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Sequiviruses are isometric aphidborne plant viruses. Dandelion yellow mosaic virus (DaYMV), genus Sequivirus, was isolated from dandelion and lettuce in Europe. Lettuce mottle virus (LeMoV), a putative sequivirus, is often found in mixed infections with Lettuce mosaic virus (LMV) in Brazil (3). DaYMV, LeMoV and LMV cause similar mosaics in field-grown lettuce. Differences in biology and sequence suggest that DaYMV and LeMoV are distinct species (2). Forty-two and 101 lettuce samples with mosaic symptoms collected from two locations near Santiago during a survey of lettuce viruses in Chile in 2002 and 2003, respectively, were analyzed for the presence of LeMoV using reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted (1) and used for RT-PCR with the specific LeMoV primers pairs Lmo3 (5′ ACATGAGCACTAGTGAGG 3′) and Lmo4 (5′ AGATAGAGCCGTCT GGCG 3′) (2). One of the 42 and three of the 101 samples produced the expected 300-bp fragment. Isometric particles of 30 nm diameter, typical of a sequivirus, were visualized by transmission electron microscopy. These samples were tested using RT-PCR for the presence of LMV and Cucumber mosaic virus (CMV), but no mixed infections were observed. One isolate, Ch36, was reamplified with the degenerate primer pairs DALE 1 (5′ GARTTCAACATGCACGCCAG 3′) and DALE 2 (5′ TTTTTCTCCCCATYCGTCAT 3′) which amplify part of the putative replicase gene (2) and produced a 563-bp fragment that was cloned on pGEM-T Easy (Promega, Madison, WI) and sequenced. The Ch36 product (EMBL Accession No. AM039965) showed 97% amino acid identity with LeMoV from Brazil, 79% with DaYMV, 72% with the sequivirus Parsnip yellow fleck virus, and 34% with the waikavirus Maize chlorotic dwarf virus. To our knowledge, this is the first report of a sequivirus in field lettuce in Chile, and although the virus was found at low incidence, this report extends the range of LeMoV to the western side of the Cordillera de Los Andes. The impact of LeMoV needs to be further analyzed in Chile, Brazil, and possibly other South American countries. References: (1) Y. D. Bertheau et al. DNA amplification by polymerase chain reaction (PCR) 1998. In: Methods for the Detection and Quantification of Erwinia carotovora subsp. atroseptica on potatoes. M. C. N. Perombelon and J. M. van der Wolff, eds. Scott. Crop Res. Inst. Occasional Publ., Dundee, 1998. (2) A. S. Jadão. Caracterização parcial e desenvolvimento de oligonucleotídeos específicos para detecção de sequivirus infectando alface. Ph.D. thesis. FCA-UNESP-Botucatu, Brazil, 2004. (3) O. Stangarlin et al. Plant Dis. 84:490, 2000.

The potyvirus Lettuce mosaic virus (LMV) is a common pathogen of lettuce crops worldwide, but it also infects other Asteraceae spp. including ornamentals (2,3,4). Cape daisies (Osteospermum sp.) are widely grown perennial ornamentals reported to be natural hosts of LMV (2,4), which causes faint leaf mosaic and sometimes mild flower breaking. A preliminary observation of mosaic symptoms prompted a large-scale survey during the spring of 2005 in Cape daisies grown in the Tunis metropolitan area and the south of Tunisia (Djerba, Medenine). Two hundred seventy-one samples (Tunis: 14 sites, 219 samples; South: 9 sites, 52 samples) were randomly collected from nurseries, roadway plantings, and home gardens and analyzed. Ninety-three samples (Tunis: 40%, South: 12%; overall: 34%) showed distinct mosaic symptoms. LMV infection was verified by immuno-tissue printing on all collected samples (1), providing evidence for even higher infection levels (Tunis: 60%; South: 25%; overall: 56%). This technique, therefore, allowed the detection of symptomless infection in a significant proportion of samples. It should however, be stressed that symptoms can be very difficult to observe in water-stressed plants, a situation frequently observed in Tunisia. Subsequent PCR analysis with LMV-specific primers (1) of a subset of 24 symptomatic and tissue-print-positive samples confirmed LMV infection in all cases. This is to our knowledge, the first report of LMV infection in Cape daisies in Tunisia. The very high rate of infection observed suggests that these popular ornamentals might constitute a reservoir of LMV as previously reported in the United States (4). References: (1) H. Fakhfakh et al. J. Plant Pathol. 83:3, 2001. (2) R. Jordan and M. Guaragna. (Abstr.) Phytopathology 96(suppl.):S56, 2006. (3) O. Le Gall. No. 399 in: Description of Plant Viruses. A. T. Jones et al., eds. CMI/AAB, Kew, Surrey, UK, 2003. (4) D. C. Opgenorth et al. Plant Dis. 75:751, 1991.

