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1.
Viruses ; 13(10)2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34696481

RESUMO

This review summarizes research on virus diseases of cereals and oilseeds in Australia since the 1950s. All viruses known to infect the diverse range of cereal and oilseed crops grown in the continent's temperate, Mediterranean, subtropical and tropical cropping regions are included. Viruses that occur commonly and have potential to cause the greatest seed yield and quality losses are described in detail, focusing on their biology, epidemiology and management. These are: barley yellow dwarf virus, cereal yellow dwarf virus and wheat streak mosaic virus in wheat, barley, oats, triticale and rye; Johnsongrass mosaic virus in sorghum, maize, sweet corn and pearl millet; turnip yellows virus and turnip mosaic virus in canola and Indian mustard; tobacco streak virus in sunflower; and cotton bunchy top virus in cotton. The currently less important viruses covered number nine infecting nine cereal crops and 14 infecting eight oilseed crops (none recorded for rice or linseed). Brief background information on the scope of the Australian cereal and oilseed industries, virus epidemiology and management and yield loss quantification is provided. Major future threats to managing virus diseases effectively include damaging viruses and virus vector species spreading from elsewhere, the increasing spectrum of insecticide resistance in insect and mite vectors, resistance-breaking virus strains, changes in epidemiology, virus and vectors impacts arising from climate instability and extreme weather events, and insufficient industry awareness of virus diseases. The pressing need for more resources to focus on addressing these threats is emphasized and recommendations over future research priorities provided.


Assuntos
Produtos Agrícolas/virologia , Grão Comestível/virologia , Doenças das Plantas/virologia , Agricultura/métodos , Austrália , Ilarvirus , Luteovirus , Doenças das Plantas/etiologia , Potyviridae , Potyvirus , Tymovirus , Viroses/epidemiologia
2.
J Appl Microbiol ; 131(4): 2072-2080, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33629458

RESUMO

AIMS: To display a short peptide (GSRSHHHHHH) at the C-terminal end of turnip yellow mosaic virus coat protein (TYMVc) and to study its assembly into virus-like particles (TYMVcHis6 VLPs). METHODS AND RESULTS: In this study, recombinant TYMVcHis6 expressed in Escherichia coli self-assembled into VLPs of approximately 30-32 nm. SDS-PAGE and Western blot analysis of protein fractions from the immobilized metal affinity chromatography (IMAC) showed that TYMVcHis6 VLPs interacted strongly with nickel ligands in IMAC column, suggesting that the fusion peptide is protruding out from the surface of VLPs. These VLPs are highly stable over a wide pH range from 3·0 to 11·0 at different temperatures. At pH 11·0, specifically, the VLPs remained intact up to 75°C. Additionally, the disassembly and reassembly of TYMVcHis6 VLPs were studied in vitro. Dynamic light scattering and transmission electron microscopy analysis revealed that TYMVcHis6 VLPs were dissociated by 7 mol l-1 urea and 2 mol l-1 guanidine hydrochloride (GdnHCl) without impairing their reassembly property. CONCLUSIONS: A 10-residue peptide was successfully displayed on the surface of TYMVcHis6 VLPs. This chimera demonstrated high stability under extreme thermal conditions with varying pH and was able to dissociate and reassociate into VLPs by chemical denaturants. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first C-terminally modified TYMVc produced in E. coli. The C-terminal tail which is exposed on the surface can be exploited as a useful site to display multiple copies of functional ligands. The ability of the chimeric VLPs to self-assemble after undergo chemical denaturation indicates its potential role to serve as a nanocarrier for use in targeted drug delivery.


