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.

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.

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.

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.

Ultrafast Collective Excited-State Dynamics of a Virus-Supported Fluorophore Antenna.

Radiation brightening was recently observed in a multifluorophore-conjugated brome mosaic virus (BMV) particle at room temperature under pulsed excitation. On the basis of its nonlinear dependence on the number of chromophores, the origins of the phenomenon were attributed to a collective relaxation. However, the mechanism remains unknown. We present ultrafast transient absorption and fluorescence spectroscopic studies which shed new light on the collective nature of the relaxation dynamics in such radiation-brightened, multifluorophore particles. Our findings indicate that the emission dynamics is consistent with a superradiance mechanism. The ratio between the rates of competing radiative and nonradiative relaxation pathways depends on the number of chromophores per virus. The findings suggest that small icosahedral virus shells provide a unique biological scaffold for developing nonclassical, deep subwavelength light sources and may open new avenues for the development of photonic probes for medical imaging applications.

Virus-Induced Gene Silencing in Sorghum Using Brome Mosaic Virus.

Sorghum [Sorghum bicolor (L.) Moench.] is a versatile crop, grown in 30 countries and a food source for nearly 500 million people globally. Although the sorghum genome is sequenced, a limited understanding of gene function prevents the improvement of resistance against almost 150 species of viruses, bacteria, fungus, and parasitic plants to improve productivity. Here, we present a Brome mosaic virus (BMV)-based virus-induced gene silencing (VIGS) to silence target genes for functional study in sorghum. This protocol achieves 100% sorghum infection with BMV by growing the plants at 18 °C instead of 22 °C. Using this method, one can achieve gene silencing in sorghum up to 100% of the inoculated plants.

Cryo-EM reconstructions of BMV-derived virus-like particles reveal assembly defects in the icosahedral lattice structure.

The increasing interest in virus-like particles (VLPs) has been reflected by the growing number of studies on their assembly and application. However, the formation of complete VLPs is a complex phenomenon, making it difficult to rationally design VLPs with desired features de novo. In this paper, we describe VLPs assembled in vitro from the recombinant capsid protein of brome mosaic virus (BMV). The analysis of VLPs was performed by Cryo-EM reconstructions and allowed us to visualize a few classes of VLPs, giving insight into the VLP self-assembly process. Apart from the mature icosahedral VLP practically identical with native virions, we describe putative VLP intermediates displaying non-icosahedral arrangements of capsomers, proposed to occur before the final disorder-order transition stage of icosahedral VLP assembly. Some of the described VLP classes show a lack of protein shell continuity, possibly resulting from too strong interaction with the cargo (in this case tRNA) with the capsid protein. We believe that our results are a useful prerequisite for the rational design of VLPs in the future and lead the way to the effective production of modified VLPs.

Hydrophobic Cargo Encapsulation into Virus Protein Cages by Self-Assembly in an Aprotic Organic Solvent.

While extensive studies of virus capsid assembly in environments mimicking in vivo conditions have led to an understanding of the thermodynamic driving forces at work, applying this knowledge to virus assembly in other solvents than aqueous buffers has not been attempted yet. In this study, Brome mosaic virus (BMV) capsid proteins were shown to preserve their self-assembly abilities in an aprotic polar solvent, dimethyl sulfoxide (DMSO). This facilitated protein cage encapsulation of nanoparticles and dye molecules that favor organic solvents, such as ß-NaYF4-based upconversion nanoparticles and BODIPY dye. Assembly was found to be robust relative to a surprisingly broad range of DMSO concentrations. Cargos with poor initial stability in aqueous solutions were readily encapsulated at high DMSO concentrations and then transferred to aqueous solvents, where they remained stable and preserved their function for months.

A viral RNA hijacks host machinery using dynamic conformational changes of a tRNA-like structure.

Viruses require multifunctional structured RNAs to hijack their host's biochemistry, but their mechanisms can be obscured by the difficulty of solving conformationally dynamic RNA structures. Using cryo­electron microscopy (cryo-EM), we visualized the structure of the mysterious viral transfer RNA (tRNA)­like structure (TLS) from the brome mosaic virus, which affects replication, translation, and genome encapsidation. Structures in isolation and those bound to tyrosyl-tRNA synthetase (TyrRS) show that this ~55-kilodalton purported tRNA mimic undergoes large conformational rearrangements to bind TyrRS in a form that differs substantially from that of tRNA. Our study reveals how viral RNAs can use a combination of static and dynamic RNA structures to bind host machinery through highly noncanonical interactions, and we highlight the utility of cryo-EM for visualizing small, conformationally dynamic structured RNAs.