Tomato black ring virus (TBRV) belongs to the nepoviruses, an important genus of phytoviruses characterized by isometric particles and by their transmission by longidorid nematodes. As for all other nepoviruses, the coat protein (CP) of TBRV is encoded by the 3′ terminal part of the viral RNA2 (positions 2801–4334). A hybrid gene driving the expression of a truncated form of the TBRV CP was constructed. It contains a frameshift deletion at position T4065 so that in the encoded protein the last 90 amino acids of the wild-type CP are replaced by 52 amino acids encoded by a different reading frame of the viral RNA. This hybrid gene was introduced into the genome of Nicotiana tabacum cv 'Xanthi' plants. When compared to control plants, progeny of some transformants expressing the mutated CP gene (CPm+ plants) showed resistance against TBRV infection. This resistance is characterized by a delay in the appearance of symptoms, a reduction in the number of infected plants and a reduction in virus accumulation.

Using recombinant proteins produced in bacteria or in infected plants, interactions between the VPg and HcPro of Lettuce mosaic potyvirus (LMV) and between LMV VPg and the lettuce translation initiation factor 4E, the cap-binding protein (eIF4E), were demonstrated in vitro. Interaction with eIF4E and HcPro both involved the same VPg central domain. The structure of this domain in the VPg context was predicted to include an amphiphilic α-helix, with the amino acids related to biological functions in various potyviruses exposed at the hydrophilic side.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Lettuce mottle virus (LeMoV) and dandelion yellow mosaic virus (DaYMV) infect lettuce in South America and Europe, respectively. LeMoV and DaYMV possess isometric particles, occur at low concentrations in plants and have narrow host ranges. Partial genome sequences of both viruses were obtained using purified viral preparations and universal primers for members of the family Sequiviridae. DaYMV and LeMoV sequences were analyzed and showed identity with other members of the family. Universal primers that detect both viruses and specific primers for LeMoV and DaYMV were designed and used in RT-PCR-based diagnostic assays. These results provide the first molecular data on the LeMoV and DaYMV genomes and suggest that LeMoV is a member of the genus Sequivirus, probably distinct from DaYMV.

Twelve Arabidopsis accessions were challenged with Plum pox potyvirus (PPV) isolates representative of the four PPV strains. Each accession supported local and systemic infection by at least some of the PPV isolates, but high variability was observed in the behavior of the five PPV isolates or the 12 Arabidopsis accessions. Resistance to local infection or long-distance movement occurred in about 40% of all the accession-isolate combinations analyzed. Except for Nd-1, all accessions showed resistance to local infection by PPV-SoC; in the Landsberg erecta (Ler) accession, this resistance was compromised by sgt1 and rar1 mutations, suggesting that it could be controlled by an R gene-mediated resistance pathway. While most of the susceptible accessions were symptomless, PPV induced severe symptoms on inflorescences in C24, Ler, and Bay-0 as early as 15 days after inoculation. Genetic analyses indicated that these interaction phenotypes are controlled by different genetic systems. The restriction of long-distance movement of PPV-El Amar and of another member of genus Potyvirus, Lettuce mosaic virus, in Col-0 requires the RTM genes, indicating for the first time that the RTM system may provide a broad range, potyvirus-specific protection against systemic infection. The restriction to PPVPS long-distance movement in Cvi-1 is controlled by a single recessive gene, designated rpv1, which was mapped to chromosome 1. The nuclear inclusion polymerase b-capsid protein region of the viral genome appears to be responsible for the ability of PPV-R to overcome rpv1-mediated resistance.