Assuntos
Tymovirus , Proteínas do Capsídeo/genética , Escherichia coli/genética , Microscopia Eletrônica de Transmissão , Proteínas Recombinantes
3.
Mol Pharm ; 17(12): 4629-4636, 2020 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-33186039

RESUMO

Nanoparticle-based prodrugs offer an effective strategy to improve the safety and delivery of small-molecule therapeutics while reducing the risk of drug resistance. Here, we conjugated a maleimide-functionalized cisplatin prodrug containing Pt(IV) to the internal and/or external surface of virus-like particles (VLPs) derived from Physalis mottle virus (PhMV) to develop a pH-sensitive drug delivery system. The internally loaded and PEGylated VLPs (Pt-PhMVCy5.5-PEG) were taken up efficiently by cancer cells where they released platinum, presumably as a reduced, DNA-reactive Pt(II) complex, rapidly under acidic conditions in vitro (>80% in 30 h). The efficacy of the VLP-based drug delivery system was demonstrated against a panel of cancer cell lines, including cell lines resistant to platinum therapy. Furthermore, Pt-PhMVCy5.5-PEG successfully inhibited the growth of xenograft MDA-MB-231 breast tumors in vivo and significantly prolonged the survival of mice compared to free cisplatin and cisplatin-maleimide. Pt-PhMVCy5.5-PEG therefore appears promising as a prodrug to overcome the limitations of conventional platinum-based drugs for cancer therapy.


Assuntos
Cisplatino/farmacocinética , Portadores de Fármacos/química , Nanopartículas/química , Neoplasias/tratamento farmacológico , Tymovirus/química , Animais , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Liberação Controlada de Fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Neoplasias/patologia , Pró-Fármacos/administração & dosagem , Pró-Fármacos/farmacocinética , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Biotechnol J ; 15(12): e2000077, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32918857

RESUMO

The controlled release of drugs using nanoparticle-based delivery vehicles is a promising strategy to improve the safety and efficacy of chemotherapy. A simple, scalable, and reproducible strategy is developed to synthesize a drug delivery system (DDS) by loading 6-maleimidocaproyl-hydrazone doxorubicin (DOX-EMCH) into the empty core of virus-like particles (VLPs) derived from Physalis mottle virus (PhMV) via a combination of chemical conjugation to cysteine residues and π-π stacking interactions with the anchored doxorubicin molecule. The DOX-EMCH prodrug features an acid-sensitive hydrazine linker that triggers the release of doxorubicin in the slightly acidic extracellular tumor microenvironment or acidic endosomal or lysosomal compartments following cellular uptake. The VLP external surface is coated with polyethylene glycol (PEG) to prevent non-specific uptake and improve biocompatibility. The DOX-PhMV-PEG particles are stable in vitro and show greater efficacy in vivo compared to free doxorubicin in a breast tumor mouse model (using MDA-MB-231 cells and nude mice): 92% of the tumor-bearing mice treated with DOX-PhMV-PEG are completely cured compared to 27% of those treated with free doxorubicin under the same conditions, representing a 3.4-fold improvement. These results lay a foundation for the further development of this biological drug delivery system for a new generation of chemotherapy products.


Assuntos
Doxorrubicina/uso terapêutico , Neoplasias , Animais , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Nus , Nanopartículas , Neoplasias/tratamento farmacológico , Polietilenoglicóis , Pró-Fármacos , Tymovirus
5.
J Biol Chem ; 295(40): 13769-13783, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-32732284

RESUMO

Single-stranded, positive-sense RNA viruses assemble their replication complexes in infected cells from a multidomain replication polyprotein. This polyprotein usually contains at least one protease, the primary function of which is to process the polyprotein into mature proteins. Such proteases also may have other functions in the replication cycle. For instance, cysteine proteases (PRO) frequently double up as ubiquitin hydrolases (DUB), thus interfering with cellular processes critical for virus replication. We previously reported the crystal structures of such a PRO/DUB from Turnip yellow mosaic virus (TYMV) and of its complex with one of its PRO substrates. Here we report the crystal structure of TYMV PRO/DUB in complex with ubiquitin. We find that PRO/DUB recognizes ubiquitin in an unorthodox way: It interacts with the body of ubiquitin through a split recognition motif engaging both the major and the secondary recognition patches of ubiquitin (Ile44 patch and Ile36 patch, respectively, including Leu8, which is part of the two patches). However, the contacts are suboptimal on both sides. Introducing a single-point mutation in TYMV PRO/DUB aimed at improving ubiquitin-binding led to a much more active DUB. Comparison with other PRO/DUBs from other viral families, particularly coronaviruses, suggests that low DUB activities of viral PRO/DUBs may generally be fine-tuned features of interaction with host factors.


