several advantages for developing subunit vaccines, such as high expression rates in E. coli, high immunogenicity and safety, and absence of pre-immunity that could interfere with the vaccine's efficacy. Cowpea chlorotic mottle virus (CCMV) is a model system that has been extensively characterized, with key advantages for its use as an epitope carrier. In the present study, three relevant epitopes from the SARS-CoV-2 Spike protein were genetically inserted into the CCMV CP and expressed in E. coli cultures, resulting in the CCMV1, CCMV2, and CCMV3 chimeras. The recombinant CP mutants were purified from the formed inclusion bodies and refolded, and their immunogenicity as a subunit vaccine was assessed in BALB/c mice. The three mutants are immunogenic as they induce high IgG antibody titers that recognize the recombinant full-length S protein. This study supports the application of CCMV CP as an attractive carrier for the clinical evaluation of vaccine candidates against SARS-CoV-2. Furthermore, it suggests that VLPs assembled from these chimeric proteins could result in antigens with better immunogenicity.