The Sharka disease caused by the potyvirus Plum pox virus (PPV) is one of the most serious viral diseases affecting stone fruit trees. The study of PPV/Prunus interaction under greenhouse controlled conditions is space, time, labor consuming. While the PPV/Prunus interactions are now quite well known at the whole plant level, few data however are available on the interactions between the virus and the Prunus host plants at the cellular level. Using a green fluorescent protein (GFP)-tagged M type PPV strain, combined to an in vitro inoculation procedure, we developed a novel tool to track PPV invasion in Prunus persica (peach) cv. GF305 and Prunus armeniaca (apricot) cv. Screara susceptible hosts. Different graft combinations were performed using in vitro-maintained healthy or GFP-tagged PPV infected 'GF305' and 'Screara'. Contact for 30 days in grafts between the inoculum and the genotype to be tested were found sufficient to allow the systemic spread of the recombinant virus: fluorescence from GFP-tagged PPV could easily be detected in the entire plant under a binocular microscope allowing quick and reliable sorting of infected plants. Using a fluorescence stereomicroscopy or confocal microscopy, GFP could also be observed in stem cross-sections especially in epidermis and pith cells. In vitro grafting inoculation with GFP-tagged PPV provides a new and powerful tool to facilitate mid-term virus maintenance. Moreover, this tool will be of special importance in the study of PPV infection dynamics in Prunus, allowing as well precise observations of cellular events related to PPV/Prunus interactions.

International audience; Full-length cDNA clones corresponding to the RNA1 and RNA2 of the Polish isolate MJ of Tomato black ring virus (TBRV, genus Nepovirus) were obtained using a direct recombination strategy in yeast, and their complete nucleotide sequences were established. RNA1 is 7358 nucleotides and RNA2 is 4633 nucleotides in length, excluding the poly(A) tails. Both RNAs contain a single open reading frame encoding polyproteins of 254thinspkDa and 149thinspkDa for RNA1 and RNA2 respectively. Putative cleavage sites were identified, and the relationships between TBRV and related nepoviruses were studied by sequence comparison.

The RNA genome of a resistance-breaking isolate of Lettuce mosaic virus (LMV-E) was engineered to express the jellyfish green fluorescent protein (GFP) or β-glucuronidase (GUS) fused to the helper-component proteinase (HC-Pro) to study LMV invasion and spread in susceptible and resistant lettuce cultivars. Virus accumulation and movement were monitored by either histochemical GUS assays or detection of GFP fluorescence under UV light. The GFP- and GUS-tagged viruses spread systemically in the susceptible lettuce cultivars Trocadero and Vanguard, where they induced attenuated symptoms, compared with the wild-type virus. Accumulation of the GFP-tagged virus was reduced but less affected than in the case of the GUS-tagged virus. Systemic movement of both recombinant viruses was very severely affected in Vanguard 75, a lettuce cultivar nearly isogenic to Vanguard but carrying the resistance gene mo12. Accumulation of the recombinant viruses in systemically infected leaves was either undetectable (GUS-tag) or erratic, strongly delayed, and inhibited by as much as 90% (GFP-tag). As a consequence, and contrary to the parental virus, the recombinant viruses were not able to overcome the protection afforded by the mo12gene. Taken together, these results indicate that GUS or GFP tagging of the HC-Pro of LMV has significant negative effects on the biology of the virus, abolishing its resistance-breaking properties and reducing its pathogenicity in susceptible cultivars.