Assuntos
Enzimas Desubiquitinantes/química , Peptídeo Hidrolases/química , Tymovirus/enzimologia , Ubiquitina/química , Proteínas Virais/química , Cristalografia por Raios X , Enzimas Desubiquitinantes/genética , Peptídeo Hidrolases/genética , Tymovirus/genética , Ubiquitina/genética , Proteínas Virais/genética
6.
Theor Appl Genet ; 133(2): 383-393, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31690991

RESUMO

KEY MESSAGE: Partially dominant resistance to Turnip yellows virus associated with one major QTL was identified in the natural allotetraploid oilseed rape cultivar Yudal. Turnip yellows virus (TuYV) is transmitted by the peach-potato aphid (Myzus persicae) and causes severe yield losses in commercial oilseed rape crops (Brassica napus). There is currently only one genetic resource for resistance to TuYV available in brassica, which was identified in the re-synthesised B. napus line 'R54'. In our study, 27 mostly homozygous B. napus accessions, either doubled-haploid (DH) or inbred lines, representing a diverse subset of the B. napus genepool, were screened for TuYV resistance/susceptibility. Partial resistance to TuYV was identified in the Korean spring oilseed rape, B. napus variety Yudal, whilst the dwarf French winter oilseed rape line Darmor-bzh was susceptible. QTL mapping using the established Darmor-bzh × Yudal DH mapping population (DYDH) revealed one major QTL explaining 36% and 18% of the phenotypic variation in two independent experiments. A DYDH line was crossed to Yudal, and reciprocal backcross (BC1) populations from the F1 with either the susceptible or resistant parent revealed the dominant inheritance of the TuYV resistance. The QTL on ChrA04 was verified in the segregating BC1 population. A second minor QTL on ChrC05 was identified in one of the two DYDH experiments, and it was not observed in the BC1 population. The TuYV resistance QTL in 'R54' is within the QTL interval on Chr A04 of Yudal; however, the markers co-segregating with the 'R54' resistance are not conserved in Yudal, suggesting an independent origin of the TuYV resistances. This is the first report of the QTL mapping of TuYV resistance in natural B. napus.


Assuntos
Brassica napus/genética , Brassica napus/virologia , Doenças das Plantas/genética , Doenças das Plantas/virologia , Tymovirus , Animais , Afídeos , Mapeamento Cromossômico , Resistência à Doença , Genótipo , Haploidia , Fenótipo , Locos de Características Quantitativas
7.
J Microbiol Biotechnol ; 29(11): 1790-1798, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31546296

RESUMO

Flock House virus (FHV), an insect RNA virus, has a bipartite genome. FHV RNA1 can be packaged in turnip yellow mosaic virus (TYMV) as long as the FHV RNA has a TYMV sequence at the 3'-end. The encapsidated FHV RNA1 has four additional nucleotides at the 5'- end. We investigated whether the recombinant FHV RNA1 could replicate in mammalian cells. To address this issue, we prepared in vitro transcribed FHV RNAs that mimicked the recombinant FHV RNA1, and introduced them into baby hamster kidney (BHK) cells. The result showed that the recombinant FHV RNA1 was capable of replication. An eGFP gene inserted into the frame with B2 gene of the FHV RNA1 was also successfully expressed. We also observed that eGFP expression at the protein level was strong at 28°C but weak at 30°C. Sequence analysis showed that the 3'-ends of the RNA1 and RNA3 replication products were identical to those of the authentic FHV RNAs. This indicates that FHV replicase correctly recognized an internally-located replication signal. In contrast, the 5'-ends of recombinant FHV RNA1 frequently had deletions, indicating random initiation of (+)-strand synthesis.


