Closely related poleroviruses depend on distinct translation initiation factors to infect Arabidopsis thaliana.

In addition to being essential for translation of eukaryotic mRNA, translation initiation factors are also key components of plant-virus interactions. In order to address the involvement of these factors in the infectious cycle of poleroviruses (aphid-transmitted, phloem-limited viruses), the accumulation of three poleroviruses was followed in Arabidopsis thaliana mutant lines impaired in the synthesis of translation initiation factors in the eIF4E and eIF4G families. We found that efficient accumulation of Turnip yellows virus (TuYV) in A. thaliana relies on the presence of eIF (iso)4G1, whereas Beet mild yellowing virus (BMYV) and Beet western yellows virus-USA (BWYV-USA) rely, instead, on eIF4E1. A role for these factors in the infectious processes of TuYV and BMYV was confirmed by direct interaction in yeast between these specific factors and the 5' viral genome-linked protein of the related virus. Although the underlying molecular mechanism is still unknown, this study reveals a totally unforeseen situation in which closely related viruses belonging to the same genus use different translation initiation factors for efficient infection of A. thaliana.

Phloem protein partners of Cucurbit aphid borne yellows virus: possible involvement of phloem proteins in virus transmission by aphids.

Poleroviruses are phytoviruses strictly transmitted by phloem-feeding aphids in a circulative and nonpropagative mode. During ingestion, aphids sample virions in sieve tubes along with sap. Therefore, any sap protein bound to virions will be acquired by the insects and could potentially be involved in the transmission process. By developing in vitro virus-overlay assays on sap proteins collected from cucumber, we observed that approximately 20 proteins were able to bind to purified particles of Cucurbit aphid borne yellows virus (CABYV). Among them, eight proteins were identified by mass spectrometry. The role of two candidates belonging to the PP2-like family (predominant lectins found in cucurbit sap) in aphid transmission was further pursued by using purified orthologous PP2 proteins from Arabidopsis. Addition of these proteins to the virus suspension in the aphid artificial diet greatly increased virus transmission rate. This shift was correlated with an increase in the number of viral genomes in insect cells and with an increase of virion stability in vitro. Surprisingly, increase of the virus transmission rate was also monitored after addition of unrelated proteins in the aphid diet, suggesting that any soluble protein at sufficiently high concentration in the diet and acquired together with virions could stimulate virus transmission.

A genomic analysis of transcytosis in the pea aphid, Acyrthosiphon pisum, a mechanism involved in virus transmission.

Aphids are the primary vectors of plant viruses. Transmission can occur via attachment to the cuticle lining of the insect (non-circulative transmission) or after internalization in the insect cells with or without replication (circulative transmission). In this paper, we have focused on the circulative and non-propagative mode during which virions enter the cell following receptor-mediated endocytosis, are transported across the cell in vesicles and released by exocytosis without replicating. The correct uptake, transport and delivery of the vesicles cargo relies on the participation of proteins from different families which have been identified in the Acyrthosiphon pisum genome. Assemblage of this annotated dataset provides a useful basis to improve our understanding of the molecules and mechanisms involved in virus transmission by A. pisum and other aphid species.

Cre/loxP-mediated chromosome engineering of the mouse genome.

Together with numerous other genome modifications, chromosome engineering offers a very powerful tool to accelerate the functional analysis of the mammalian genome. The technology, based on the Cre/loxP system, is used more and more in the scientific community in order to generate new chromosomes carrying deletions, duplications, inversions and translocations in targeted regions of interest. In this review, we will present the basic principle of the technique either in vivo or in vitro and we will briefly describe some applications to provide highly valuable genetic tools, to decipher the mammalian genome organisation and to analyze human diseases in the mouse.

Differential epitope tagging of actin in transformed Drosophila produces distinct effects on myofibril assembly and function of the indirect flight muscle.

We have tested the impact of tags on the structure and function of indirect flight muscle (IFM)-specific Act88F actin by transforming mutant Drosophila melanogaster, which do not express endogenous actin in their IFMs, with tagged Act88F constructs. Epitope tagging is often the method of choice to monitor the fate of a protein when a specific antibody is not available. Studies addressing the functional significance of the closely related actin isoforms rely almost exclusively on tagged exogenous actin, because only few antibodies exist that can discriminate between isoforms. Thereby it is widely presumed that the tag does not significantly interfere with protein function. However, in most studies the tagged actin is expressed in a background of endogenous actin and, as a rule, represents only a minor fraction of the total actin. The Act88F gene encodes the only Drosophila actin isoform exclusively expressed in the highly ordered IFM. Null mutations in this gene do not affect viability, but phenotypic effects in transformants can be directly attributed to the transgene. Transgenic flies that express Act88F with either a 6x histidine tag or an 11-residue peptide derived from vesicular stomatitis virus G protein at the C terminus were flightless. Overall, the ultrastructure of the IFM resembled that of the Act88F null mutant, and only low amounts of C-terminally tagged actins were found. In contrast, expression of N-terminally tagged Act88F at amounts comparable with that of wild-type flies yielded fairly normal-looking myofibrils and partially reconstituted flight ability in the transformants. Our findings suggest that the N terminus of actin is less sensitive to modifications than the C terminus, because it can be tagged and still polymerize into functional thin filaments.