International audience; Lettuce mosaic virus (LMV) is an economically important Potyvirus causing a severe disease of commercial lettuce crops. Based on molecular data, three phylogenetic groups of isolates have previously been discriminated, reflecting their geographical origin (Western Europe-California, Greece, or Yemen). Sequence information for the entire coat protein domain was only available for one of the Western Europe-California phylogenetic group. We have now sequenced the 3′ terminal region of the genome LMV-Gr4, -Gr5 and -GrB, isolates which belong to the Greek phylogenetic group and of LMV-Yar, the sole known representative of the third LMV phylogenetic group. The region sequenced encodes the last 62 amino-acids of the polymerase and the entire coat protein of the four isolates, plus the 3′ non-translated region of LMV-Gr5 and -Yar. The Greek and Yemenite isolates studied are all very aggressive on lettuce, are able to overcome the resistance genes mo1 1 and mo1 2 and belong to the two phylogenetic groups which have so far been only partially characterised. As for other Potyviruses, the core and the C-terminal regions of the coat protein are highly conserved among all isolates whereas the N-terminus is more variable. No amino acid change in the coat protein or carboxy-terminal part of the polymerase could be related to the resistance-breaking properties of the isolates analysed. The sequences obtained provide the basis for the rapid typing of LMV isolates using the restriction pattern of segments of cDNA amplified by PCR.

International audience; A full length cDNA copy of the genomic RNA of lettuce mosaic virus (LMV) was constructed under the control of an enhanced CaMV 35S promoter and of the NOS terminator. This construct was found infectious when inoculated to lettuce plants. The intron II of the bean nitrite reductase gene was engineered into the LMV FL cDNA in order to relieve possible deleterious effects of viral sequences to Escherichia coli cells and to evaluate the effects of the presence of the intron on the FL cDNA infectivity. The intron-less FL cDNA was found to be as stable as its intron-containing counterpart in E. coli. Sequence analysis of progeny RNA derived from plants inoculated with the intron-containing FL cDNA demonstrated that the inserted intron was perfectly spliced out. The symptoms induced in lettuce by either the intron-less or the intron-containing constructs were identical to those caused by the wild-type virus. However a slight delay in the establishment of infection in lettuce and a more obvious lag in Nicotiana benthamiana were observed with the intron-containing FL cDNA.

International audience; A virus-induced, viral RNA-specific, RNA-dependent-RNA-poly-merase activity has been observed in vitro associated with membrane extracts of plants of three different species after infection with grapevine chrome mosaic nepovirus (GCMV). The products of this activity are full-length, positive sense GCMV RNAs present in double-stranded structures. As has been demonstrated for a relative to nepoviruses, cowpea mosaic comovirus, this activity probably corresponds to the nepoviral replication complex.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Recessive resistance to lettuce mosaic virus (LMV) is conferred in lettuce by the mo1 gene, encoding the eukaryotic translation initiation factor 4E (eIF4E). The C terminus of the viral cylindrical inclusion helicase (CI-Cter), together with the VPg, is involved directly in overcoming mo1 resistance. In this study, recombinant LMV VPg and CI-Cter proteins from wild-type or resistance-breaking isolates were expressed and purified from Escherichia coli. The allelic forms of eIF4E from susceptible or resistant lettuce cultivars were produced similarly and these proteins were used in ELISA-based assays to demonstrate the in vitro binding of the various forms of LMV CI-Cter to both lettuce eIF4E and LMV VPg proteins. All combinations tested displayed significant and specific interactions, and the interaction between the C-terminal part of the LMV CI and eIF4E was confirmed in vivo in bimolecular fluorescence complementation assays. Higher interaction signals for both CI–eIF4E and CI–VPg were observed for LMV-E, indicating that the eIF4E interaction network involving CI and VPg appears to be stronger in the case of this resistance-breaking isolate. This could suggest the need for a minimal interaction threshold for infection success in resistant lettuce, but more precise measurement of the interaction parameters linking eIF4E, VPg and CI is needed in order to reinforce such a hypothesis.

Transgenic plants of the grapevine rootstock 110 Richter (Vitis Berlandieri × V. rupestris) were regenerated from embryogenic cultures co-cultivated with a disarmed LBA4404 strain of Agrobacterium tumefaciens harbouring a binary vector which contained chimeric genes for hygromycin resistance (HPT), kanamycin resistance (NPT II), β-glucuronidase (GUS) and the coat protein of grapevine chrome mosaic nepovirus (GCMV-CP). The best rate of transformation was obtained by selection of putative embryogenic tissues on 16 μg/ml hygromycin. GUS activity was readily observed in somatic embryos and in transformed plants derived from them. Southern blot analysis demonstrated the integration of the T-DNA sequence into the plant genome. High levels of GCMV-CP expression were detected by ELISA in somatic embryos, leaves and roots of the transformed plants, and confirmed by Western blot.

