Thermal remodelling of Alternanthera mosaic virus virions and virus-like particles into protein spherical particles

The present work addresses the thermal remodelling of flexible plant viruses with a helical structure and virus-like particles (VLPs). Here, for the first time, the possibility of filamentous Alternanthera mosaic virus (AltMV) virions’ thermal transition into structurally modified spherical particles (SP) has been demonstrated. The work has established differences in formation conditions of SP from virions (SPV) and VLPs (SPVLP) that are in accordance with structural data (on AltMV virions and VLPs). SP originate from AltMV virions through an intermediate stage. However, the same intermediate stage was not detected during AltMV VLPs’ structural remodelling. According to the biochemical analysis, AltMV SPV consist of protein and do not include RNA. The structural characterisation of AltMV SPV/VLP by circular dichroism, intrinsic fluorescence spectroscopy and thioflavin T fluorescence assay has been performed. AltMV SPV/VLP adsorption properties and the availability of chemically reactive surface amino acids have been analysed. The revealed characteristics of AltMV SPV/VLP indicate that they could be applied as protein platforms for target molecules presentation and for the design of functionally active complexes.

Download Full-text

Encapsidation of DNA, a protein and a fluorophore into virus-like particles by the capsid protein of cucumber mosaic virus

Journal of General Virology ◽

10.1099/vir.0.040170-0 ◽

2012 ◽

Vol 93 (5) ◽

pp. 1120-1126 ◽

Cited By ~ 20

Author(s):

Xiaoyun Lu ◽

Jeremy R. Thompson ◽

Keith L. Perry

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Capsid Protein ◽

Plant Viruses ◽

Insect Vector ◽

Virus Like Particles ◽

Protein Cage ◽

Transmission Electron ◽

Lower Size

An important property of some spherical plant viruses is their ability to reassemble in vitro from native capsid protein (CP) and RNA into infectious virus-like particles (VLPs). Virions of cucumber mosaic virus (CMV) are stabilized by protein–RNA interactions and the nucleic acid is essential for assembly. This study demonstrated that VLPs will form in the presence of both ssDNA and dsDNA oligonucleotides, and with a lower size limit of 20 nt. Based on urea disruption assays, assembled VLPs from CMV CP and RNA (termed ReCMV) exhibited a level of stability similar to that of virions purified from plants, whilst VLPs from CMV CP and a 20mer exhibited comparable or greater stability. Fluorescent labelling of VLPs was achieved by the encapsidation of an Alexa Fluor 488-labelled 45mer oligonucleotide (ReCMV-Alexa488-45) and confirmed by transmission electron and confocal microscopy. Using ssDNA as a nucleating factor, encapsidation of fluorescently labelled streptavidin (53 kDa) conjugated to a biotinylated oligonucleotide was observed. The biological activity and stability of ReCMV and ReCMV-Alexa488-45 was confirmed in infectivity assays and insect vector feeding assays. This work demonstrates the utility of CMV CP as a protein cage for use in the growing repertoire of nanotechnological applications.

Download Full-text

Further studies on the ultrafiltration of plant viruses

Parasitology ◽

10.1017/s0031182000024513 ◽

1941 ◽

Vol 33 (3) ◽

pp. 320-330 ◽

Cited By ~ 5

Author(s):

Kenneth M. Smith ◽

W. D. MacClement

Keyword(s):

Mosaic Virus ◽

Particle Diameter ◽

Potato Virus X ◽

Great Difficulty ◽

Plant Viruses ◽

Spherical Particles ◽

Tobacco Necrosis Virus ◽

Necrosis Virus ◽

End Point ◽

Consistent Manner

An account is given of ultrafiltration studies with 9 plant viruses. It is shown that 3 of these viruses filter in a consistent manner and appear to have approximately spherical particles. These three are Lycopersicum virus 4 (tomato bushy stunt virus), Nicotiana virus 11 (tobacco necrosis virus) and Nicotiana virus 12 (tobacco ringspot virus). The filtration end-point of 40 mμ is the same in each case and from this a particle diameter of 13–20 mμ is calculated. There is a peculiarity, however, in the filtration curve of tobacco necrosis virus which shows itself in a “bench” or “shelf” and which suggests either a polydisperse system or some degree of dissymmetry of particle shape.Great difficulty was experienced in filtering Nicotiana virus 1 (tobacco mosaic virus) and its strains. A value of 13–20 mμ was obtained for the particle diameter of the type virus and this agrees well with measurements obtained by other methods. The filtration results, however, suggest that the infective units are not of the same length and that this variability may be considerable. Similar difficulty was experienced in filtering Solanum virus 1 (potato virus X), another rod-shaped virus; the end-point was found to be 100 mμ, from which a particle diameter of 33–50 mμ is calculated. It was not possible to obtain a definite filtration end-point for Cucumis virus 1 (cucumber mosaic virus), probably because of the low initial concentration of virus in the extracted sap.