A sequence required for -1 ribosomal frameshifting located four kilobases downstream of the frameshift site.

Programmed ribosomal frameshifting allows one mRNA to encode regulate expression of, multiple open reading frames (ORFs). The polymerase encoded by ORF 2 of Barley yellow dwarf virus (BYDV) is expressed via minus one (-1) frameshifting from the overlapping ORF 1. Previously, this appeared to be mediated by a 116 nt RNA sequence that contains canonical -1 frameshift signals including a shifty heptanucleotide followed by a highly structured region. However, unlike known -1 frameshift signals, the reporter system required the zero frame stop codon and did not require a consensus shifty site for expression of the -1 ORF. In contrast, full-length viral RNA required a functional shifty site for frameshifting in wheat germ extract, while the stop codon was not required. Increasing translation initiation efficiency by addition of a 5' cap on the naturally uncapped viral RNA, decreased the frameshift rate. Unlike any other known RNA, a region four kilobases downstream of the frameshift site was required for frameshifting. This included an essential 55 base tract followed by a 179 base tract that contributed to full frameshifting. The effects of most mutations on frameshifting correlated with the ability of viral RNA to replicate in oat protoplasts, indicating that the wheat germ extract accurately reflected control of BYDV RNA translation in the infected cell. However, the overall frameshift rate appeared to be higher in infected cells, based on immunodetection of viral proteins. These findings show that use of short recoding sequences out of context in reporter constructs may overlook distant signals. Most importantly, the remarkably long-distance interaction reported here suggests the presence of a novel structure that can facilitate ribosomal frameshifting.

Virus-induced gene silencing in transgenic plants expressing the minor capsid protein of Beet western yellows virus.

Transgenic Nicotiana benthamiana expressing the minor coat protein P74 of the phloem-limited Beet western yellows virus (BWYV) exhibited an unusual spatial pattern of post-transcriptional gene silencing (PTGS) when infected with BWYV or related viruses. Following infection, transgenic P74 and its mRNA accumulated to only low levels, 21 to 23 nucleotide RNAs homologous to the transgene appeared, and the transgene DNA underwent methylation. The infecting viral RNA, however, was not subject to significant silencing but multiplied readily and produced P74 in the phloem tissues, although the P74 encoded by the transgene disappeared from the phloem as well as the nonvascular tissues.

Cloning full-length cDNA of grapevine chrome mosaic nepovirus.

Full-length cDNA copies of the genomic RNAs of grapevine chrome mosaic virus were obtained and cloned in Escherichia coli by a one-step procedure. The cloning protocol included size selections by agarose-gel electrophoresis of both the single-stranded and the double-stranded full-length cDNAs. First-strand cDNA synthesis was primed with oligodeoxythymidine while second-strand synthesis was primed with specific synthetic oligodeoxynucleotides, allowing cloning of the 3' poly(A) and of the last 5' nucleotides of the viral RNA template. For the 7.2-kb and 4.4-kb viral RNAs, up to 20% and 80%, respectively, of the clones were found to be full-length. Even for large templates, this procedure allows fast and efficient cloning of full-length cDNAs.

A reinvestigation provides no evidence for sugar residues on structural proteins of poleroviruses and argues against a role for glycosylation of virus structural proteins in aphid transmission.

Poleroviruses are strictly transmitted by aphids. Glycosylation of Turnip yellows virus (TuYV) was previously reported and this modification was supposed to be required for aphid transmission. Using different approaches based on (i) a lectin-binding assay, (ii) use of specific complex glycan antibodies and (iii) mass spectrometry, we found no evidence that the structural proteins of TuYV and Cucurbit aphid-borne yellow virus (CABYV) carry glycan residues. Moreover, mutation of each of the four potential N-glycosylation sites of the structural protein sequences of CABYV indicated that, unless more than one site on the structural protein is glycosylated, N-glycosylation is not involved in aphid transmission. These results did not corroborate the previous hypothesis for the role of glycosylation in aphid transmission. They, however, revealed the presence of a glycosylated plant protein in purified polerovirus suspensions, whose function in aphid transmission should be further investigated.

Posterior midgut and hindgut are both sites of acquisition of Cucurbit aphid-borne yellows virus in Myzus persicae and Aphis gossypii.

