RESUMO
The Lolium latent virus (LoLV) major coat protein sequence contains a typical chloroplast transit peptide (cTP) domain. In infected Nicotiana benthamiana leaf tissue, LoLV coat proteins can be detected at the chloroplast. In transient expression, several N-terminal deletions of the CP sequence, increasing in length, result in disruption of the domain functionality, markedly affecting intracellular localization. A yeast two-hybrid-based study using LoLV CP as bait identified several potentially interacting Arabidopsis host proteins, most of them with chloroplast-linked pathways. One of them, an ankyrin repeat protein, was studied in detail. The N. benthamiana homologue (NbANKr) targets chloroplasts, is able to co-localize with LoLV CP at chloroplast membranes in transient expression and shows a robust interaction with LoLV CP in vivo by BiFC, which has been confirmed by yeast two-hybrid data. Silencing NbANKr genes in N. benthamiana plants, prior to challenging with LoLV by mechanical inoculation, affects LoLV infection, significantly reducing the level of viral RNA in young leaves, compared to levels in control plants, and suggesting an inhibition of virus movement. Silencing of NbANKr has no obvious effect on plant phenotype, but is able to interfere with LoLV infection, opening the way for a new strategy for virus infection control.
Assuntos
Repetição de Anquirina , Proteínas do Capsídeo/genética , Cloroplastos/virologia , Nicotiana/virologia , Proteínas de Plantas/genética , Vírus de Plantas/genética , Proteínas de Cloroplastos/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Doenças das Plantas/virologia , Folhas de Planta/virologia , Sinais Direcionadores de Proteínas/genética , RNA Viral/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
The Cucumber mosaic virus (CMV) is a three-component isodiametric plant virus with an extremely wide host range, present worldwide. A pseudorecombinant form has been described, deriving from the RNA3 component of the CMV-S strain, carrying the coat protein (CP) gene, and the RNA 1, 2 components of the CMV-D strain. The CP gene was then engineered to express one or two copies of a synthetic peptide derived from many hypervariable region 1 (HVR1) sequences of the Hepatitis C virus (HCV) envelope protein E2 (the so-called R9 mimotope). Study of the symptoms pattern displayed in tobacco by these chimeric CMV particles, together with determination of their structural characteristics, assessed by circular dichroism (CD) spectroscopy and electron microscopy, revealed a possible relationship between the biological behavior and the structural properties of virus components.
Assuntos
Epitopos/química , Hepacivirus/genética , Recombinação Genética , Proteínas do Envelope Viral/química , Vírion , Dicroísmo Circular , Cucumovirus/química , Cucumovirus/genética , Cucumovirus/metabolismo , Cucumovirus/ultraestrutura , Epitopos/genética , Epitopos/metabolismo , Hepacivirus/química , Hepacivirus/metabolismo , Microscopia Eletrônica , Doenças das Plantas/virologia , Folhas de Planta/virologia , Nicotiana/virologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Vírion/química , Vírion/genética , Vírion/metabolismo , Vírion/ultraestruturaRESUMO
Chimeric plant viruses are emerging as promising vectors for use in innovative vaccination strategies. In this context, cucumber mosaic virus (CMV) has proven to be a suitable carrier of the hepatitis C virus (HCV)-derived R9 mimotope. In the present work, a new chimeric CMV, expressing on its surface the HCV-derived R10 mimotope, was produced but lost the insert after the first passage on tobacco. A comparative analysis between R10- and R9-CMV properties indicated that R9-CMV stability was related to structural features typical of the foreign insert. Thus, in order to combine high virus viability with strong immuno-stimulating activity, we doubled R9 copies on each of the 180 coat protein (CP) subunits of CMV. One of the chimeras produced by this approach (2R9-CMV) was shown to systemically infect the host, stably maintaining both inserts. Notably, it was strongly recognized by sera of HCV-infected patients and, as compared with R9-CMV, displayed an enhanced ability to stimulate lymphocyte IFN-gamma production. The high immunogen levels achievable in plants or fruits infected with 2R9-CMV suggest that this chimeric form of CMV may be useful in the development of oral vaccines against HCV.