Fragments of the putative non-structural proteins (44K and 46K) encoded by RNA2 of grapevine chrome mosaic nepovirus (GCMV) were expressed as fusion proteins in Escherichia coli and used to raise specific antisera. All three proteins encoded by GCMV RNA2 (viral coat protein, and the 44K and 46K proteins) were detected by immunoblotting in subcellular fractions prepared from the leaves of infected Chenopodium quinoa plants, confirming a previously proposed model of the GCMV RNA2-encoded polyprotein. In addition to the 44K protein, one of the antisera detected a 90K protein presumably representing a precursor of the 44K and 46K proteins. Whereas the 44K and coat proteins could be detected in both soluble and membrane fractions, the 46K protein was found to be specific to the membrane fraction. Analysis of the kinetics of accumulation of the proteins showed that the 44K and 46K proteins were very transient whereas the coat protein was more stable and could be detected up to 21 days after inoculation. These results provide the first direct in vivo data supporting the maturation map of the GCMV RNA2 polyprotein deduced from in vitro experiments.

International audience; The genus Nepovirus (family Comoviridae) was known both for a good level of homogeneity and for the presence of atypical members. In particular, the atypical members of the genus differed by the number of capsid protein (CP) subunits. While typical nepoviruses have a single CP subunit with three structural domains, atypical nepoviruses have either three small CP subunits, probably corresponding to the three individual domains, or a large and a small subunit, probably containing two and one structural domains, respectively. These differences are corroborated by hierarchical clustering based on sequences derived from both genomic RNAs. Therefore, these atypical viruses are now classified in two distinct genera, Cheravirus (three CP subunits; type species Cherry rasp leaf virus) and Sadwavirus (two CP subunits; type species Satsuma dwarf virus).

International audience; Characterization of a seemingly new spherical virus isolated from severely affected plum trees in south-western France indicated that its divided genome is composed of two single-stranded, polyadenylated RNAs of approximately 7.4 and 3.7 kb. Its particles are composed of three coat protein subunits of approximately 23, 23.5, and 24.5 kDa. Partial sequencing of the genomic RNAs indicated that this new virus, tentatively named stocky prune virus (StPV), is distantly related to the two sequenced cheraviruses, cherry rasp leaf virus (CRLV) and apple latent spherical virus (ALSV). StPV should be regarded as a new member in the unassigned genus Cheravirus.

Knowledge regarding the plant-pathogen interactions is increasing steadily, with the common claim that this knowledge will help improving the control of diseases. Various ways to achieve this goal using biotechnologies will be discussed in the light of some examples in the field of plant viruses, as well as some of the biosafety issues associated. First, soon after the achievement of gene transfer to plants, it became evident that transgenic plants expressing portions of the virus genome were resistant to virus infection in a manner reminiscent of cross-protection, but without some of the adverse effects associated with inoculation of mild virus strains. Second, the recent isolation of natural virus resistance genes potentially enables their transfer to other plant species. However there are two classes of natural resistance genes, dominant and recessive, which differ so much in the mechanisms associated that the prospects to use them in plant biotechnology differ broadly, as well as the expected properties of the resulting virus resistance in the field.

The nucleotide sequences of RNAs 1 and 2 of a German isolate of Raspberry ringspot virus (RpRSV) infecting grapevine (RpRSV-Grapevine), as well as partial sequences of another grapevine isolate from Switzerland (RAC815) were determined. The sequences of the protease-polymerase region encoded by RNA1, and the movement protein and coat protein genes encoded by RNA 2, of these isolates were compared with those of other isolates available in databases. The coat proteins of the grapevine isolates formed a sister group to all those from other RpRSV isolates, but whether this resulted from divergence or recombination was uncertain.

