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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 -
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 -
Nature Communications Sep 2023Observing proteins as they perform their tasks has largely remained elusive, which has left our understanding of protein function fundamentally incomplete. To enable...
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.
Topics: Cryoelectron Microscopy; Bromovirus; Capsid; Capsid Proteins; Motion
PubMed: 37704664
DOI: 10.1038/s41467-023-41444-x -
Viruses Aug 2023Ecological strategies for resource utilisation are important features of pathogens, yet have been overshadowed by stronger interest in genetic mechanisms underlying...
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.
Topics: Ecosystem; Bromovirus; Genetic Drift; High-Throughput Nucleotide Sequencing; Host Specificity
PubMed: 37632121
DOI: 10.3390/v15081779 -
Viruses Mar 2023The 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...
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.
Topics: Bromovirus; Aptamers, Peptide; Capsid Proteins; Capsid; Nanoparticles
PubMed: 36992405
DOI: 10.3390/v15030697 -
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 -
Molecular Plant Pathology Feb 2023Infection of viruses from the genera Bromovirus, Potyvirus, and Potexvirus in Nicotiana benthamiana induces significant up-regulation of the genes that encode the HSP70...
Infection of viruses from the genera Bromovirus, Potyvirus, and Potexvirus in Nicotiana benthamiana induces significant up-regulation of the genes that encode the HSP70 family, including binding immunoglobulin protein 2 (BiP2). Three up-regulated genes were knocked down and infection assays with these knockdown lines demonstrated the importance of the BiP2 gene for potyvirus infection but not for infection by the other tested viruses. Distinct symptoms of cucumber mosaic virus (CMV) and potato virus X (PVX) were observed in the BiP2 knockdown line at 10 days postagroinfiltration. Interestingly, following inoculation with either soybean mosaic virus (SMV) or pepper mottle virus (PepMoV) co-expressing green fluorescent protein (GFP), neither crinkle symptoms nor GFP signals were observed in the BiP2 knockdown line. Subsequent reverse transcription-quantitative PCR analysis demonstrated that knockdown of BiP2 resulted in a significant decrease of SMV and PepMoV RNA accumulation but not PVX or CMV RNA accumulation. Further yeast two-hybrid and co-immunoprecipitation analyses validated the interaction between BiP2 and nuclear inclusion protein b (NIb) of SMV. Together, our findings suggest the crucial role of BiP2 as a proviral host factor necessary for potyvirus infection. The interaction between BiP2 and NIb may be the critical factor determining susceptibility in N. benthamiana, but further studies are needed to elucidate the underlying mechanism.
Topics: Nicotiana; Proviruses; RNA; Potyvirus; Cytomegalovirus Infections; Plant Diseases
PubMed: 36416097
DOI: 10.1111/mpp.13284 -
Journal of Virology Nov 2022Although 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...
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. 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., ). 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.
Topics: 5' Untranslated Regions; Alfalfa mosaic virus; Bromovirus; RNA Transport; RNA, Viral; Plant Viral Movement Proteins
PubMed: 36314818
DOI: 10.1128/jvi.00988-22 -
Proceedings of the National Academy of... Sep 2022Understanding the pathways by which simple RNA viruses self-assemble from their coat proteins and RNA is of practical and fundamental interest. Although RNA-protein...
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.
Topics: Bromovirus; Capsid; RNA Viruses; RNA, Viral; Virion
PubMed: 36122222
DOI: 10.1073/pnas.2206292119 -
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