Assuntos
Proteínas do Capsídeo/imunologia , Cucumovirus/metabolismo , Epitopos/metabolismo , Vetores Genéticos/metabolismo , Hepacivirus/imunologia , Peptídeos/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Vírus Reordenados/metabolismo , Vacinas contra Hepatite Viral/genética , Sequência de Aminoácidos , Especificidade de Anticorpos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Cucumovirus/genética , Cucumovirus/crescimento & desenvolvimento , Epitopos/química , Epitopos/genética , Hepatite C/imunologia , Humanos , Soros Imunes/imunologia , Interferon gama/biossíntese , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Vacinas contra Hepatite Viral/imunologiaRESUMO
The Cucumber mosaic virus Ixora isolate (CMV) coat protein gene (CP) was placed under the transcriptional control of the duplicated subgenomic CP promoter of a Potato virus X (PVX)-based vector. In vitro RNA transcripts were inoculated onto Nicotiana benthamiana plants and recombinant CMV capsid proteins were identified on Western blots probed with CMV antibodies 5-7 days post-inoculation. PVX-produced CMV CP subunits were capable of assembling into virus-like particles (VLPs), which were visualized by electron microscopy. We further used the PVX/CMVCP system for transient expression of recombinant CMV CP constructs containing different neutralizing epitopes of Newcastle disease virus (NDV) engineered into the internal betaH-betaI (motif 5) loop. Both crude plant extracts and purified VLPs were immunoreactive with CMV antibodies as well as with epitope-specific antibodies to NDV, thus confirming the surface display of the engineered NDV epitope. Our study demonstrates the potential of PVX/CMVCP as an expression tool and as a presentation system for promising epitopes.
Assuntos
Proteínas do Capsídeo/biossíntese , Cucumovirus/imunologia , Epitopos/biossíntese , Expressão Gênica , Vetores Genéticos , Potexvirus/genética , Western Blotting , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Cucumovirus/genética , Epitopos/genética , Microscopia Imunoeletrônica , Modelos Moleculares , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/imunologia , RNA Viral/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Recombinação Genética , Nicotiana/química , Nicotiana/genética , Virossomos/isolamento & purificação , Virossomos/ultraestruturaRESUMO
A vaccine against Hepatitis C virus (HCV) is urgently needed due to the unsatisfactory clinical response to current therapies. We evaluated the immunological properties of a chimeric Cucumber mosaic virus (CMV), a plant virus engineered to express on its surface a synthetic peptide derived from many HVR1 sequences of the HCV envelope protein E2 (R9 mimotope). Evidence was obtained that the chimeric R9-CMV elicits a specific humoral response in rabbits. Furthermore, in patients with chronic HCV infection, purified preparations of R9-CMV down-modulated the lymphocyte surface density of CD3 and CD8, and induced a significant release of interferon (IFN)-gamma, interleukin (IL)-12 p70 and IL-15 by lymphomonocyte cultures. Finally, an R9 mimotope-specific CD8 T-cell response, as assessed by intracellular IFN-gamma production, was achieved in the majority of the patients studied. Our results open up new prospects for the development of effective vaccines against HCV infection. Moreover, the wide edible host range of CMV makes the production of an edible vaccine conceivable.
Assuntos
Quimera/imunologia , Cucumovirus/genética , Cucumovirus/imunologia , Epitopos/imunologia , Hepacivirus/imunologia , Vacinas contra Hepatite Viral/imunologia , Adulto , Idoso , Animais , Células Cultivadas , Quimera/genética , Doença Crônica , Citocinas/metabolismo , Epitopos/genética , Feminino , Hepacivirus/genética , Hepatite C/genética , Hepatite C/imunologia , Hepatite C/virologia , Humanos , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Coelhos , Linfócitos T/metabolismo , Linfócitos T/virologiaRESUMO
Cucumber mosaic virus (CMV) is a three component isodiametric plant virus which is common worldwide and has an extremely wide host range. A pseudorecombinant was made, derived from the RNA3 component of the CMV-S strain, carrying the coat protein (CP) gene, and the RNA1,2 components of the CMV-D strain. This system developed mild mosaic and vein clearing in Xanthi tobacco three weeks after inoculation. The CP gene was then engineered in three different positions, to encode a Hepatitis C virus (HCV) epitope. The selected peptide was the so-called R9 mimotope, a synthetic surrogate derived from a consensus profile of many hypervariable region 1 (HVR1) sequences of the putative HCV envelope protein E2. Serum samples from 60 patients with chronic hepatitis C displayed a significant immunoreactivity to crude plant extracts infected with the chimeric CMV. These results suggest that further investigation should be made into a possible vaccine function for the CMV-HCV mimotope system.
