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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., (Copyright 2010 Elsevier Inc. All rights reserved.)

Beet western yellows virus (BWYV) is a Polerovirus that relies on the aphid Myzus persicae for its transmission, in a persistent-circulative mode. To be transmitted, the virus must cross the midgut and the accessory salivary glands (ASG) epithelial barriers in a transcytosis mechanism where vector receptors interact with virions. In this paper, we report in vitro interaction experiments between BWYV and aphid components. Using the M. persicae clone from Colmar, we showed that a set of aphid polypeptides, separated by SDS-PAGE or 2D electrophoresis (2DE), can bind in vitro to purified wild type or mutant particles. Using subcellular fractionation, we showed that the 65-kDa polypeptide identified as symbionin is a soluble protein whereas the other polypeptides seem to be associated more or less strongly to the membrane. We hypothesize that three polypeptides, identified by mass spectrometry as Rack-1, GAPDH3, and actin, may be involved in the epithelial transcytosis of virus particles in the aphid vector.

It appears from grafting experiments that the androgenic hormone (AH) of terrestrial isopods has a narrow species-specificity [J. Crust. Biol. 19 (1999) 684], even if AH of different species shared common epitopes [Gen. Comp. Endocrinol. 125 (2002) 218]. To date only the glycosylated AH of the woodlouse Armadillidium vulgare has been deciphered by us [Ann. New York Acad. Sci. 839 (1998) 111; Eur. J. Biochem. 262 (1999) 727] and [Biochem. Biophys. Res. Commun. 264 (1999) 419] have confirmed the primary structure of this protein. We reported in the present paper the characterization of the AH in another species Porcellio scaber by a combination of microsequencing, mass spectrometry, and molecular cloning. We found only one glycoform which consisted of two peptide chains, A and B, of 31 and 44 amino acids, respectively, with A chain carrying on Asn18 a N-glycosylated moiety, size of which has been determined by MALDI-MS measurements. The expected structure of the glycosylation was proposed. The deduced amino acid sequence of the AH precursor was mainly identical to the one obtained independently by another group [Zool. Sci. 20 (2003) 75]. We also showed that AH gene is exclusively expressed in androgenic glands. Sequence comparison with A. vulgare and P. scaber (population of Japan) AH was discussed.

Structural studies of biomolecules using nuclear magnetic resonance (NMR) rely on the availability of samples enriched in (13)C and (15)N isotopes. While (13)C/(15)N-labeled proteins are generally obtained by overexpression in transformed Escherichia coli cells cultured in the presence of an expensive mixture of labeled precursors, those of the photoautotrophic cyanobacterium Anabaena sp. PCC 7120 can be uniformly labeled by growing them in medium containing Na(15)NO(3) and NaH(13)CO(3) as the sole nitrogen and carbon sources. We report here a novel vector-host system suitable for the efficient preparation of uniformly (13)C/(15)N-labeled proteins in Anabaena sp. PCC 7120. The 24-kDa N-terminal domain of the E. coli gyrase B subunit, used as a test protein, was cloned into the pRL25C shuttle vector under the control of the tac promoter. The transformed Anabaena cells were grown in the presence of the labeled mineral salts and culture conditions were optimized to obtain over 90% of (13)C and (15)N enrichment in the constitutively expressed 24-kDa polypeptide. The yield of purified 24-kDa protein after dual isotope labeling under anaerobic conditions was similar to that obtained with E. coli cells bearing a comparable expression vector and cultured in parallel in a commercially available labeling medium. Furthermore, as probed by NMR spectroscopy and mass spectrometry, the 24-kDa N-terminal domain expressed in Anabaena was identical to the E. coli sample, demonstrating that it was of sufficient quality for 3D-structure determination. Because the Anabaena system was far more advantageous taking into consideration the expense for the labels that were necessary, these results indicate that Anabaena sp. PCC 7120 is an economic alternative for the (13)C/(15)N-labeling of soluble recombinant proteins destined for structural studies., (Copyright 2001 Academic Press.)