The genetically anchored physical map of peach is a valuable tool for identifying loci controlling economically important traits in Prunus. Breeding for disease resistance is a key component of most breeding programs. The identification of loci for pathogen resistance in peach provides information about resistance loci, the organization of resistance genes throughout the genome, and permits comparison of resistance regions among other genomes in the Rosaceae. This information will facilitate the breeding of resistant species of Prunus. A candidate gene approach was implemented for locating resistance loci in the genome of peach. Candidate genes representing NBS-LRR, kinase, transmembrane domain classes, as well as, pathogen response (PR) proteins and resistance-associated transcription factors were hybridized to a peach BAC library and mapped by using the peach physical map database and the Genome Database for Rosaceae (GDR). A resistance map for Prunus was generated and currently contains 42 map locations for putative resistance regions distributed among 7 of the 8 linkage groups.

Plum pox virus (PPV), the causative agent of sharka disease in Prunoideae, is one of the most serious problems affecting stone fruit production in Europe and America. Resistance to PPV was previously described in a Prunus davidiana clone, P1908, and introduced into peach (Prunus persica) genotypes. Genetic resistance to PPV displays a complex pattern of quantitative inheritance. An analysis of quantitative trait loci (QTLs) for resistance was performed on an F1 interspecific peach population obtained from a cross between the susceptible nectarine cultivar Summergrand and P. davidiana. The hybrids were graft-inoculated with PPV in duplicate following a classical procedure. The incidence of infection was evaluated four times, over two vegetative cycles, by symptom observation and enzyme-linked immunoadsorbent assays (ELISA). Restriction of systemic downward movement of the PPV virus was also evaluated by testing the susceptible rootstocks. Using both analysis of variance and non-parametric tests, six genomic regions involved in PPV resistance were detected. Depending on the scoring data considered, between 22 and 51% of the phenotypic variance could be explained by the quantitative model. One QTL, located in the distal region of linkage group 1, maps in a genomic region that is syntenic to the location of a resistance gene previously identified in the apricot cv. Goldrich. Some QTLs appeared to be temporally specific, reflecting the environmental dependence of PPV-resistance scoring. Candidate gene fragments were amplified by PCR, isolated and mapped on the peach interspecific linkage map. We report here the co-localization of three analogues of virus resistance genes with two distinct genomic regions linked to PPV resistance in P. davidiana.

International audience; LMV-Common and LMV-Most are two seed-borne types of Lettuce mosaic virus (LMV), genus Potyvirus. LMV-Most, but not LMV-Common, overcomes the resistance afforded to lettuce by two recessive genes, mo1 1 and mo1 2. An RT-PCR-based assay thought to be specific for LMV-Most also amplified LMV-Tn2, previously typified as LMV-Common. The sequence of selected regions along the genome indicated that LMV-Tn2 is a natural recombinant between LMV-Most and LMV-Common isolates, with a putative recombination site located within the P3 coding region. This is the first evidence of a naturally occurring LMV recombinant isolate.

Lettuce mosaic virus (LMV)-Most isolates can infect and are seed-borne in cultivars containing the mo1 gene. A reverse transcription and polymerase chain reaction (RT-PCR)-based test was developed for the specific detection of LMV-Most isolates. Based on the complete genome sequences of three LMV isolates belonging respectively to the Most type, the Common type and neither of these two types, three different assays were compared: (i) presence of a diagnostic restriction site in the region of the genome encoding the variable N-terminus of the capsid protein, in the 3' end of the genome, (ii) RT-PCR using primers designed to amplify a cDNA corresponding to a portion of the P1 coding region, in the 5' end of the genome and (iii) RT-PCR using primers designed to amplify a central region of the genome. The assays were performed against a collection of 21 isolates from different geographical origins and representing the molecular variability of LMV. RT-PCR of the central region of the genome was preferred because its results are expected to be less affected by natural recombination between LMV isolates, and it allows sensitive detection of LMV-Most in situations of single as well as mixed contamination.