Download Full-text

Cowpea Mosaic Virus Nanoparticles and Empty Virus-Like Particles Show Distinct but Overlapping Immunostimulatory Properties

Journal of Virology ◽

10.1128/jvi.00129-19 ◽

2019 ◽

Vol 93 (21) ◽

Cited By ~ 12

Author(s):

Chao Wang ◽

Veronique Beiss ◽

Nicole F. Steinmetz

Keyword(s):

Mosaic Virus ◽

Tumor Regression ◽

Expression System ◽

Plant Viruses ◽

Cowpea Mosaic Virus ◽

Wild Type ◽

Virus Like Particles ◽

Virus Rna ◽

Tumor Mouse Model ◽

Cell Expression

ABSTRACT Cowpea mosaic virus (CPMV) is a plant virus that has been developed for multiple biomedical and nanotechnology applications, including immunotherapy. Two key platforms are available: virus nanoparticles (VNPs) based on the complete CMPV virion, including the genomic RNA, and virus-like nanoparticles (VLPs) based on the empty CPMV (eCPMV) virion. It is unclear whether these platforms differ in terms of immunotherapeutic potential. We therefore compared their physicochemical properties and immunomodulatory activities following in situ vaccination of an aggressive ovarian tumor mouse model (ID8-Defb29/Vegf-A). In physicochemical terms, CPMV and eCPMV were very similar, and both significantly increased the survival of tumor-bearing mice and showed promising antitumor efficacy. However, they demonstrated distinct yet overlapping immunostimulatory effects due to the presence of virus RNA in wild-type particles, indicating their suitability for different immunotherapeutic strategies. Specifically, we found that the formulations had similar effects on most secreted cytokines and immune cells, but the RNA-containing CPMV particles were uniquely able to boost populations of potent antigen-presenting cells, such as tumor-infiltrating neutrophils and activated dendritic cells. Our results will facilitate the development of CPMV and eCPMV as immunotherapeutic vaccine platforms with tailored responses. IMPORTANCE The engagement of antiviral effector responses caused by viral infection is essential when using viruses or virus-like particles (VLPs) as an immunotherapeutic agent. Here, we compare the chemophysical and immunostimulatory properties of wild-type cowpea mosaic virus (CPMV) (RNA containing) and eCPMV (RNA-free VLPs) produced from two expression systems (agrobacterium-based plant expression system and baculovirus-insect cell expression). CPMV and eCPMV could each be developed as novel adjuvants to overcome immunosuppression and thus promote tumor regression in ovarian cancer (and other tumor types). To our knowledge, this is the first study to define the immunotherapeutic differences between CPMV and eCPMV, which is essential for the further development of biomedical applications for plant viruses and the selection of rational combinations of immunomodulatory reagents.

Download Full-text

Identification of Tomato Infecting Viruses That Co-Isolate with Nanovesicles Using a Combined Proteomics and Electron-Microscopic Approach

Nanomaterials ◽

10.3390/nano11081922 ◽

2021 ◽

Vol 11 (8) ◽

pp. 1922

Author(s):

Ramila Mammadova ◽

Immacolata Fiume ◽

Ramesh Bokka ◽

Veronika Kralj-Iglič ◽

Darja Božič ◽

...