Assuntos
Fibroblastos/virologia , Genes Reporter/genética , Nodaviridae/fisiologia , RNA Viral/metabolismo , Animais , Linhagem Celular , Cricetinae , Expressão Gênica , Genoma Viral/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Nodaviridae/genética , RNA Viral/genética , Tymovirus/genética , Replicação Viral
8.
Plant Dis ; 103(9): 2246-2251, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31287777

RESUMO

Naranjilla (Solanum quitoense Lam.) and tamarillo (S. betaceum Cav.) are two important perennial solanaceous crops grown in Ecuador for the fresh market and juice production. Viruses infecting tamarillo and naranjilla are currently poorly studied, and no clean stock program exists in Ecuador. Here, we report a new virus, provisionally named as naranjilla mild mosaic virus (NarMMV) (genus Tymovirus, family Tymoviridae), isolated from naranjilla grown in an orchard in Pichincha Province, Ecuador. The complete genome of the virus consists of 6,348 nucleotides and encodes three open reading frames typical for members of the genus Tymovirus. Phylogenetically, Chiltepin yellow mosaic virus, Eggplant mosaic virus, and the recently characterized naranjilla chlorotic mosaic virus (NarCMV) were found to be the closest relatives of NarMMV. Unlike NarCMV, the new virus induced mild mosaic in naranjilla and more severe symptoms in tamarillo. Similar to NarCMV, NarMMV was unable to systemically infect potato. Virus surveys found NarMMV prevalent in naranjilla production areas of two provinces of Ecuador, especially where hybrid cultivars of naranjilla were cultivated. NarMMV was also found in field-grown tamarillo. The new virus cross-reacted with antibodies developed against NarCMV. Hence, this antibody will be useful for its field diagnosis using enzyme-linked immunosorbent assay or immunocapture reverse transcription polymerase chain reaction in future virus-free certification programs.


Assuntos
Solanum , Tymovirus , Equador , Genoma Viral/genética , Filogenia , Prevalência , Solanum/virologia , Tymovirus/classificação , Tymovirus/genética , Tymovirus/fisiologia
9.
Nat Nanotechnol ; 14(7): 712-718, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31110265

RESUMO

Large doses of chemical pesticides are required to achieve effective concentrations in the rhizosphere, which results in the accumulation of harmful residues. Precision farming is needed to improve the efficacy of pesticides, but also to avoid environmental pollution, and slow-release formulations based on nanoparticles offer one solution. Here, we tested the mobility of synthetic and virus-based model nanopesticides by combining soil column experiments with computational modelling. We found that the tobacco mild green mosaic virus and cowpea mosaic virus penetrate soil to a depth of at least 30 cm, and could therefore deliver nematicides to the rhizosphere, whereas the Physalis mosaic virus remains in the first 4 cm of soil and would be more useful for the delivery of herbicides. Our experiments confirm that plant viruses are superior to synthetic mesoporous silica nanoparticles and poly(lactic-co-glycolic acid) for the delivery and controlled release of pesticides, and could be developed as the next generation of pesticide delivery systems.


Assuntos
Agricultura/métodos , Preparações de Ação Retardada/metabolismo , Vírus do Mosaico/metabolismo , Praguicidas/metabolismo , Microbiologia do Solo , Comovirus/metabolismo , Nanopartículas/metabolismo , Solo/química , Vírus do Mosaico do Tabaco/metabolismo , Tymovirus/metabolismo
10.
Plant Sci ; 284: 99-107, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31084885

RESUMO

Phloem-mobile mRNAs are assumed to contain sequence elements directing RNA to the phloem translocation pathway. One of such elements is represented by tRNA sequences embedded in untranslated regions of many mRNAs, including those proved to be mobile. Genomic RNAs of a number of plant viruses possess a 3'-terminal tRNA-like structures (TLSs) only distantly related to genuine tRNAs, but nevertheless aminoacylated and capable of interaction with some tRNA-binding proteins. Here, we elaborated an experimental system for analysis of RNA phloem transport based on an engineered RNA of Potato virus X capable of replication, but not encapsidation and movement in plants. The TLSs of Brome mosaic virus, Tobacco mosaic virus and Turnip yellow mosaic virus were demonstrated to enable the phloem transport of foreign RNA. A miRNA precursor, pre-miR390b, was also found to render RNA competent for the phloem transport. In line with this, sequences of miRNA precursors were identified in a Cucurbita maxima phloem transcriptome, supporting the hypothesis that, at least in some cases, miRNA phloem signaling can involve miRNA precursors. Collectively, the data presented here suggest that RNA molecules can be directed into the phloem translocation pathway by structured RNA elements such as those of viral TLSs and miRNA precursors.


