Previous resistance analyses of Arabidopsis thaliana mutants knocked out for eukaryotic translation initiation factors showed that disruption of the At-eIF(iso)4E or both the AteIF(iso)4G1 and At-eIF(iso)4G2 genes resulted in resistance against turnip mosaic virus (TuMV). This study selected TuMV virulent variants that overcame this resistance and showed that two independent mutations in the region coding for the viral genome-linked protein (VPg) were sufficient to restore TuMV virulence in At-eIF(iso)4E and At-eIF(iso)4G1¾At-eIF(iso)4G2 knockout plants. As a VPg-eIF(iso)4E interaction has been shown previously to be critical for TuMV infection, a systematic analysis of the interactions between A. thaliana eIF4Es and VPgs of virulent and avirulent TuMVs was performed. The results suggest that virulent TuMV variants may use an eIF4F-independent pathway.The eukaryotic translation initiation factors eIF4E and eIF4G play a key role during virus infection in plants ( Robaglia & Caranta, 2006). During mRNA translation, eIF4E provides the cap-binding function and is associated with eIF4G, the scaffold for the other components of the translation initiation complex, to form the eIF4F complex (Marcotrigiano et al., 1999). In plants, a second eIF4F complex called eIF(iso)4F is present, and results from the assembly of eIF(iso)4E and eIF(iso)4G isoforms (Browning, 2004). These two complexes perform essentially the same task in translation, but have different affinities for certain classes of mRNA substrates and are probably involved in different cellular events (Gallie & Browning, 2001). Plant genes encoding proteins from the eIF4F and eIF(iso)4F complexes belong to small gene families. In Arabidopsis thaliana, three genes encode the eIF4E subfamily proteins (eIF4E1, eIF4E2 and eIF4E3), one encodes eIF(iso)4E and one a non-canonical eIF4E-like protein, known as novel cap-binding protein (nCBP). A single gene encodes eIF4G, whilst two genes encode the eIF(iso)4G subfamily proteins [eIF(iso)4G1 and eIF(iso)4G2] ( Robaglia & Caranta, 2006). Viruses that depend on these factors to perform their infectious cycle include members of the genus Potyvirus, the largest and the most diverse genus of plant viruses (Adams et al., 2005;Shukla et al., 1994). Potyviruses are characterized by a positive-sense, single-stranded RNA genome of about 10 kb that encodes at least ten multifunctional proteins. The genome is 39-polyadenylated and covalently linked at its 59 end to a virus-encoded protein (VPg) (Murphy et al., 1991;Revers et al., 1999).Potyviruses may selectively use either the eIF4E or eIF(iso)4E isoform in A. thaliana to achieve a successful infection (Sato et al., 2005). We have demonstrated that potyviruses, including turnip mosaic virus (TuMV), selectively use members of the eIF4G family in a fashion that parallels the selective recruitment of eIF4E isoforms (Nicaise et al., 2007;Sato et al., 2005 ; data not shown).In order to confirm that each single amino acid change in the VPg cistron of TuMV determined its virulence on the At...
The eukaryotic translation initiation factor 4E (eIF4E) (the cap-binding protein) is involved in natural resistance against several potyviruses in plants. In lettuce, the recessive resistance genes mo1 1 and mo1 2 against Lettuce mosaic virus (LMV) are alleles coding for forms of eIF4E unable, or less effective, to support virus accumulation. A recombinant LMV expressing the eIF4E of a susceptible lettuce variety from its genome was able to produce symptoms in mo1 1 or mo1 2 varieties. In order to identify the eIF4E amino acid residues necessary for viral infection, we constructed recombinant LMV expressing eIF4E with point mutations affecting various amino acids and compared the abilities of these eIF4E mutants to complement LMV infection in resistant plants. Three types of mutations were produced in order to affect different biochemical functions of eIF4E: cap binding, eIF4G binding, and putative interaction with other virus or host proteins. Several mutations severely reduced the ability of eIF4E to complement LMV accumulation in a resistant host and impeded essential eIF4E functions in yeast. However, the ability of eIF4E to bind a cap analogue or to fully interact with eIF4G appeared unlinked to LMV infection. In addition to providing a functional mutational map of a plant eIF4E, this suggests that the role of eIF4E in the LMV cycle might be distinct from its physiological function in cellular mRNA translation.
The carbohydrate chains of the bronchial-mucus glycoproteins of six cystic fibrosis patients with blood group 0 were released by alkaline borohydride treatment. Low-molecular-mass, monosialyl oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by high-performance liquid chromatography. Structural characterization was performed by 500-MHz 'H-NMR spectroscopy in combination with quantitative sugar analysis. The established structures range in size from tetra-up to heptasaccharides. They are all sialyl analogs of neutral oligosaccharides that were characterized previously [Lamblin G., Boersma A., Lhermitte M., Roussel P., Mutsaers J. Human bronchial mucus is secreted as a gelatinous layer on the surface of the tracheobronchial airway epithelium and is continuously moved towards the pharynx where it is swallowed. It acts as a medium for protection and lubrication of the mucosa and for transport of inhaled particles such as dust, pollen, viruses and bacteria to clear the bronchial epithelium.Bronchial mucins, as most mucins [l], contain about 70% carbohydrate which is 0-glyosidically linked to the peptide backbone as neutral and acidic oligosaccharide chains. The acid functions stem from the presence of sialic and/or sulfate residues [2].Any change in the structure of these oligosaccharides may modify the rheological properties of the bronchial mucus and lead to a non-efficient mucociliary clearance and to destruction phenomena found in cystic fibrosis or other bronchial diseases where hypersecretion of mucus is a pre-eminent feature of the pathological process. Increased adherence of inhaled particles may be a concomitant phenomenon.Since it is difficult to obtain the amount of normal human bronchial secretion required for detailed carbohydrate structural analysis [3], we have taken mucins secreted by patients suffering from cystic fibrosis (CF) for our structural studies. In the future, the results of the structural characterization of their carbohydrates will be compared to mucins secreted by patients with other chronic bronchial hypersecretion diseases.Previously, we have isolated and characterized several lowmolecular-weight oligosaccharides that were obtained from the carbohydrate material released from the mucins occurring in the sputum of six C F patients with blood group 0 [4-61. The structures of 20 neutral and 5 sialyl-oligosaccharides were determined by 500-MHz 'H-NMR spectroscopy in conjunction with sugar composition analysis. The present paper deals with the determination of the structure of another 13 sialyloligosaccharides isolated from the same pool of CF bronchial mucins. MATERIALS AND METHODS Isolation and purijkation of human bronchial sialyl-oligosaccharidesMucin glycoproteins were isolated from the sputum of six patients (blood group 0) suffering from cystic fibrosis as described [2]. Briefly, gelatinous bronchial mucus from the sputum was solubilized by the action of 2-mercaptoethanol (1 %, by vol.) and fractionated on an Ecteola-cellulose column with stepwi...
The capacity of Lettuce mosaic virus to overcome the lettuce resistance conferred by the mo1(1) and mo1(2) alleles of the gene for eukaryotic translation initiation factor 4E (eIF4E) was analysed using reverse genetics. Mutations in the virus genome-linked protein (VPg) allowed mo1(1) only to be overcome, but mutations in the C-terminal portion of the cylindrical inclusion (CI) protein allowed both alleles to be overcome. Site-directed mutagenesis pinpointed a key role of the amino acid at position 621 in the virulence. This is the first example of the involvement of a potyviral CI protein in the breaking of an eIF4E-mediated resistance.
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