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The interest in plant-derived virus-like particles (pVLPs) for the design of a new generation of nanocarriers is based on their lack of infection for humans, their immunostimulatory properties to fight cancer cells, and their capability to contain and release cargo molecules. Asparaginase (ASNase) is an FDA-approved drug to treat acute lymphoblastic leukemia (LLA); however, it exhibits high immunogenicity which often leads to discontinuation of treatment. In previous work, we encapsulated ASNase into bacteriophage P22-based VLPs through genetic-directed design to form the ASNase-P22 nanobioreactors. In this work, a commercial ASNase was encapsulated into brome mosaic virus-like particles (BMV-VLPs) to form stable ASNase-BMV nanobioreactors. According to our results, we observed that ASNase-BMV nanobioreactors had similar cytotoxicity against MOLT-4 and Reh cells as the commercial drug. In vivo assays showed a higher specific anti-ASNase IgG response in BALB/c mice immunized with ASNase encapsulated into BMV-VLPs compared with those immunized with free ASNase. Nevertheless, we also detected a high and specific IgG response against BMV capsids on both ASNase-filled capsids (ASNase-BMV) and empty BMV capsids. Despite the fact that our in vivo studies showed that the BMV-VLPs stimulate the immune response either empty or with cargo proteins, the specific cytotoxicity against leukemic cells allows us to propose ASNase-BMV as a potential novel formulation for LLA treatment where in vitro and in vivo evidence of functionality is provided. [ABSTRACT FROM AUTHOR]

Wheat breeders are developing new virus-resistant varieties; however, it is assumed that only a few viruses or well-known viruses are present in the field. New sequencing technology is allowing for better determination of natural field virus populations. For three years, 2019-2021, Kansas wheat field surveys were conducted to determine the constituents of natural field virus populations using nanopore sequencing. During analysis, brome mosaic virus (BMV) was identified for the first time in Kansas but was in association with other wheat viruses. Brome mosaic virus was identified from 29 out of 47 different Kansas counties sampled and 44% of the total samples. BMV was found co-infected with wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) in 27.8% of the samples, with WSMV only (13.9%) and co-infected with WSMV + TriMV + High Plains wheat mosaic emaravirus (HPWMoV) (13.9%). RNA genomes of Kansas BMV isolates had 99.4 to 100% nucleotide and amino acid sequence identity, respectively, to each other. RNA2a possessed relatively high divergence (p = 0.01) compared to RNA1a and RNA3a (p = 0.004). Coding regions of all BMV RNAs were considered negative for purifying selection pressure as nonsynonymous and synonymous nucleotide ratio was less than one (dNs/dS >1). The identification of BMV in Kansas virus populations adds another layer of complexity to plant breeding. This work provides information to improve tools to aid in monitoring, detecting, and determining the variation within BMV. [ABSTRACT FROM AUTHOR]

Wheat breeders are developing new virus-resistant varieties; however, it is assumed that only a few viruses or well-known viruses are present in the field. New sequencing technology is allowing for better determination of natural field virus populations. For three years, 2019-2021, Kansas wheat field surveys were conducted to determine the constituents of natural field virus populations using nanopore sequencing. During analysis, brome mosaic virus (BMV) was identified for the first time in Kansas but was in association with other wheat viruses. Brome mosaic virus was identified from 29 out of 47 different Kansas counties sampled and 44% of the total samples. BMV was found co-infected with wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) in 27.8% of the samples, with WSMV only (13.9%) and co-infected with WSMV + TriMV + High Plains wheat mosaic emaravirus (HPWMoV) (13.9%). RNA genomes of Kansas BMV isolates had 99.4 to 100% nucleotide and amino acid sequence identity, respectively, to each other. RNA2a possessed relatively high divergence (π = 0.01) compared to RNA1a and RNA3a (π = 0.004). Coding regions of all BMV RNAs were considered negative for purifying selection pressure as nonsynonymous and synonymous nucleotide ratio was less than one (dNs/dS >1). The identification of BMV in Kansas virus populations adds another layer of complexity to plant breeding. This work provides information to improve tools to aid in monitoring, detecting, and determining the variation within BMV.