Assuntos
MicroRNAs/metabolismo , Floema/metabolismo , RNA de Plantas/metabolismo , RNA de Transferência/metabolismo , Bromovirus/metabolismo , Cucurbita/metabolismo , Cucurbita/virologia , MicroRNAs/fisiologia , Floema/fisiologia , Potexvirus/metabolismo , RNA de Transferência/fisiologia , Vírus do Mosaico do Tabaco/metabolismo , Tymovirus/metabolismo
11.
ACS Appl Mater Interfaces ; 11(20): 18213-18223, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31074602

RESUMO

One of the greatest challenges in nanomedicine is the low efficiency with which nanoparticles are delivered to lesions such as tumors in vivo. Here, we show that Physalis mottle virus (PhMV)-like nanoparticles can be developed as bimodal contrast agents to achieve long circulation, specific targeting capability, and efficient delivery to tumors in vivo. The self-assembling coat protein nanostructure offers various opportunities to modify the internal and external surfaces separately. After loading the internal cavity of the particles with the fluorescent dye Cy5.5 and paramagnetic Gd(III) complexes, we modified the outer surface by PEGylation and conjugation with targeting peptides. Using this combined approach, we were able to monitor a human prostate tumor model for up to 10 days by near-infrared fluorescence and magnetic resonance imaging, with up to 6% of the injection dose remaining. Our results show that PhMV-like nanoparticles provide a promising and innovative platform for the development of next-generation diagnostic and therapeutic agents.


Assuntos
Carbocianinas , Meios de Contraste , Gadolínio , Imageamento por Ressonância Magnética , Nanopartículas , Neoplasias Experimentais/diagnóstico por imagem , Imagem Óptica , Tymovirus/química , Animais , Carbocianinas/química , Carbocianinas/farmacologia , Meios de Contraste/química , Meios de Contraste/farmacologia , Gadolínio/química , Gadolínio/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Nanopartículas/uso terapêutico , Células PC-3
12.
Arch Virol ; 164(7): 1753-1760, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31025116

RESUMO

The expression of several structural proteins from a wide variety of viruses in heterologous cell culture systems results in the formation of virus-like particles (VLPs). These VLPs structurally resemble the wild-type virus particles and have been used to study viral assembly process and as antigens for diagnosis and/or vaccine development. Tomato blistering mosaic virus (ToBMV) is a tymovirus that has a 6.3-kb positive-sense ssRNA genome. We have employed the baculovirus expression vector system (BEVS) for the production of tymovirus-like particles (tVLPs) in insect cells. Two recombinant baculoviruses containing the ToBMV wild-type coat protein (CP) gene or a modified short amino-terminal deletion (Δ2-24CP) variant were constructed and used to infect insect cells. Both recombinant viruses were able to express ToBMV CP and Δ2-24CP from infected insect cells that self-assembled into tVLPs. Therefore, the N-terminal residues (2-24) of the native ToBMV CP were shown not to be essential for self-assembly of tVLPs. We also constructed a third recombinant baculovirus containing a small sequence coding for the major epitope of the chikungunya virus (CHIKV) envelope protein 2 (E2) replacing the native CP N-terminal 2-24 amino acids. This recombinant virus also produced tVLPs. In summary, ToBMV VLPs can be produced in a baculovirus/insect cell heterologous expression system, and the N-terminal residues 2-24 of the CP are not essential for this assembly, allowing its potential use as a protein carrier that facilitates antigen purification and might be used for diagnosis.