The Comoviridae are plant viruses that share many structural and functional features with animal and human viruses of the family Picornaviridae. Keywords: comovirus; nepovirus; fabavirus; polyprotein; VPg

The region of the RNA 2 coding for the putative helper/movement protein and the coat protein (CP) of each of six isolates of Raspberry ringspot virus was sequenced and these sequences were compared with the published sequence of the Scottish type isolate. Minimal differences were detected among the putative translations of the helper/movement proteins, however, multiple alignment and phylogenetic analysis of the putative CPs separated the English and Scottish serotypes into two distinct clades. Superimposing the amino acid sequences of the CPs of these two serotypes on the 3D model for the CP of a comovirus/nepovirus, showed that eight of the differences identified between the two serotypes occurred on the surface of the protein. Inspection of the recently reported structure of the capsid protein of Tobacco ringspot virus, the type member of the genus Nepovirus, indicated identical locations for these differences. The change of H (Scottish isolates) to R (English isolates) at position 219 in the amino acid sequences of the viruses occurred on an exposed, erect surface loop. The potential role of this change, and other unique differences between the amino acid sequences of the two serotypes, in the specificity of nematode transmission of the virus is discussed.

International audience; The complete nucleotide sequences of the genomic RNAs of the 0 and E isolates of lettuce mosaic potyvirus (LMV) have been determined. These two isolates differ by their behavior towards two lettuce resistance genes and by their seed transmission properties. LMV-0 is unable to induce disease in lettuce carrying either one of the mol(1) and mol(2) recessive resistance genes, whereas LMV-E is able to induce disease in the same plants. The genomes of these two isolates are 10080 nucleotides (nt) in length, excluding the poly(A) tract, and encode polyproteins of 3255 amino acids (aa). The open reading frame is flanked by a 5' non-coding region of 103 nt and a 3' non-coding region of 212 nucleotides. Ten proteins were predicted. The P3 protein. with 377 aa,is the longest potyviral P3 protein characterized to date while the P1 protein, with 437 aa, is among the longest pi proteins reported. Sequence comparisons between the two isolates demonstrated only limited sequence difference. The overall nucleotide and amino acid sequence identities between LMV-0 and LMV-E are 94 and 97% respectively. The greatest variability occurs in the P1 and in the variable N-terminal region of the coat protein, while the NIa protease domain, the NIb protein, the C-terminus of the helper component protease and the 3' non-coding region are extensively conserved. While this sequence analysis does not allow direct identification of determinants involved in the resistance breaking or in the seed transmissibility properties, these data are a first step towards the characterization of these determinants.

We have performed a systematic search for recombination in the region encoding coat protein and the 3' non-translated region in natural isolates of potyviruses, the largest group of plant RNA viruses. The presence of recombination, and the localization of the cross-over points, were confirmed statistically, by three different methods. Recombination was detected or suspected in 18 out of 109 potyvirus isolates tested, belonging to four out of eight virus species, and was most prevalent in potato virus Y, clear in bean common mosaic virus, and possible in bean yellow mosaic and zucchini yellow mosaic viruses. Recombination was not detected in the four other potyvirus species tested, including plum pox virus, despite the availability of numerous sequences for this last species. Though it was not specifically researched, no evidence for inter-specific recombination was found. For several reasons, including the fact that only a minor portion of the genome was analysed, the above figures certainly represent an underestimate of the extent of recombination among isolates of potyviruses, which might thus be a common phenomenon.

In grapevine chrome mosaic and tomato black ring viruses (GCMV and TBRV), as in many other nepoviruses, the 3′ non-translated regions (3′NTR) are identical between the two genomic RNAs. We have investigated the structure of the 3′NTR of two recombinant isolates which contain GCMV RNA-1 and TBRV RNA-2. In these isolates, the 3′NTR of RNA-1 was transferred to RNA-2, thus restoring the 3′ identity. The transfer occurred within three passages, and probably contributes to the spread of randomly appearing mutations from one genomic RNA to the other. The site of recombination is near the 3′ end of the open reading frame.

International audience; The amino acid sequences of the regions encoding the structural proteins of eleven nepoviruses and five comoviruses, two genera of the familyComoviridae, have been aligned. The properties predicted by computer analysis (three-dimensional-3D-structure, hydrophobicity) are also correlated along this alignment, and aligned to the experimentally determined 3D structure of two comoviruses. It can thus be assumed that the 3D structure of the unique nepovirus coat protein matches that of the bipartite protomer found in the comovirus particles. In this model, the spatial locations of two amino-acid motifs characteristic of nepoviruses are in close vicinity, at the external surface of the virion. The coat proteins of nepoviruses and comoviruses may thus share a common evolutionary origin. A phylogenetic analysis was made using the multiple alignment, allowing a better understanding of the molecular relationships between these two groups of viruses.