The interest in plant-derived virus-like particles (pVLPs) for the design of a new generation of nanocarriers is based on their lack of infection for humans, their immunostimulatory properties to fight cancer cells, and their capability to contain and release cargo molecules. Asparaginase (ASNase) is an FDA-approved drug to treat acute lymphoblastic leukemia (LLA); however, it exhibits high immunogenicity which often leads to discontinuation of treatment. In previous work, we encapsulated ASNase into bacteriophage P22-based VLPs through genetic-directed design to form the ASNase-P22 nanobioreactors. In this work, a commercial ASNase was encapsulated into brome mosaic virus-like particles (BMV-VLPs) to form stable ASNase-BMV nanobioreactors. According to our results, we observed that ASNase-BMV nanobioreactors had similar cytotoxicity against MOLT-4 and Reh cells as the commercial drug. In vivo assays showed a higher specific anti-ASNase IgG response in BALB/c mice immunized with ASNase encapsulated into BMV-VLPs compared with those immunized with free ASNase. Nevertheless, we also detected a high and specific IgG response against BMV capsids on both ASNase-filled capsids (ASNase-BMV) and empty BMV capsids. Despite the fact that our in vivo studies showed that the BMV-VLPs stimulate the immune response either empty or with cargo proteins, the specific cytotoxicity against leukemic cells allows us to propose ASNase-BMV as a potential novel formulation for LLA treatment where in vitro and in vivo evidence of functionality is provided.

Field-grown wheat (Triticum aestivum L.) plants can be co-infected by multiple viruses, including wheat streak mosaic virus (WSMV), Triticum mosaic virus (TriMV), brome mosaic virus (BMV), and barley stripe mosaic virus (BSMV). These viruses belong to four different genera in three different families and are, hence, genetically divergent. However, the impact of potential co-infections with two, three, or all four of them on the viruses themselves, as well as the wheat host, has yet to be examined. This study examined bi-, tri-, and quadripartite interactions among these viruses in wheat for disease development and accumulation of viral genomic RNAs, in comparison with single virus infections. Co-infection of wheat by BMV and BSMV resulted in BMV-like symptoms with a drastic reduction in BSMV genomic RNA copies and coat protein accumulation, suggesting an antagonism-like effect exerted by BMV toward BSMV. However, co-infection of either BMV or BSMV with WSMV or TriMV led to more severe disease than singly infected wheat, but with a decrease or no significant change in titers of interacting viruses in the presence of BMV or BSMV, respectively. These results were in stark contrast with exacerbated disease phenotype accompanied with enhanced virus titers caused by WSMV and TriMV co-infection. Co-infection of wheat by WSMV, TriMV, and BMV or BSMV resulted in enhanced synergistic disease accompanied by increased accumulation of TriMV and BMV but not WSMV or BSMV. Quadripartite interactions in co-infected wheat by all four viruses resulted in very severe disease synergism, leading to the death of the most infected plants, but paradoxically, a drastic reduction in BSMV titer. Our results indicate that interactions among different viruses infecting the same plant host are more complex than previously thought, do not always entail increases in virus titers, and likely involve multiple mechanisms. These findings lay the foundation for additional mechanistic dissections of synergistic interactions among unrelated plant viruses. [ABSTRACT FROM AUTHOR]