Assuntos
Baculoviridae/genética , Proteínas do Capsídeo/biossíntese , Tymovirus/crescimento & desenvolvimento , Tymovirus/genética , Proteínas do Envelope Viral/biossíntese , Montagem de Vírus/genética , Animais , Proteínas do Capsídeo/genética , Linhagem Celular , Vírus Chikungunya/genética , Expressão Gênica/genética , Lycopersicon esculentum/virologia , Mariposas/citologia , Proteínas do Envelope Viral/genética
13.
Vet Microbiol ; 229: 20-27, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30642595

RESUMO

The aim of the present study is to use Physalis mottle virus (PhMV) coat protein (CP) as a scaffold to display the neutralizing epitopes of Infectious bursal disease virus (IBDV) VP2. For this, three different chimeric constructs were synthesized by replacing the N-terminus of PhMV CP with tandem repeats of neutralizing epitopes of IBDV VP2 and expressed in Escherichia coli. Expression analysis revealed that all the three recombinant chimeric coat protein subunits are soluble in nature and self-assembled into virus-like particles (VLPs) as evidenced through sucrose density gradient ultracentrifugation. The chimeric VLPs were characterized by various biochemical and biophysical techniques and found that they are stable and structurally sound. When the chimeric VLPs were used as coating antigen, they were able to detect IBDV antibodies. These results indicated that the chimeric VLPs can be used as potential vaccine candidates for the control of IBDV, which needs to be further evaluated in animal models.


Assuntos
Anticorpos Antivirais/sangue , Infecções por Birnaviridae/veterinária , Vírus da Doença Infecciosa da Bursa/imunologia , Animais , Anticorpos Neutralizantes , Infecções por Birnaviridae/sangue , Infecções por Birnaviridae/virologia , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Galinhas , Epitopos , Tymovirus , Proteínas Estruturais Virais
14.
Viruses ; 10(8)2018 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-30081487

RESUMO

A novel virus with distinct genome features was discovered by high throughput sequencing in a symptomatic blackcurrant plant. The virus, tentatively named Ribes americanum virus A (RAVA), has distinct genome organization and molecular features bridging genera in the order Tymovirales. The genome consists of 7106 nucleotides excluding the poly(A) tail. Five open reading frames were identified, with the first encoding a putative viral replicase with methyl transferase (MTR), AlkB, helicase, and RNA dependent RNA polymerase (RdRp) domains. The genome organization downstream of the replicase resembles that of members of the order Tymovirales with an unconventional triple gene block (TGB) movement protein arrangement with none of the other four putative proteins exhibiting significant homology to viral proteins. Phylogenetic analysis using replicase conserved motifs loosely placed RAVA within the Betaflexiviridae. Data strongly suggest that RAVA is a novel virus that should be classified as a species in a new genus in the Betaflexiviridae or a new family within the order Tymovirales.


Assuntos
Genoma Viral , Ribes/virologia , Tymovirus/classificação , Tymovirus/genética , Vírus de DNA , Flexiviridae/classificação , Sequenciamento de Nucleotídeos em Larga Escala , Fases de Leitura Aberta , Filogenia , Doenças das Plantas/virologia , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Tymovirus/isolamento & purificação , Proteínas Virais/genética
15.
Methods Mol Biol ; 1776: 609-627, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29869269

RESUMO

Stem cells can interact and respond to the extracellular nanoscale environment. Viral nanoparticles have been utilized as building blocks to control cell growth and differentiation. By integrating stem cell research and virus nanoparticle chemistry together, a systematic analysis of the effects of nanotopography on stem cell differentiation can be accomplished. The fabrication of thin films of the viral nanoparticles is particularly valuable for such studies. Here, we describe two methods to fabricate plant virus-based thin films and procedures to study the osteogenic differentiation of mesenchymal stem cells on plant virus-based substrates. The method makes use of wild-type tobacco mosaic virus (wt-TMV), RGD-modified TMV (TMV-RGD), turnip yellow mosaic virus (TYMV), cowpea mosaic virus (CPMV), turnip vein clearing virus (TVCV), and potato virus X (PVX) for development of bone tissue engineering biomaterials.