Keyword(s):

Electron Microscopy ◽

Mosaic Virus ◽

Protein Identification ◽

Plant Viruses ◽

Size Exclusion ◽

Tomato Mosaic Virus ◽

High Yield ◽

Plant Resources ◽

Electron Microscopic ◽

Viral Particles

Plant-derived nanovesicles (NVs) have attracted interest due to their anti-inflammatory, anticancer and antioxidative properties and their efficient uptake by human intestinal epithelial cells. Previously we showed that tomato (Solanum lycopersicum L.) fruit is one of the interesting plant resources from which NVs can be obtained at a high yield. In the course of the isolation of NVs from different batches of tomatoes, using the established differential ultracentrifugation or size-exclusion chromatography methods, we occasionally observed the co-isolation of viral particles. Density gradient ultracentrifugation (gUC), using sucrose or iodixanol gradient materials, turned out to be efficient in the separation of NVs from the viral particles. We applied cryogenic transmission electron microscopy (cryo-TEM), scanning electron microscopy (SEM) for the morphological assessment and LC–MS/MS-based proteomics for the protein identification of the gradient fractions. Cryo-TEM showed that a low-density gUC fraction was enriched in membrane-enclosed NVs, while the high-density fractions were rich in rod-shaped objects. Mass spectrometry–based proteomic analysis identified capsid proteins of tomato brown rugose fruit virus, tomato mosaic virus and tomato mottle mosaic virus. In another batch of tomatoes, we isolated tomato spotted wilt virus, potato virus Y and southern tomato virus in the vesicle sample. Our results show the frequent co-isolation of plant viruses with NVs and the utility of the combination of cryo-TEM, SEM and proteomics in the detection of possible viral contamination.

Download Full-text

Towards plant resistance to viruses using protein-only RNase P

Nature Communications ◽

10.1038/s41467-021-21338-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Anthony Gobert ◽

Yifat Quan ◽

Mathilde Arrivé ◽

Florent Waltz ◽

Nathalie Da Silva ◽

...

Keyword(s):

Mosaic Virus ◽

Virus Replication ◽

Yield Loss ◽

Rnase P ◽

Plant Viruses ◽

Yellow Mosaic Virus ◽

Resistance To Viruses ◽

Target Plant

AbstractPlant viruses cause massive crop yield loss worldwide. Most plant viruses are RNA viruses, many of which contain a functional tRNA-like structure. RNase P has the enzymatic activity to catalyze the 5′ maturation of precursor tRNAs. It is also able to cleave tRNA-like structures. However, RNase P enzymes only accumulate in the nucleus, mitochondria, and chloroplasts rather than cytosol where virus replication takes place. Here, we report a biotechnology strategy based on the re-localization of plant protein-only RNase P to the cytosol (CytoRP) to target plant viruses tRNA-like structures and thus hamper virus replication. We demonstrate the cytosol localization of protein-only RNase P in Arabidopsis protoplasts. In addition, we provide in vitro evidences for CytoRP to cleave turnip yellow mosaic virus and oilseed rape mosaic virus. However, we observe varied in vivo results. The possible reasons have been discussed. Overall, the results provided here show the potential of using CytoRP for combating some plant viral diseases.

Download Full-text

Virus-Like Particles Produced Using the Brome Mosaic Virus Recombinant Capsid Protein Expressed in a Bacterial System

International Journal of Molecular Sciences ◽

10.3390/ijms22063098 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3098

Author(s):

Aleksander Strugała ◽

Jakub Jagielski ◽

Karol Kamel ◽

Grzegorz Nowaczyk ◽

Marcin Radom ◽

...

Keyword(s):

Ionic Strength ◽

Mosaic Virus ◽

Capsid Protein ◽

Self Assembly ◽

Environmental Changes ◽

Expression System ◽

Brome Mosaic Virus ◽

Virus Like Particles ◽

Assembly Principles ◽

The Impact

Virus-like particles (VLPs), due to their nanoscale dimensions, presence of interior cavities, self-organization abilities and responsiveness to environmental changes, are of interest in the field of nanotechnology. Nevertheless, comprehensive knowledge of VLP self-assembly principles is incomplete. VLP formation is governed by two types of interactions: protein–cargo and protein–protein. These interactions can be modulated by the physicochemical properties of the surroundings. Here, we used brome mosaic virus (BMV) capsid protein produced in an E. coli expression system to study the impact of ionic strength, pH and encapsulated cargo on the assembly of VLPs and their features. We showed that empty VLP assembly strongly depends on pH whereas ionic strength of the buffer plays secondary but significant role. Comparison of VLPs containing tRNA and polystyrene sulfonic acid (PSS) revealed that the structured tRNA profoundly increases VLPs stability. We also designed and produced mutated BMV capsid proteins that formed VLPs showing altered diameters and stability compared to VLPs composed of unmodified proteins. We also observed that VLPs containing unstructured polyelectrolyte (PSS) adopt compact but not necessarily more stable structures. Thus, our methodology of VLP production allows for obtaining different VLP variants and their adjustment to the incorporated cargo.