Field-grown wheat (Triticum aestivum L.) plants can be co-infected by multiple viruses, including wheat streak mosaic virus (WSMV), Triticum mosaic virus (TriMV), brome mosaic virus (BMV), and barley stripe mosaic virus (BSMV). These viruses belong to four different genera in three different families and are, hence, genetically divergent. However, the impact of potential co-infections with two, three, or all four of them on the viruses themselves, as well as the wheat host, has yet to be examined. This study examined bi-, tri-, and quadripartite interactions among these viruses in wheat for disease development and accumulation of viral genomic RNAs, in comparison with single virus infections. Co-infection of wheat by BMV and BSMV resulted in BMV-like symptoms with a drastic reduction in BSMV genomic RNA copies and coat protein accumulation, suggesting an antagonism-like effect exerted by BMV toward BSMV. However, co-infection of either BMV or BSMV with WSMV or TriMV led to more severe disease than singly infected wheat, but with a decrease or no significant change in titers of interacting viruses in the presence of BMV or BSMV, respectively. These results were in stark contrast with exacerbated disease phenotype accompanied with enhanced virus titers caused by WSMV and TriMV co-infection. Co-infection of wheat by WSMV, TriMV, and BMV or BSMV resulted in enhanced synergistic disease accompanied by increased accumulation of TriMV and BMV but not WSMV or BSMV. Quadripartite interactions in co-infected wheat by all four viruses resulted in very severe disease synergism, leading to the death of the most infected plants, but paradoxically, a drastic reduction in BSMV titer. Our results indicate that interactions among different viruses infecting the same plant host are more complex than previously thought, do not always entail increases in virus titers, and likely involve multiple mechanisms. These findings lay the foundation for additional mechanistic dissections of synergistic interactions among unrelated plant viruses.

Virus-induced gene silencing (VIGS) is a rapid and powerful method to evaluate gene function, especially for species like hexaploid wheat that have large, redundant genomes and are difficult and time-consuming to transform. The Brome mosaic virus (BMV)-based VIGS vector is widely used in monocotyledonous species but not wheat. Here we report the establishment of a simple and effective VIGS procedure in bread wheat using BMVCP5, the most recently improved BMV silencing vector, and wheat genes PHYTOENE DESATURASE (TaPDS) and PHOSPHATE2 (TaPHO2) as targets. Time-course experiments revealed that smaller inserts (~100 nucleotides, nt) were more stable in BMVCP5 and conferred higher silencing efficiency and longer silencing duration, compared with larger inserts. When using a 100-nt insert and a novel coleoptile inoculation method, BMVCP5 induced extensive silencing of TaPDS transcript and a visible bleaching phenotype in the 2nd to 5th systemically-infected leaves from nine to at least 28 days post inoculation (dpi). For TaPHO2, the ability of BMVCP5 to simultaneously silence all three homoeologs was demonstrated. To investigate the feasibility of BMV VIGS in wheat roots, ectopically expressed enhanced GREEN FLUORESCENT PROTEIN (eGFP) in a transgenic wheat line was targeted for silencing. Silencing of eGFP fluorescence was observed in both the maturation and elongation zones of roots. BMVCP5 mediated significant silencing of eGFP and TaPHO2 mRNA expression in roots at 14 and 21 dpi, and TaPHO2 silencing led to the doubling of inorganic phosphate concentration in the 2nd through 4th systemic leaves. All 54 wheat cultivars screened were susceptible to BMV infection. BMVCP5-mediated TaPDS silencing resulted in the expected bleaching phenotype in all eight cultivars examined, and decreased TaPDS transcript was detected in all three cultivars examined. This BMVCP5 VIGS technology may serve as a rapid and effective functional genomics tool for high-throughput gene function studies in aerial and root tissues and in many wheat cultivars.