Members of the family Luteoviridae ('luteovirids') rely strictly on aphid vectors for plant-to-plant transmission. This interaction operates according to a persistent and circulative manner, which implies that the virions are being endocytosed and exocytosed across two epithelial barriers (alimentary tract and accessory salivary glands) in the vector's body. In several luteovirid-aphid vector species combinations, the route of virions in the insect has been investigated ultrastructurally by transmission electron microscopy (TEM). Here, we used TEM to follow the route of Cucurbit aphid-borne yellows virus (CABYV; genus Polerovirus) in its two efficient vector species, Myzus persicae and Aphis gossypii. We demonstrated that CABYV particles are acquired from the gut lumen to the haemocoel through two different sites in both aphid species, i.e. the posterior midgut (as for Beet western yellows virus in M. persicae) and the hindgut (as for Barley yellow dwarf virus complex in cereal aphids). This 'dual' tissue specificity of CABYV represents an original situation among viruses in the family Luteoviridae examined so far by TEM. A variety of virion-containing structures (e.g. clathrin-coated and tubular vesicles, endosome-like bodies) are found in intestinal cells of both types in both aphids. Release of virus particles from midgut and hindgut cells into the haemolymph was confirmed by immunotrapping using CABYV-specific antibodies. In accessory salivary glands, transport of CABYV virions across the cells was similar in each aphid species, and occurred by a transcytosis mechanism involving formation of tubular and coated vesicles before release of free virions in the salivary canal.

Translational frameshifting mediated by a viral sequence in plant cells.

It has been proposed that the polymerase gene of barley yellow dwarf virus and related viruses is expressed by a ribosomal frameshift event during translation. The 5' end of this gene overlaps with the 3' end of an upstream gene that is in a different reading frame. The region of overlap is similar to sequences in retro- and coronaviruses that are known to express their polymerase genes by frameshifting. This overlap region includes a "shifty" heptanucleotide, followed by a highly structured region that may contain a pseudoknot. Sequences of 115 or 144 base pairs that span this region from barley yellow dwarf virus (PAV serotype) genomic RNA were introduced into a plasmid, so that a reporter gene could be expressed in plant cells only if a minus one (-1) frameshift event occurred. Frameshifting was detected at a rate of approximately 1%. This frameshifting was abolished when the stop codon at the 3' end of the upstream open reading frame was deleted. A sequence expected to form a strong stem-loop immediately upstream of the frameshift site was unnecessary for frameshifting, and initiation at AUG codons within the stem-loop appeared to be inhibited. Like viruses that infect hosts in other kingdoms, plant viruses also can induce frameshifting in translation of their genes.

Nucleotide sequence of Hungarian grapevine chrome mosaic nepovirus RNA1.

The nucleotide sequence of the RNA1 of hungarian grapevine chrome mosaic virus, a nepovirus very closely related to tomato black ring virus, has been determined from cDNA clones. It is 7212 nucleotides in length excluding the 3' terminal poly(A) tail and contains a large open reading frame extending from nucleotides 216 to 6971. The presumably encoded polyprotein is 2252 amino acids in length with a molecular weight of 250 kDa. The primary structure of the polyprotein was compared with that of other viral polyproteins, revealing the same general genetic organization as that of other picorna-like viruses (comoviruses, potyviruses and picornaviruses), except that an additional protein is suspected to occupy the N-terminus of the polyprotein.

Precise mapping and in vitro translation of a trifunctional subgenomic RNA of barley yellow dwarf virus.

The barley yellow dwarf virus genome consists of a single 5.7-kb RNA encoding six open reading frames (ORFs). The four ORFs in the 3' half of the genome were proposed to be expressed via subgenomic mRNAs. Here, we show that the PAV serotype of barley yellow dwarf virus generates two subgenomic RNAs of 2.9 and 0.8 kb in infected plants and protoplasts. The 5' end of the larger subgenomic RNA (sgRNA1) was precisely mapped to base 2769 by ribonuclease protection and primer extension methods. Synthetic sgRNA1, containing the exact 5' and 3' ends, was generated by in vitro transcription, translated in vitro, and shown to serve as a message for three ORFs. Translation initiated at the first two AUG codons in sgRNA1, yielding the 22-kDa viral coat protein (CP) and a 17-kDa protein encoded by an overlapping, out-of-frame ORF. In addition, readthrough of the amber stop codon of the CP ORF gave rise to a 72-kDa fusion product of the CP ORF and an in-frame 50K ORF immediately 3' of the CP termination codon. The 0.8-kb sgRNA2 was present in greater abundance than sgRNA1 and likely serves as a message for a 6.7K ORF at the 3' end of the genome. Sequences that may control subgenomic RNA synthesis and the translational events are compared with those of other plant viruses.

Genetically engineered resistance against grapevine chrome mosaic nepovirus.

