Discovery of a Small Non-AUG-Initiated ORF in Poleroviruses and Luteoviruses That Is Required for Long-Distance Movement.

Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5' end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement.ES and WAM were financed through a Gutenberg Chair (Région Alsace) grant awarded to WAM. Work in the AEF lab was funded by grants from the Wellcome Trust (088789) and the UK Biotechnology and Biological Research Council (BBSRC) (BB/J007072/1 and BB/J015652/1). WAM was also funded by a Fulbright Foundation Research Scholarship and grant number 5R01GM067104-09 from the NIH Institute of General Medical Sciences.This is the final version. It was first published by PLOS at http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1004868#ack

PLoS Pathog

Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5' end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement

Les polerovirus (Mode d'action de leur suppresseur et approche génétique du tropisme phloémien)

Le RNA silencing est un mécanisme utilisé par les plantes comme moyen de lutte antivirale. Pour contrecarrer ce système de défense, les phytovirus ont développé des protéines dites suppresseurs de RNA silencing capables d inhiber une ou plusieurs des étapes de la cascade réactionnelle du RNA silencing. Ce mémoire présente l étude du mode d action de l une de ces protéines, la protéine P0 des polerovirus. Cette protéine possède un motif F-box par lequel elle interagit avec les protéines SKP dans un complexe E3 ubiquitine ligase. Par différentes approches d expression ectopiques, nous avons montré qu elle est capable d inhiber le RNA silencing en induisant la déstabilisation d une des protéines essentielles de ce mécanisme, la protéine AGO1. La deuxième partie de ce mémoire décrit une approche génétique visant à élucider les mécanismes impliqués dans le tropisme phloémien des polerovirus. Si elle n a pas apporté de réponse à la question initiale, cette analyse a permis d identifier certains des facteurs impliqués dans la défense contre les polerovirus chez la plante modèle Arabidopsis thaliana. Notamment, l implication de la protéine DCL2 dans le clivage du génome viral a pu être mise en évidence. Cette découverte indique une probable inhibition de l activité de la protéine DCL4 lors de l infection virale, cette protéine ayant été caractérisée à plusieurs reprises comme agissant de manière prioritaire à DCL2. Ces données suggèrent l existence potentielle d un deuxième suppresseur de RNA silencing codé par les polerovirus.RNA silencing is a mechanism used by plants as an antiviral defense. As a counterpart, plant viruses have developed proteins that can suppress this mechanism. This manuscript describes the study of the mode of action of the one encoded by polerovirus, the P0 protein. This protein carries an F-box motif which allows it to interact with SKP protein in an E3 ubibiquitine ligase complex. Using different approaches where P0 was expressed ectopically, we found that this viral protein is able to suppress RNA silencing by destabilization of AGO1, one of the major actors of RNA silencing. The second part of this manuscript describes a genetic approach used in order to elucidate the mechanisms that restrict poleroviruses into phloem cells. If no clear answer to this question could be drawn, these experiments allowed us to identify some cellular factors implicated in antiviral response against poleroviruses in the model plant Arabidopsis thaliana. In particular, the implication of DCL2 in the cleavage of the viral genome has been underlined. This discovery indicates a possible inhibition of the DCL4 activity during viral infection because this protein has been identified several times as the first actor in antiviral defense, acting primarily to DCL2. This data suggests that poleroviruses might encode a second suppressor of RNA silencing.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Role of vector-transmission proteins

International audienceMost phytoviruses rely on vectors for their spread and survival. Although a great variety of virus vectors have been described, there are relatively few different mechanisms mediating virus transmission by vectors: virions can either be internalized into vector cells where replication may or may not take place or they can simply be adsorbed on the vector's surface or cuticle. Virus transmission by vectors requires tight associations between viral proteins, generally capsid proteins, and vector compounds, usually referred to as receptors. This review will focus on the viral determinants involved in virus transmission. Only the best-known models for which molecular data are available are described

Les polérovirus (étude fonctionnelle de la protéine PO, suppresseur de RNA silencing, et nouvelle analyse de leur tropisme phloémien)

Le mécanisme de RNA silencing représente un mécanisme de défense antiviral majeur chez les plantes qui est déclenché par la présence d'ARN double brin. Reconnu par les protéines Dicer, cet ARN est coupé en siRNA qui seront intégrés dans un complexe RISC (RNA induced silencing complex) et serviront de guide pour le clivage des ARN homologues. Les phytovirus codent pour diverses protéines suppresseurs afin d'interférer avec ce mécanisme de défense.La protéine P0 des polérovirus, virus à ARN simple brin de polarité positive, est capable d'inhiber le RNA silencing en induisant la déstabilisation des protéines ARGONAUTEs (AGO), les principaux composants du complexe RISC. La protéine P0 interagit aussi avec les protéines SKP-like de plantes grâce à son motif F-box-like, une interaction qui semble requise à sa fonction de suppresseur. Plusieurs protéines P0 mutées, provenant de deux polérovirus différents, ont été analysées. Leur activité de suppresseur du silencing, leur interaction avec les protéines SKP et leur capacité à déstabiliser les protéines AGO ont été testées et leur localisation subcellulaire a été déterminée. Ces expériences ont permis d'identifier des domaines importants pour la fonction des protéines et ont révélé des différences étonnantes entre les deux protéines P0.Le tropisme phloémien des polérovirus a été analysé en utilisant des plantes transgéniques, exprimant le cDNA complet du Turnip yellows virus sous le contrôle du promoteur constitutif 35S. Les résultats obtenus donnent de nouvelles indications sur le silencing induit contre les polérovirus et soulèvent des questions intéressantes quant au mécanisme de synthèse de l'ARN subgénomique de ces virus.RNA silencing is a major antiviral defense mechanism in plants. Viral double-stranded RNAs are cleaved into siRNA duplexes by Dicer proteins and subsequently incorporated into the RNA induced silencing complex (RISC), which guides sequence specific degradation of homologous RNAs. In order to counteract this plant defense, most plant viruses evolved silencing suppressor proteins.Poleroviruses, a group of single-stranded RNA plant viruses belonging to the Luteoviridae family, encode the strong silencing suppressor P0. This protein interacts with plant SKP-like proteins through an F-box-like motif, which appears important for its function. Recently, it was demonstrated that P0 destabilizes ARGONAUTE (AGO) proteins, key members of RISC. Their degradation leads to the inhibition of the silencing mechanism.In this work we designed several mutants of P0 proteins from two different poleroviruses. The mutant proteins were tested for their silencing suppressor activity, SKP interaction, their ability to induce AGO destabilization and their subcellular localization. These experiments allowed to determine several domains required for P0's function and revealed striking differences between the two P0 proteins.The particular phloem-limited tropism of the Luteoviridae was analyzed in the second part of this thesis. The use of transgenic Arabidopsis plants, expressing a full-length Turnip yellows virus cDNA clone under the control of the constitutive 35S promoter, led to interesting conclusions regarding the silencing response induced against poleroviruses. The results presented here also raise questions about the mechanism of subgenomic RNA synthesis.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Viral determinants involved in luteovirus - aphid interactions

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Phloem specific Virus-Induced Gene-Silencing using a Recombinant polerovirus

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Etude fonctionnelle d’un isolat cloné du virus de la jaunisse modérée de la betterave ou BMYV

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Poleroviruses: New insights into the mystery of phloem restriction

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