Virus-induced gene silencing (VIGS) is a rapid and powerful method to evaluate gene function, especially for species like hexaploid wheat that have large, redundant genomes and are difficult and time-consuming to transform. The Brome mosaic virus (BMV)-based VIGS vector is widely used in monocotyledonous species but not wheat. Here we report the establishment of a simple and effective VIGS procedure in bread wheat using BMVCP5, the most recently improved BMV silencing vector, and wheat genes PHYTOENE DESATURASE (TaPDS) and PHOSPHATE2 (TaPHO2) as targets. Time-course experiments revealed that smaller inserts (~100 nucleotides, nt) were more stable in BMVCP5 and conferred higher silencing efficiency and longer silencing duration, compared with larger inserts. When using a 100-nt insert and a novel coleoptile inoculation method, BMVCP5 induced extensive silencing of TaPDS transcript and a visible bleaching phenotype in the 2nd to 5th systemically-infected leaves from nine to at least 28 days post inoculation (dpi). For TaPHO2 , the ability of BMVCP5 to simultaneously silence all three homoeologs was demonstrated. To investigate the feasibility of BMV VIGS in wheat roots, ectopically expressed enhanced GREEN FLUORESCENT PROTEIN (eGFP) in a transgenic wheat line was targeted for silencing. Silencing of eGFP fluorescence was observed in both the maturation and elongation zones of roots. BMVCP5 mediated significant silencing of eGFP and TaPHO2 mRNA expression in roots at 14 and 21 dpi, and TaPHO2 silencing led to the doubling of inorganic phosphate concentration in the 2nd through 4th systemic leaves. All 54 wheat cultivars screened were susceptible to BMV infection. BMVCP5-mediated TaPDS silencing resulted in the expected bleaching phenotype in all eight cultivars examined, and decreased TaPDS transcript was detected in all three cultivars examined. This BMVCP5 VIGS technology may serve as a rapid and effective functional genomics tool for high-throughput gene function studies in aerial and root tissues and in many wheat cultivars. [ABSTRACT FROM AUTHOR]

Ovarian cancer is the most lethal malignancy affecting the female reproductive system. Identification and removal of all ovarian intraperitoneal tumor deposits during the intraoperative surgery is important towards preventing cancer recurrence and ultimately improving patient survival. Herein, we investigate the effectiveness of virus mimicking nanoparticles, derived from genome-depleted plant-infecting brome mosaic virus, and doped with near infrared (NIR) brominated cyanine dye BrCy106-NHS, for targeted NIR fluorescence imaging of intraperitoneal ovarian tumors. We refer to these nanoparticles as optical viral ghosts (OVGs). We functionalized the OVGs with antibodies against HER2 receptor, a biomarker over-expressed in ovarian cancers. We injected functionalized OVGs, non-functionalized OVGs, and non-encapsulated BrCy106-NHS intravenously in mice implanted with ovarian intraperitoneal tumors. Tumors were extracted at 2, 6, and 24 h post-injection, and quantitatively analyzed using NIR fluorescence imaging. Fluorescence emission from tumors associated with the injection of the functionalized OVGs continued to increase between 2 and 24 h post-injection. At 24 h timepoint, the average spectrally-integrated fluorescence emission from homogenized tumors containing functionalized-OVGs was about 3.5 and 19.5 times higher than those containing non-functionalized OVGs or non-encapsulated BrCy106-NHS, respectively. Similarly, by using the functionalized-OVGs, the imaging signal-to-noise ratio at 24 h timepoint was enhanced by approximately threefold and sevenfold as compared to non-functionalized OVGs and the non-encapsulated dye, respectively. These functionalized virus-mimicking NIR nano-constructs could potentially be used for intraoperative visualization of ovarian tumors implants. [ABSTRACT FROM AUTHOR]

We experimentally register stimulated low-frequency Raman scattering (SLFRS) in the suspension of brome mosaic virus (BMV) in phosphate buffer with very high conversion efficiency. We identify two components of the SLFRS spectrum as the breathing and quadrupole modes of BMV and determine damping characteristics and gain factors for these modes. We show that, using the core–shell model for BMV and taking into account the influence of the environment, the acoustic properties of individual components of such a composite nanosystem can be determined. Thus, we define the sound velocity in the RNA core of BMV, in view of spectral characteristics of SLFRS. [ABSTRACT FROM AUTHOR]

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.