Assuntos
Células-Tronco Mesenquimais/química , Nanopartículas/química , Osteogênese/genética , Engenharia Tecidual/métodos , Adesão Celular/genética , Diferenciação Celular/genética , Comovirus/química , Comovirus/genética , Potexvirus/química , Potexvirus/genética , Vírus do Mosaico do Tabaco/química , Vírus do Mosaico do Tabaco/genética , Tymovirus/química , Tymovirus/genética
16.
Virus Res ; 252: 13-21, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29730306

RESUMO

Turnip yellow mosaic virus (TYMV) was able to enter animal cells when the spherical plant virus was conjugated with Tat, a cell penetrating peptide (CPP). Tat was chemically attached to the surface lysine residues of TYMV using hydrazone chemistry. Baby hamster kidney (BHK) cells were incubated with either unmodified or Tat-conjugated TYMV and examined by flow cytometry and confocal microscopic analyses. Tat conjugation was shown to be more efficient than Lipofectamine in allowing TYMV to enter the mammalian cells. Tat-assisted-transfection was also associated with less loss of cell viability than lipofection. Among the CPPs tested (Tat, R8, Pep-1 and Pen), it was observed that R8 and Pen were also effective while Pep-1 was not. We also examined if the internal space of TYMV can be used to load fluorescein dye as a model cargo. When TYMV is treated by freezing and thawing, the virus is known to convert into a structure with a 6-8 nm hole and release viral RNA. When the resultant pot-like particles were reacted with fluorescein-5-maleimide using interior sulfhydryl groups as conjugation sites, about 145 fluorescein molecules were added per particle. The fluorescein-loaded TYMV particles were conjugated with Tat and introduced into BHK cells, again with higher transfection efficiency compared to lipofection. Our studies demonstrate the potential of modified TYMV as an efficient system for therapeutic cargo delivery to mammalian cells.


Assuntos
Capsídeo/química , Peptídeos Penetradores de Células/química , Sistemas de Liberação de Medicamentos , Tymovirus , Animais , Linhagem Celular , Cricetinae , RNA Viral/metabolismo , Transfecção
17.
Plant Dis ; 102(5): 911-918, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-30673388

RESUMO

Naranjilla ("little orange"), also known as lulo (Solanum quitoense Lam.), is a perennial shrub species cultivated in the Andes for fresh fruit and juice production. In 2015, a naranjilla plant exhibiting stunting, mosaic, and chlorotic spots was sampled in the Pastaza province of Ecuador and maintained under greenhouse conditions. An infectious agent was mechanically transmitted to indicator plants and was subjected to biological and molecular characterization. Spherical particles approximately 30 nm in diameter, composed of a single 20-kDa capsid protein, were observed under an electron microscope in infected naranjilla plants. High-throughput sequencing conducted on inoculated Nicotiana benthamiana plants produced a single sequence contig sharing the closest relationship with several tymoviruses. The entire 6,245-nucleotide genome of a new tymovirus was amplified using reverse-transcription polymerase chain reaction and resequenced with the Sanger methodology. The genome had three open reading frames typical of tymoviruses, and displayed a whole-genome nucleotide identity level with the closest tymovirus, Eggplant mosaic virus, at 71% (90% coverage). This tymovirus from naranjilla was able to systemically infect eggplant, tamarillo, N. benthamiana, and naranjilla. In naranjilla, it produced mosaic, chlorotic spots, and stunting, similar to the symptoms observed in the original plant. The virus was unable to infect potato and tobacco and unable to systemically infect pepper plants, replicating only in inoculated leaves. We concluded that this virus represented a new tymovirus infecting naranjilla, and proposed the tentative name Naranjilla chlorotic mosaic virus (NarCMV).


Assuntos
Doenças das Plantas/virologia , Folhas de Planta/virologia , Solanum/virologia , Tymovirus/genética , Genoma Viral , Filogenia
18.
Biomacromolecules ; 18(12): 4141-4153, 2017 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-29144726