Nepoviruses are a group of isometric plant viruses with a genome divided between two-single-stranded, positive-sense, RNA molecules. They are usually transmitted by nematodes and a number of them have significant economic impact, especially in perennial crops such as grapevine and fruit trees. Like all other picorna-like viruses, nepoviruses express their coat protein (CP) as part of a larger polyprotein which is further processed by a virus-encoded protease, a feature which poses specific problems when trying to express the viral coat protein in transgenic plants. A hybrid gene, driving the high-level expression of the CP of grapevine chrome mosaic nepovirus (GCMV) has been constructed and transferred to the genome of tobacco plants. Progeny of CP-expressing transformants show resistance against GCMV. When compared to control plants, fewer inoculated plants become infected and those that become infected accumulate reduced levels of viral RNAs. This protection was also shown to be efficient when plants are inoculated with purified viral RNA.

Substitution of flight muscle-specific actin by human (beta)-cytoplasmic actin in the indirect flight muscle of Drosophila.

The human (beta)-cytoplasmic actin differs by only 15 amino acids from Act88F actin which is the only actin expressed in the indirect flight muscle (IFM) of Drosophila melanogaster. To test the structural and functional significance of this difference, we ectopically expressed (beta)-cytoplasmic actin in the IFM of Drosophila that lack endogenous Act88F. When expression of the heterologous actin was regulated by approximately 1.5 kb of the 5' promoter region of the Act88F gene, little (beta)-cytoplasmic actin accumulated in the IFM of the flightless transformants. Including Act88F-specific 5' and 3' untranslated regions (UTRs) yielded transformants that expressed wild-type amounts of (beta)-cytoplasmic actin. Despite the assembly of (beta)-cytoplasmic actin containing thin filaments to which endogenous myosin crossbridges attached, sarcomere organization was deficient, leaving the transformants flightless. Rather than affecting primarily actin-myosin interactions, our findings suggest that the (beta)-cytoplasmic actin isoform is not competent to interact with other actin-binding proteins in the IFM that are involved in the organization of functional myofibrils.

Nucleotide sequence and genetic organization of Hungarian grapevine chrome mosaic nepovirus RNA2.

The complete nucleotide sequence of hungarian grapevine chrome mosaic nepovirus (GCMV) RNA2 has been determined. The RNA sequence is 4441 nucleotides in length, excluding the poly(A) tail. A polyprotein of 1324 amino acids with a calculated molecular weight of 146 kDa is encoded in a single long open reading frame extending from nucleotides 218 to 4190. This polyprotein is homologous with the protein encoded by the S strain of tomato black ring virus (TBRV) RNA2, the only other nepovirus sequenced so far. Direct sequencing of the viral coat protein and in vitro translation of transcripts derived from cDNA sequences demonstrate that, as for comoviruses, the coat protein is located at the carboxy terminus of the polyprotein. A model for the expression of GCMV RNA2 is presented.

32P- and biotin-labelled in vitro transcribed cRNA probes for the detection of potato spindle tuber viroid and chrysanthemum stunt viroid.

Replacing nick-translated DNA probes by in vitro transcribed complementary RNA (cRNA) probes considerably increased the sensitivity of dot-blot detection tests of potato spindle tuber viroid and chrysanthemum stunt viroid. As compared to the limit of detection of 5-10 pg of viroid obtained with 32P-labelled DNA probes, cRNA probes allow the detection of less than 1 pg of pure viroid. When labelled with biotin by incorporation of biotin-labelled ribonucleotides, the cRNA probes have a limit of detection of approximately 5 pg of purified viroid.

Effects of point mutations in the major capsid protein of beet western yellows virus on capsid formation, virus accumulation, and aphid transmission.

Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected approximately 90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a "reverse" substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid.

Effects of point mutations in the readthrough domain of the beet western yellows virus minor capsid protein on virus accumulation in planta and on transmission by aphids.

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.

Aphid transmission of beet western yellows luteovirus requires the minor capsid read-through protein P74.

Beet western yellows luteovirus is obligately transmitted by the aphid Myzus persicae in a circulative, non-propagative fashion. Virus movement across the epithelial cells of the digestive tube into the hemocoel and from the hemocoel into the accessory salivary glands is believed to occur by receptor-mediated endocytosis and exocytosis. Virions contain two types of protein; the major 22 kDa capsid protein and the minor read-through protein, P74, which is composed of the major capsid protein fused by translational read-through to a long C-terminal extension called the read-through domain. Beet western yellows virus carrying various mutations in the read-through domain was tested for its ability to be transmitted to test plants by aphids fed on agro-infected plants and semi-purified or purified virus preparations. The results establish that the read-through domain carries determinants that are essential for aphid transmission. The findings also reveal that the read-through domain is important for accumulation of the virus in agro-infected plants.