Self‐propelled nanomotors based on virus structure were designed, fabricated and characterized. Capsids from plant virus were covered on one hemisphere with platinum layer to promote motion in the presence of hydrogen peroxide. The Janus viral nanomotors (JVNs) with 25–30 nm diameter from brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) showed motion speeds of up to 9 μm/sec, depending of the peroxide fuel concentration in the medium. The JVNs can carry and deliver the chemo‐therapeutic drug Tamoxifen, and they are able to internalize into breast tumor cells without any functionalization. The therapeutic payload is released by a pH‐driven delivery action of the nanomotor. Here, the first proof of concept of a self‐propelled virus‐like nanoparticle is demonstrated. The attractive capabilities of nanomotors based on virus‐like nanoparticles (VLPs) pave the way to diverse biomedical applications. The first self‐propelled nanomotors based on a virus structure have been designed. Virus capsids were covered on one hemisphere with a Pt layer to promote motion in the presence of H2O2. The nanomotor can carry and deliver the drug, and they are able to internalize into tumor cells. [ABSTRACT FROM AUTHOR]

Positive-strand RNA viruses generally assemble RNA replication complexes on rearranged host membranes. Alphaviruses, other members of the alpha-like virus superfamily, and many other positive-strand RNA viruses invaginate host membrane into vesicular RNA replication compartments, known as spherules, whose interior is connected to the cytoplasm. Brome mosaic virus (BMV) and its close relative, cowpea chlorotic mottle virus (CCMV), form spherules along the endoplasmic reticulum. BMV spherule formation and RNA replication can be fully reconstituted in S. cerevisiae, enabling many studies identifying host factors and viral interactions essential for these processes. To better define and understand the conserved, core pathways of bromovirus RNA replication, we tested the ability of CCMV to similarly support spherule formation and RNA replication in yeast. Paralleling BMV, we found that CCMV RNA replication protein 1a was the only viral factor necessary to induce spherule membrane rearrangements and to recruit the viral 2a polymerase (2apol) to the endoplasmic reticulum. CCMV 1a and 2apol also replicated CCMV and BMV genomic RNA2, demonstrating core functionality of CCMV 1a and 2apol in yeast. However, while BMV and CCMV 1a/2apol strongly replicate each others’ genomic RNA3 in plants, neither supported detectable CCMV RNA3 replication in yeast. Moreover, in contrast to plant cells, in yeast CCMV 1a/2apol supported only limited replication of BMV RNA3 (<5% of that by BMV 1a/2apol). In keeping with this, we found that in yeast CCMV 1a was significantly impaired in recruiting BMV or CCMV RNA3 to the replication complex. Overall, we show that many 1a and 2apol functions essential for replication complex assembly, and their ability to be reconstituted in yeast, are conserved between BMV and CCMV. However, restrictions of CCMV RNA replication in yeast reveal previously unknown 1a-linked, RNA-selective host contributions to the essential early process of recruiting viral RNA templates to the replication complex. [ABSTRACT FROM AUTHOR]

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.

Background: Breast cancer is the most common malignant tumor disease and the leading cause of female mortality. The evolution of nanomaterials science opens the opportunity to improve traditional cancer therapies, enhancing therapy efficiency and reducing side effects., Methods and Major Results: Herein, protein cages conceived as enzymatic nanoreactors were designed and produced by using virus-like nanoparticles (VLPs) from Brome mosaic virus (BMV) and containing the catalytic activity of glucose oxidase (GOx) enzyme. The GOx enzyme was encapsulated into the BMV capsid (VLP-GOx), and the resulting enzymatic nanoreactors were coated with human serum albumin (VLP-GOx@HSA) for breast tumor cell targeting. The effect of the synthesized GOx nanoreactors on breast tumor cell lines was studied in vitro. Both nanoreactor preparations VLP-GOx and VLP-GOx@HSA showed to be highly cytotoxic for breast tumor cell cultures. Cytotoxicity for human embryonic kidney cells was also found. The monitoring of nanoreactor treatment on triple-negative breast cancer cells showed an evident production of oxygen by the catalase antioxidant enzyme induced by the high production of hydrogen peroxide from GOx activity., Conclusions and Implications: The nanoreactors containing GOx activity are entirely suitable for cytotoxicity generation in tumor cells. The HSA functionalization of the VLP-GOx nanoreactors, a strategy designed for selective cancer targeting, showed no improvement in the cytotoxic effect. The GOx containing enzymatic nanoreactors seems to be an interesting alternative to improve the current cancer therapy. In vivo studies are ongoing to reinforce the effectiveness of this treatment strategy., (© 2023 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Viruses are widely used to fabricate nanomaterials in the field of nanotechnology. Plant viruses are of great interest to the nanotechnology field because of their symmetry, polyvalency, homogeneous size distribution, and ability to self-assemble. This homogeneity can be used to obtain the high uniformity of the templated material and its related properties. In this paper, the variety of nanomaterials generated in rod-like and spherical plant viruses is highlighted for the cowpea chlorotic mottle virus (CCMV), cowpea mosaic virus (CPMV), brome mosaic virus (BMV), and tobacco mosaic virus (TMV). Their recent studies on developing nanomaterials in a wide range of applications from biomedicine and catalysts to biosensors are reviewed. [ABSTRACT FROM AUTHOR]