The genome of cowpea mosaic virus (CPMV) is divided among two positive strand RNA molecules. B-RNA is able to replicate independently from M-RNA in cowpea protoplasts. Replication of mutant B-transcripts could not be supported by co-inoculated wild-type B-RNA, indicating that B-RNA cannot be efficiently replicated in trans. Hence replication of a B-RNA molecule is tightly linked to its translation and/or at least one of the replicative proteins functions in cis only. Remarkably also for efficient replication of M-RNA one of its translation products was found to be required in cis. This 58K protein possibly helps in directing the B-RNA-encoded replication complex to the M-RNA. In order to identify the viral polymerase the CPMV B-RNA-specific proteins have been produced individually in cowpea protoplasts using CaMV 35S promoter based expression vectors. Only protoplasts transfected with a vector containing the 200K coding sequence were able to support replication of co-transfected M-RNA. Despite this, CPMV-specific RNA polymerase activity could not be detected in extracts of these protoplasts using a poly(A)/oligo(U) assay. These results indicate that, in contrast to the poliovirus polymerase, the CPMV polymerase is not able to accept oligo(U) as a primer and in addition support the concept that translation and replication are linked.

Cowpea mosaic virus (CPMV) B-RNA encodes the viral proteins required for viral RNA replication while M-RNA does so for the capsid proteins and functions required in cell-to-cell movement of the virus. Accordingly, B-RNA can replicate by itself, whereas M-RNA can only replicate in the presence of B-RNA. We have made heterologous sequence insertions at different positions in the open reading frame of B-RNA, leaving the 5' and 3' non-coding ends intact. None of these mutant B-RNAs were able to replicate. Furthermore, it was not possible to support replication of these mutant B-RNAs by co-inoculating wild-type B-RNA as a helper, indicating that B-RNA can not be replicated in trans. In contrast, replication of M-RNA must occur in trans, as the viral replicative proteins are encoded by B-RNA. Mutant M-RNA transcripts containing 5' and 3' non-coding regions of B-RNA are still efficiently replicated in protoplasts if co-inoculated with B-RNA, indicating that in cis or in trans replication of the CPMV RNAs is not primarily determined by the non-coding regions. Remarkably, for replication of M-RNA, the N-terminal domain of the 58K protein encoded by M-RNA was found to be required.

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.

In vitro translation of RNA-2 of each of two closely related nepoviruses, tomato black ring virus (TBRV) and grapevine chrome mosaic virus (GCMV), in a rabbit reticulocyte lysate resulted in the synthesis of single polypeptides of 150K and 146K respectively. Processing of these polyproteins occurred after the addition of translation products of homologous RNA-1. The positions of the cleavage products within the polyproteins were determined. From the N to the C terminus, M r values for the proteins were 50K, 46K and 59K for TBRV and 44K, 46K and 56K for GCMV. TBRV RNA-1 translation products also cleaved the polyproteins encoded by GCMV RNA-2 which suggests that the cleavage sites in the two polyproteins are similar.

Cowpea mosaic virus M RNA is translated in vitro as well as in vivo into two C-coterminal polyproteins of Mr 105K and 95K. Initiation of translation of the 95K protein gene occurs at an AUG codon at position 512 of M RNA, 351 nucleotides downstream of the initiation codon of the 105K protein gene at position 161. By employing an in vitro transcription and translation system it was determined that this 351 nucleotide sequence has the capacity to direct ribosomes to initiate translation at a downstream start codon. This effect is independent of the position of this sequence in an mRNA. Furthermore, evidence has been obtained that scanning ribosomes can bypass the AUG at position 161. Thus, both leaky scanning and internal entry are mechanisms for the initiation of translation of the 95K protein gene.

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.

International audience

International audience; 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.

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.