RESUMO
Virus-like particles (VLP) of the cowpea chlorotic mottle virus (CCMV), a plant virus, have been shown to be safe and noncytotoxic vehicles for delivering various cargos, including nucleic acids and peptides, and as scaffolds for presenting epitopes. Thus, CCMV-VLP have acquired increasing attention to be used in fields such as gene therapy, drug delivery, and vaccine development. Regardless of their production method, most reports purify CCMV-VLP through a series of ultracentrifugation steps using sucrose density gradient ultracentrifugation, which is a complex and time-consuming process. Here, the use of anion exchange chromatography is described as a one-step protocol for purification of CCMV-VLP produced by the insect cell-baculovirus expression vector system (IC-BEVS).
Assuntos
Bromovirus , Bromovirus/genética , Animais , Baculoviridae/genética , Vetores Genéticos/genética , Cromatografia por Troca Iônica/métodos , Vírion/isolamento & purificação , Vírion/genética , Vírion/metabolismoRESUMO
Plant viruses such as brome mosaic virus and cowpea chlorotic mottle virus are effectively purified through PEG precipitation and sucrose cushion ultracentrifugation. Increasing ionic strength and an alkaline pH cause the viruses to swell and disassemble into coat protein subunits. The coat proteins can be reassembled into stable virus-like particles (VLPs) that carry anionic molecules at low ionic strength and through two-step dialysis from neutral pH to acidic buffer. VLPs have been extensively studied due to their ability to protect and deliver cargo, particularly RNA, while avoiding degradation under physiological conditions. Furthermore, chemical functionalization of the surface of VLPs allows for the targeted drug delivery. VLPs derived from plants have demonstrated great potential in nanomedicine by offering a versatile platform for drug delivery, imaging, and therapeutic applications.
Assuntos
Vírus de Plantas , Vírus de Plantas/genética , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Vírion/química , Vírion/genética , Bromovirus/química , Bromovirus/genética , RNA/química , Concentração de Íons de Hidrogênio , RNA Viral/genéticaRESUMO
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.
Assuntos
Bromovirus , Penaeidae , Vírus da Síndrome da Mancha Branca 1 , Animais , Vírus da Síndrome da Mancha Branca 1/genética , Bromovirus/genética , RNA de Cadeia Dupla/genéticaRESUMO
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.
Assuntos
Bromovirus , Vírus de Plantas , Bromovirus/genética , Proteínas do Capsídeo/metabolismo , Capsídeo , Concentração OsmolarRESUMO
Enzymatic nanoreactors were obtained by galactose-1-phosphate uridylyl-transferase (GALT) encapsulation into plant virus capsids by a molecular self-assembly strategy. The aim of this work was to produce virus-like nanoparticles containing GALT for an enzyme-replacement therapy for classic galactosemia. The encapsulation efficiency and the catalytic constants of bio-nanoreactors were determined by using different GALT and virus coat protein ratios. The substrate affinity of nanoreactors was slightly lower than that of the free enzyme; the activity rate was 16 % of the GALT free enzyme. The enzymatic nanoreactors without functionalization were internalized into different cell lines including fibroblast and kidney cells, but especially into hepatocytes. The enzymatic nanoreactors are an innovative enzyme preparation with potential use for the treatment of classic galactosemia.
Assuntos
Bromovirus/metabolismo , Proteínas do Capsídeo/química , Composição de Medicamentos/métodos , UTP-Hexose-1-Fosfato Uridililtransferase/química , Animais , Proteínas do Capsídeo/isolamento & purificação , Linhagem Celular , Endocitose , Fluoresceína-5-Isotiocianato/química , Galactosemias/tratamento farmacológico , Galactosemias/patologia , Humanos , Cinética , Camundongos , Nanotecnologia , UTP-Hexose-1-Fosfato Uridililtransferase/metabolismo , UTP-Hexose-1-Fosfato Uridililtransferase/uso terapêuticoRESUMO
Different types of gold nanoparticles have been synthesized that show great potential in medical applications such as medical imaging, bio-analytical sensing and photothermal cancer therapy. However, their stability, polydispersity and biocompatibility are major issues of concern. For example, the synthesis of gold nanorods, obtained through the elongated micelle process, produce them with a high positive surface charge that is cytotoxic, while gold nanoshells are unstable and break down in a few weeks due to the Ostwald ripening process. In this work, we report the self-assembly of the capsid protein (CP) of cowpea chlorotic mottle virus (CCMV) around spherical gold nanoparticles, gold nanorods and gold nanoshells to form virus-like particles (VLPs). All gold nanoparticles were synthesized or treated to give them a negative surface charge, so they can interact with the positive N-terminus of the CP leading to the formation of the VLPs. To induce the protein self-assembly around the negative gold nanoparticles, we use different pH and ionic strength conditions determined from a CP phase diagram. The encapsidation with the viral CP will provide the nanoparticles better biocompatibility, stability, monodispersity and a new biological substrate on which can be introduced ligands toward specific cells, broadening the possibilities for medical applications.