Aim. Viral diseases are one of the factors governing yield of cereal crops which is a key element to guaranteeing food security in Ukraine. This work assesses spread of most harmful viruses infecting cereals in agriecosystems using different diagnostic approaches. Methods. Viruses were detected in collected samples using double-antibody sandwich enzyme-linked immunosorbent assay with commercial test systems specific to 12 cereal viruses. Transmission electron microscopy was used for direct detection of virus particles. Results. To evaluate the spread of cereal viruses, we have carried out a 15-year monitoring of cereal crops in different regions of Ukraine. For virus screening, we collected plants with typical virus-like symptoms (mosaic, leaf discoloration, etc.). We have identified arthropod-transmitted viruses: Barley yellow dwarf virus-PAV (BYDV-PAV), Barley yellow dwarf virus-MAV (BYDV-MAV), Cereal yellow dwarf virus-RPV (CYDV-RPV), Wheat streak mosaic virus (WSMV) and Wheat dwarf virus (WDV). Brome mosaic virus (BMV) and Barley stripe mosaic virus (BSMV) were sporadically detected in the commercial fields under cereals, as well as several soil-borne viruses, including Wheat spindle streak mosaic virus (WSSMV), Soil-borne cereal mosaic virus (SBCMV), Soil-borne wheat mosaic virus (SBWMV) and Barley mild mosaic virus (BaMMV). Conclusions. Barley yellow dwarf virus-PAV, Wheat streak mosaic virus and Wheat dwarf virus are the most spread viruses endangering cereals’ cultivation in Ukraine. During the last 15 years, these viruses gained not only in terms of spread but also in the context of their economic effect. [ABSTRACT FROM AUTHOR]

Conventional time of flight ion detectors are based on secondary electron multipliers encountering a significant loss in detection efficiency, sensitivity and resolution with protein mass above 50 kDa. In this work we employ a silicon nanomembrane detector in a Matrix-Assisted Laser Desorption/Ionization coupled to time of flight (MALDI-TOF) mass spectrometer. The operating principle relies on phonon-assisted field emission with excellent performance in the high mass range from 0.001–2 MDa. In addition to the analysis of standard proteins the nanomembrane detector (NMD) has the potential for the detection and structural investigation of complex macromolecular assemblies through non-covalent interactions. In order to investigate this hypothesis, the N-terminal capping/methyltransferase domain (CAP) of the Brome Mosaic Virus (BMV) 1a replication protein by MALDI-TOF-NMD is analyzed. The signals detected at the high m / z -ratios of 912.6/982.7 (× 10 3 ) and 1333.3 (× 10 3 ) could be modified species of CAP-tricta/tetractamer and the octadecamer. For the first time, the NMD is applied to detect biologically complex macromolecular protein assemblies. Hence, this technology overcomes the limitations of conventional TOF-detectors and increases the analytical range of MALDI-TOF. This technology will be a future alternative for the structural analysis of intact virus capsids that will complement other MS-based techniques such as native mass spectrometry. [ABSTRACT FROM AUTHOR]