RESUMO

Platform technologies based on plant virus nanoparticles (VNPs) and virus-like particles (VLPs) are attracting the attention of researchers and clinicians because the particles are biocompatible, biodegradable, noninfectious in mammals, and can readily be chemically and genetically engineered to carry imaging agents and drugs. When the Physalis mottle virus (PhMV) coat protein is expressed in Escherichia coli, the resulting VLPs are nearly identical to the viruses formed in vivo. Here, we isolated PhMV-derived VLPs from ClearColi cells and carried out external and internal surface modification with fluorophores using reactive lysine-N-hydroxysuccinimide ester and cysteine-maleimide chemistries, respectively. The uptake of dye-labeled particles was tested in a range of cancer cells and monitored by confocal microscopy and flow cytometry. VLPs labeled internally on cysteine residues were taken up with high efficiency by several cancer cell lines and were colocalized with the endolysosomal marker LAMP-1 within 6 h, whereas VLPs labeled externally on lysine residues were taken up with lower efficiency, probably reflecting differences in surface charge and the propensity to bind to the cell surface. The infusion of dye and drug molecules into the cavity of the VLPs revealed that the photosensitizer (PS), Zn-EpPor, and the drugs crystal violet, mitoxantrone (MTX), and doxorubicin (DOX) associated stably with the carrier via noncovalent interactions. We confirmed the cytotoxicity of the PS-PhMV and DOX-PhMV particles against prostate cancer, ovarian and breast cancer cell lines, respectively. Our results show that PhMV-derived VLPs provide a new platform technology for the delivery of imaging agents and drugs, with preferential uptake into cancer cells. These particles could therefore be developed as multifunctional tools for cancer diagnosis and therapy.


Assuntos
Portadores de Fármacos/química , Indicadores e Reagentes/química , Nanopartículas/química , Preparações Farmacêuticas/química , Tymovirus/química , Animais , Linhagem Celular , Linhagem Celular Tumoral , Doxorrubicina/química , Células HeLa , Humanos , Lisina/química , Maleimidas/química , Camundongos , Mitoxantrona/química , Neoplasias/diagnóstico , Neoplasias/diagnóstico por imagem , Fármacos Fotossensibilizantes/química , Células RAW 264.7
19.
PLoS Pathog ; 13(11): e1006714, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29117247

RESUMO

The positive-strand RNA virus Turnip yellow mosaic virus (TYMV) encodes an ovarian tumor (OTU)-like protease/deubiquitinase (PRO/DUB) protein domain involved both in proteolytic processing of the viral polyprotein through its PRO activity, and in removal of ubiquitin chains from ubiquitylated substrates through its DUB activity. Here, the crystal structures of TYMV PRO/DUB mutants and molecular dynamics simulations reveal that an idiosyncratic mobile loop participates in reversibly constricting its unusual catalytic site by adopting "open", "intermediate" or "closed" conformations. The two cis-prolines of the loop form a rigid flap that in the most closed conformation zips up against the other side of the catalytic cleft. The intermediate and closed conformations also correlate with a reordering of the TYMV PRO/DUB catalytic dyad, that then assumes a classical, yet still unusually mobile, OTU DUB alignment. Further structure-based mutants designed to interfere with the loop's mobility were assessed for enzymatic activity in vitro and in vivo, and were shown to display reduced DUB activity while retaining PRO activity. This indicates that control of the switching between the dual PRO/DUB activities resides prominently within this loop next to the active site. Introduction of mutations into the viral genome revealed that the DUB activity contributes to the extent of viral RNA accumulation both in single cells and in whole plants. In addition, the conformation of the mobile flap was also found to influence symptoms severity in planta. Such mutants now provide powerful tools with which to study the specific roles of reversible ubiquitylation in viral infection.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Peptídeo Hidrolases/metabolismo , Tymovirus/enzimologia , Arabidopsis/virologia , Domínio Catalítico/fisiologia , Enzimas Desubiquitinantes/química , Peptídeo Hidrolases/química , Tymovirus/química
20.
Virus Res ; 241: 131-136, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28756104

RESUMO

Application of a severe water deficit to Arabidopsis thaliana plants infected with a mutant of Turnip yellows virus (TuYV, Family Luteoviridae) triggers a significant alteration of several plant phenology traits and strongly reduces the transmission efficiency of the virus by aphids. Although virus accumulation in water-stressed plants was similar to that in plants grown under well-watered conditions, virus accumulation was reduced in aphids fed on plants under water deficit. These results suggest alteration of the aphid feeding behavior on plants under water deficit.


Assuntos
Afídeos/fisiologia , Arabidopsis/virologia , Comportamento Alimentar/fisiologia , Insetos Vetores/virologia , Doenças das Plantas/virologia , Tymovirus/crescimento & desenvolvimento , Privação de Água/fisiologia , Animais , Afídeos/virologia , Secas
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