Download Full-text

Genome-Wide Identification and Expression Analysis of the Histone Deacetylase Gene Family in Wheat (Triticum aestivum L.)

Plants ◽

10.3390/plants10010019 ◽

2020 ◽

Vol 10 (1) ◽

pp. 19

Author(s):

Peng Jin ◽

Shiqi Gao ◽

Long He ◽

Miaoze Xu ◽

Tianye Zhang ◽

...

Keyword(s):

Abiotic Stress ◽

Gene Family ◽

Mosaic Virus ◽

Stress Responses ◽

Viral Infections ◽

Phylogenetic Analyses ◽

Plant Viruses ◽

Gene Family Evolution ◽

Yellow Mosaic Virus ◽

Wheat Yellow Mosaic Virus

Histone acetylation is a dynamic modification process co-regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Although HDACs play vital roles in abiotic or biotic stress responses, their members in Triticumaestivum and their response to plant viruses remain unknown. Here, we identified and characterized 49 T. aestivumHDACs (TaHDACs) at the whole-genome level. Based on phylogenetic analyses, TaHDACs could be divided into 5 clades, and their protein spatial structure was integral and conserved. Chromosomal location and synteny analyses showed that TaHDACs were widely distributed on wheat chromosomes, and gene duplication has accelerated the TaHDAC gene family evolution. The cis-acting element analysis indicated that TaHDACs were involved in hormone response, light response, abiotic stress, growth, and development. Heatmaps analysis of RNA-sequencing data showed that TaHDAC genes were involved in biotic or abiotic stress response. Selected TaHDACs were differentially expressed in diverse tissues or under varying temperature conditions. All selected TaHDACs were significantly upregulated following infection with the barley stripe mosaic virus (BSMV), Chinese wheat mosaic virus (CWMV), and wheat yellow mosaic virus (WYMV), suggesting their involvement in response to viral infections. Furthermore, TaSRT1-silenced contributed to increasing wheat resistance against CWMV infection. In summary, these findings could help deepen the understanding of the structure and characteristics of the HDAC gene family in wheat and lay the foundation for exploring the function of TaHDACs in plants resistant to viral infections.

Download Full-text

Chloroplast Hsp70 Isoform Is Required for Age-Dependent Tissue Preference of Bamboo mosaic virus in Mature Nicotiana benthamiana Leaves

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-01-17-0012-r ◽

2017 ◽

Vol 30 (8) ◽

pp. 631-645 ◽

Cited By ~ 3

Author(s):

Ying Wen Huang ◽

Chung Chi Hu ◽

Ching Hsiu Tsai ◽

Na Sheng Lin ◽

Yau Heiu Hsu

Keyword(s):

Mosaic Virus ◽

Nicotiana Benthamiana ◽

Transit Peptide ◽

Plant Viruses ◽

Age Dependent ◽

Efficient Replication ◽

Underlying Mechanisms ◽

Lines Of Evidence ◽

Suitable Environment

Plant viruses may exhibit age-dependent tissue preference in their hosts but the underlying mechanisms are not well understood. In this study, we provide several lines of evidence to reveal the determining role of a protein of the Nicotiana benthamiana chloroplast Hsp70 (NbcpHsp70) family, NbcpHsp70-2, involved in the preference of Bamboo mosaic virus (BaMV) to infect older tissues. NbcpHsp70 family proteins were identified in complexes pulled down with BaMV replicase as the bait. Among the isoforms of NbcpHsp70, only the specific silencing of NbcpHsp70-2 resulted in the significant decrease of BaMV RNA in N. benthamiana protopalsts, indicating that NbcpHsp70-2 is involved in the efficient replication of BaMV RNA. We further identified the age-dependent import regulation signal contained in the transit peptide of NbcpHsp70-2. Deletion, overexpression, and substitution experiments revealed that the signal in the transit peptide of NbcpHsp70-2 is crucial for both the import of NbcpHsp70-2 into older chloroplasts and the preference of BaMV for infecting older leaves of N. benthamiana. Together, these data demonstrated that BaMV may exploit a cellular age-dependent transportation mechanism to target a suitable environment for viral replication.

Download Full-text