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International audience; En plus d'être essentiel pour la traduction de l'ARNm eucaryote, facteurs d'initiation de traduction sont également des éléments clés des interactions plante-virus. Afin de répondre à l'implication de ces facteurs dans le cycle infectieux de poleroviruses (transmis par les pucerons, les virus limité au phloème), l'accumulation de trois poleroviruses a été suivie en lignées mutantes d'Arabidopsis thaliana ayant une déficience dans la synthèse des facteurs d'initiation de traduction dans le eIF4E et eIF4G familles. Nous avons constaté que l'accumulation efficace de virus de la jaunisse du navet (TuYV) chez A. thaliana repose sur la présence de eIF (iso) 4G1, alors betterave doux virus jaunissement (BMYV) et betteraves jaunes de l'Ouest virus USA (BWYV-USA) comptent, au lieu , sur eIF4E1. Un rôle de ces facteurs dans les processus infectieux de TuYV et BMYV a été confirmée par une interaction directe dans la levure entre ces facteurs spécifiques et l'extrémité 5 'du génome viral lié à une protéine du virus apparenté. Bien que le mécanisme moléculaire sous-jacente est encore inconnue, cette étude révèle une situation totalement imprévue dans lequel des virus appartenant au même genre d'utilisation différents facteurs d'initiation de traduction pour une infection efficace de A. thaliana étroitement liés.

BGPI : équipe 2; International audience; A fluorescent viral clone of the polerovirus Turnip yellows virus (TuYV) was engineered by introducing the Enhanced Green Fluorescent Protein (EGFP) sequence into the non-structural domain sequence of the readthrough protein, a minor capsid protein. The resulting recombinant virus, referred to as TuYV-RTGFP, was infectious in several plant species when delivered by agroinoculation and invaded efficiently non-inoculated leaves. As expected for poleroviruses, which infect only phloem cells, the fluorescence emitted by TuYV-RTGFP was restricted to the vasculature of infected plants. In addition, TuYV-RTGFP was aphid transmissible and enabled the observation of the initial sites of infection in the phloem after aphid probing in epidermal cells. The aphid-transmitted virus moved efficiently to leaves distant from the inoculation sites and importantly retained the EGFP sequence in the viral genome. This work reports on the first engineered member in the Luteoviridae family that can be visualized by fluorescence emission in systemic leaves of different plant species after agroinoculation or aphid transmission

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., Author Summary In order to maximize coding capacity, RNA viruses often encode overlapping genes and use unusual translational control mechanisms. Plant viruses express proteins required for movement of the virus through the plant, often from non-canonically translated open reading frames (ORFs). Viruses in the economically important Luteoviridae family are confined to the phloem (vascular) tissue, probably due to their specialized phloem-specific movement proteins. These proteins are translated from one viral mRNA, sgRNA1, via initiation at more than one AUG codon to express overlapping genes, and by ribosomal read-through of a stop codon. Here, we describe yet another gene translated from sgRNA1, ORF3a. Translation of ORF3a initiates at a non-standard (not AUG) start codon. We found that ORF3a is not required for viral genome replication, but is required for long-distance movement of the virus in the plant. The movement function could be restored in trans by providing the ORF3a product, P3a, from another viral or plasmid vector. P3a localizes in the Golgi apparatus and adjacent to the plasmodesmata, supporting a role in intercellular movement. In summary, we used a powerful bioinformatic tool to discover a cryptic gene whose product is required for movement of a phloem-specific plant virus, revealing multiple levels of translational control that regulate expression of four proteins from a single mRNA.

Aphid transmission of poleroviruses is highly specific, but the viral determinants governing this specificity are unknown. We used a gene exchange strategy between two poleroviruses with different vectors, Beet western yellows virus (BWYV) and Cucurbit aphid-borne yellows virus (CABYV), to analyze the role of the major and minor capsid proteins in vector specificity. Virus recombinants obtained by exchanging the sequence of the readthrough domain (RTD) between the two viruses replicated in plant protoplasts and in whole plants. The hybrid readthrough protein of chimeric viruses was incorporated into virions. Aphid transmission experiments using infected plants or purified virions revealed that vector specificity is driven by the nature of the RTD. BWYV and CABYV have specific intestinal sites in the vectors for endocytosis: the midgut for BWYV and both midgut and hindgut for CABYV. Localization of hybrid virions in aphids by transmission electron microscopy revealed that gut tropism is also determined by the viral origin of the RTD.

Point mutations were introduced into or near five conserved sequence motifs of the readthrough domain of the beet western yellows virus minor capsid protein P74. The mutant virus was tested for its ability to accumulate efficiently in agroinfected plants and to be transmitted by its aphid vector, Myzus persicae . The stability of the mutants in the agroinfected and aphid-infected plants was followed by sequence analysis of the progeny virus. Only the mutation Y201D was found to strongly inhibit virus accumulation in planta following agroinfection, but high accumulation levels were restored by reversion or pseudoreversion at this site. Four of the five mutants were poorly aphid transmissible, but in three cases successful transmission was restored by pseudoreversion or second-site mutations. The same second-site mutations in the nonconserved motif PVT(32-34) were shown to compensate for two distinct primary mutations (R24A and E59A/D60A), one on each side of the PVT sequence. In the latter case, a second-site mutation in the PVT motif restored the ability of the virus to move from the hemocoel through the accessory salivary gland following microinjection of mutant virus into the aphid hemocoel but did not permit virus movement across the epithelium separating the intestine from the hemocoel. Successful movement of the mutant virus across both barriers was accompanied by conversion of A59 to E or T, indicating that distinct features of the readthrough domain in this region operate at different stages of the transmission process.

International audience; The nucleotide sequence of the genomic RNA (5641 nt) of beet western yellows virus (BWYV) isolated from lettuce has been determined and its genetic organization deduced. The sequence of the 3'terminal 2208 nt of RNA of a second BWYV isolate, obtained from sugarbeet, was also determined and was found to be very similar but not identical to that of the lettuce isolate. The complete sequence of BWYV RNA contains six long open reading frames (ORFs). A cluster of three of these ORFs, including the coat protein cistron, display extensive amino acid sequence homology with corresponding ORFs of a second luteovirus, the PAV isolate of barley yellow dwarf virus (BYDV) (1,2). The ORF corresponding to the putative viral RNA-dependant RNA polymerase, on the other hand, resembles that of southem bean mosaic virus. There is circumstantial evidence that expression of the BWYV RNA polymerase ORF may involve a translational frameshift mechanism. The ORF immediately following the coat protein cisuon may be translated by in-frame readthrough of the coat protein cisnon amber termination codon. Similar mechanisms have been proposed for expression of the corresponding ORFs of BYDV(PAV) (1).