Assuntos
Bromovirus/metabolismo , Proteínas do Capsídeo/química , Ouro/química , Nanopartículas Metálicas/química , Nanoconchas/química , Vírion/metabolismo , LigantesRESUMO
Virus-like particles (VLPs) are being used for therapeutic developments such as vaccines and drug nanocarriers. Among these, plant virus capsids are gaining interest for the formation of VLPs because they can be safely handled and are noncytotoxic. A paradigm in virology, however, is that plant viruses cannot transfect and deliver directly their genetic material or other cargos into mammalian cells. In this work, we prepared VLPs with the CCMV capsid and the mRNA-EGFP as a cargo and reporter gene. We show, for the first time, that these plant virus-based VLPs are capable of directly transfecting different eukaryotic cell lines, without the aid of any transfecting adjuvant, and delivering their nucleic acid for translation as observed by the presence of fluorescent protein. Our results show that the CCMV capsid is a good noncytotoxic container for genome delivery into mammalian cells.
Assuntos
Bromovirus/genética , Técnicas de Transferência de Genes , Vírus de Plantas/genética , Vacinas de Partículas Semelhantes a Vírus/genética , Animais , Proteínas do Capsídeo/genética , Linhagem Celular , Células Eucarióticas/virologia , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Transfecção/métodos , Montagem de Vírus/genéticaRESUMO
Obtaining pure and soluble viral capsid proteins (CPs) has been a major challenge in the fields of science and technology in recent decades. In many cases, the CPs can self-assemble in the absence of a viral genome, resulting in non-infectious, empty virus-like particles (VLPs) which can be safely handled. The use of VLPs has found great potential in biotechnology and health purposes. In addition, VLPs are a good model system to study protein-protein interactions at the molecular level. In this work, an optimized strategy for the heterologous expression of the Cowpea chlorotic mottle virus (CCMV) CP based in Escherichia coli is described. The method is efficient, inexpensive and it consistently produces higher yields and greater purity levels than those reported so far. Additionally, one of the main advantages of this method is the prevention of the formation of inclusion bodies, thus allowing to directly obtain high amounts of the CP in a soluble and functionally active state with the capacity to readily form VLPs in vitro. The CCMV CP self-assembly pH dependence was also investigated, providing guidelines to easily modulate the process.
Assuntos
Bromovirus/genética , Proteínas do Capsídeo/isolamento & purificação , Proteínas do Capsídeo/metabolismo , Multimerização Proteica , Virossomos/metabolismo , Proteínas do Capsídeo/genética , Escherichia coli/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Virossomos/genéticaRESUMO
This work shows, for the first time, the encapsulation of a highly relevant protein in the biomedical field into virus-like particles (VLPs). A bacterial CYP variant was effectively encapsulated in VLPs constituted of coat protein from cowpea chlorotic mottle virus (CCMV). The catalytic VLPs are able to transform the chemotherapeutic pro-drug, tamoxifen, and the emerging pro-drug resveratrol. The chemical nature of the products was identified, confirming similar active products than those obtained with human CYP. The enzymatic VLPs remain stable after the catalytic reaction. The potential use of these biocatalytic nanoparticles as targeted CYP carriers for the activation of chemotherapy drugs is discussed.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Pró-Fármacos/metabolismo , Ativação Metabólica , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Bromovirus/química , Bromovirus/ultraestrutura , Proteínas do Capsídeo/química , Sistema Enzimático do Citocromo P-450/genética , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Humanos , Cinética , Nanocápsulas , Nanopartículas/metabolismo , Pró-Fármacos/administração & dosagem , Resveratrol , Estilbenos/metabolismo , Tamoxifeno/administração & dosagem , Tamoxifeno/metabolismo , Vírion/metabolismoRESUMO
Symptom development in a susceptible sunflower line inoculated with Sunflower chlorotic mottle virus (SuCMoV) was followed in the second pair of leaves at different post-inoculation times: before symptom expression (BS), at early (ES) and late (LS) symptom expression. Sugar and starch increases and photoinhibition were observed as early effects BS, and were maintained or enhanced later on, however, chlorophyll loss was detected only at LS. Photoinhibition correlated with a drastic decrease in D1 protein level. The progress of infection was accompanied by decreasing levels of apoplastic reactive oxygen species (ROS). In infected leaves, higher antioxidant enzyme activities (superoxide dismutase, SOD; ascorbate peroxidase, APX; glutathione reductase, GR) were observed from BS. The purpose of this work was to evaluate whether the early increases in carbohydrate accumulation may participate in SuCMoV symptom expression. Similar effects on photoinhibition, apoplastic ROS generation and antioxidant activity were generated when healthy leaves were treated with sugars. These results suggest that photoinhibitory processes and lower apoplastic superoxide levels induced by SuCMoV infection may be modulated by sugar increases.