Positive-strand RNA viruses replicate their genomes in membrane-bound replication compartments. Brome mosaic virus (BMV) replicates in vesicular invaginations of the endoplasmic reticulum membrane. BMV has served as a productive model system to study processes like virus-host interactions, RNA replication and recombination. Here we present multiple lines of evidence showing that the structure of the viral RNA replication compartments plays a fundamental role and that recruitment of parental RNAs to a common replication compartment is a limiting step in intermolecular RNA recombination. We show that a previously defined requirement for an RNA recruitment element on both parental RNAs is not to function as a preferred crossover site, but in order for individual RNAs to be recruited into the replication compartments. Moreover, modulating the form of the replication compartments from spherular vesicles (spherules) to more expansive membrane layers increased intermolecular RNA recombination frequency by 200- to 1000-fold. We propose that intermolecular RNA recombination requires parental RNAs to be recruited into replication compartments as monomers, and that recruitment of multiple RNAs into a contiguous space is much more common for layers than for spherules. These results could explain differences in recombination frequencies between viruses that replicate in association with smaller spherules versus larger double-membrane vesicles and convoluted membranes. [ABSTRACT FROM AUTHOR]

The article presents a study which examines the replication of positive (+)-strand RNA viruses by brome mosaic virus (BMV) and their dependence on endoplasmic reticulum (ER) luminal thiol oxidase ERO1. Topics include the permeabilization of ER membranes by BMV RNA replication protein 1a, the complexities of transmembrane in positive-strand RNA virus replication, and how their interaction can lead to the development of virus control treatment.

Brome mosaic virus (BMV) has been successfully loaded with different types of nanoparticles. However, studies concerning its application as a nanoparticle carrier demand high-purity virions in large amounts. Existing BMV purification protocols rely on multiple differential ultracentrifugation runs of the initially purified viral preparation. Herein, we describe an alternative method for BMV purification based on ion-exchange chromatography and size-exclusion chromatography (SEC) yielding 0.2 mg of virus from 1 g of plant tissue. Our method is of similar efficiency to previously described protocols and can easily be scaled up. The method results in high-quality BMV preparations as confirmed by biophysical analyses, including cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), static light scattering (SLS), and circular dichroism (CD) measurements and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy. Our results revealed that purified BMV capsids are stable and monodisperse and can be used for further downstream applications. In this work, we also characterize secondary structure and size fluctuations of the BMV virion at different pH values. [ABSTRACT FROM AUTHOR]

Magnaporthe oryzae is a devastating plant pathogen, which has a detrimental impact on rice production worldwide. Despite its agronomical importance, some newly-emerging pathotypes often overcome race-specific disease resistance rapidly. It is thus desirable to develop a novel strategy for the long-lasting resistance of rice plants to ever-changing fungal pathogens. Brome mosaic virus (BMV)-induced RNA interference (RNAi) has emerged as a useful tool to study host-resistance genes for rice blast protection. Planta-generated silencing of targeted genes inside biotrophic pathogens can be achieved by expression of M. oryzae-derived gene fragments in the BMV-mediated gene silencing system, a technique termed host-induced gene silencing (HIGS). In this study, the effectiveness of BMV-mediated HIGS in M. oryzae was examined by targeting three predicted pathogenicity genes, MoABC1, MoMAC1 and MoPMK1. Systemic generation of fungal gene-specific small interfering RNA (siRNA) molecules induced by inoculation of BMV viral vectors inhibited disease development and reduced the transcription of targeted fungal genes after subsequent M. oryzae inoculation. Combined introduction of fungal gene sequences in sense and antisense orientation mediated by the BMV silencing vectors significantly enhanced the efficiency of this host-generated trans-specific RNAi, implying that these fungal genes played crucial roles in pathogenicity. Collectively, our results indicated that BMV-HIGS system was a great strategy for protecting host plants against the invasion of pathogenic fungi. [ABSTRACT FROM AUTHOR]