Arch Virol (2006) 151: 967–984 DOI 10.1007/s00705-005-0669-8

Glycosylation of beet western yellows virus proteins is implicated in the aphid transmission of the virus P. Seddas and S. Boissinot Institut National de la Recherche Agronomique, Unit´e de Recherche Biologie des Interactions Virus/Vecteur, Colmar, France Received July 1, 2005; accepted September 24, 2005 c Springer-Verlag 2005 Published online November 30, 2005


Summary. Beet western yellows virus relies on the aphid M. persicae for its transmission in a persistent and circulative mode. To be transmitted, the virus must cross the midgut and the accessory salivary gland epithelial barriers by a transcytosis mechanism where vector receptors interact with virions. The aphid and the peptidic viral determinants implicated in this interaction mechanism have been studied. In this paper, we report that the coat and the readthrough proteins that constitute the capsid of this virus are glycosylated. Modification of the glucidic core of these structural viral proteins by oxidation with sodium metaperiodate or deglycosylation with N-glycosidase F or α-D-galactosidase abrogates the aphid transmission of the virus. Aphid transmission could also be inhibited by lectins directed against α-D-galactose when aphids were allowed to acquire virus on artificial membranes. These results suggest that the glucidic cores of the capsid proteins of beet western yellows virus contain α-D-galactose residues that are implicated in virus-aphid interaction and promote aphid transmission of the virus. Introduction Beet western yellows virus (BWYV), a member of the genus Polerovirus, family Luteoviridae [40], relies on the aphid Myzus persicae for its transmission. The virions containing a plus-sense RNA genome of about 5.7 kb are 25 nm icosahedral particles composed of the major coat protein (CP) of 22 kDa encoded by ORF 3, and the minor readthrough (RT) protein of 74 kDa encoded by ORF 3 and ORF 5. The downstream ORF 5 is expressed by a stop codon readthrough mechanism indicating that the CP protein and the N-terminal extremity of RT protein possess the same 203 amino acid sequence [39]. As found with all luteoviruses, BWYV is transmitted by aphids in a persistent, circulative and

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non-propagative manner [65]. In order to be aphid transmitted, virions must cross the midgut cells and the accessory salivary glands (ASG) cells of its vector [24, 47] and this involves transcytosis events based on a receptor-mediated mechanism [23–25, 49, 53]. A far-Western blot approach was used to determine which vector components interact with virus particles. Several proteins of M. persicae were found to bind in vitro to potato leafroll virus (PLRV) [61]. Recently, Seddas et al. [53] identified three proteins from M. persicae that are implicated in the transcytosis of BWYV in its vector: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH3) which may act as a receptor of BWYV, a receptor for activated C kinase 1 (Rack-1), and actin which may be implicated in the enhancement of transcytosis of the wild type BWYV compared to deleted or modified RT mutants. In the aphid vector Sitobion avenae, Li et al. [35] showed that two proteins of ASG cells, SaM35 and SaM50, bind specifically to purified particles of the MAV-strain of barley yellow dwarf virus (BYDV-MAV). In the leafhopper Nilaparvata lugens, Zhou et al. [66] showed the existence of a 32-kDa membrane protein, a potential receptor of rice ragged stunt virus (RRSV). In the thrip Frankliniella occidentalis, a 50-kDa protein localized in the midgut brush border and a 94-kDa protein, believed to represent a receptor involved in virus circulation in the vector, have been shown to bind in vitro to particles of tomato spotted wilt virus (TSWV) [5, 30, 41]. The viral determinants of the BWYV/M. persicae interaction are localized on the capsid of BWYV [9, 10]. The RT protein of BWYV does not seem to be required for the transport of virions across midgut cells, although the presence of RT protein greatly enhances the efficiency of this process [47]. In luteoviruses, the RT protein and more specifically the conserved RT domain (RTD), composed of the 226 first N-terminal amino acids of the protein [39], stabilizes the virions in the haemolymph and during their transport to the ASG [8, 10, 11, 13]. This involves the binding of RT protein to Buchnera GroEL also known as symbionin [62]. The molecular basis and chemical nature of the specific interactions between BWYV and aphid proteins have not yet been elucidated. Glycosylation is one of the most important co- and post-translational protein modifications. Glycosylation of proteins occurs either through a glycosidic bond to an asparagine side chain (N-glycosylation [33, 56], a serine or threonine side chain (O-glycosylation [31]) or via a glycosyl-phosphatidylinositol (GPI) anchor [19]. Many functions have been suggested for protein glycosylation, including signalling for intracellular targeting, protection from proteolytic breakdown, control of correct folding and maintenance of protein stability and configuration [17, 18, 38, 50]. In enveloped animal viruses like human immunodeficiency virus, dengue viruses and hepatitis C virus, glycosylation induces a cascade of events initiated by the interaction of viral envelope glycoproteins with specific entry receptors and co-receptors which then triggers virus-cell membrane fusion [2, 51]. There is little information on glycosylation of plant virus proteins. Carbohydrates were reported to be present on virions of the seed-transmitted barley stripe

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mosaic virus (BSMV) and cowpea mosaic virus (CPMV) [45] but this was not confirmed in a subsequent study of the capsid protein of a CPMV isolate [1]. The large structural protein of lettuce necrotic yellow virus (LNYV) was reported to be glycosylated with complex oligosaccharides containing beta-N-acetylchitobiose N-linked to asparagine residues [15]. The capsid proteins of potato virus X (PVX) and plum pox virus (PPV) have been found to be O-glycosylated [6, 20, 58]. Recently, the two envelope membrane proteins of TSWV were shown to be N-glycosylated [44]. In the present study, we demonstrate that the CP and RT proteins of BWYV are glycosylated and that this glycosylation plays a role in the BWYV/aphid interaction mechanism that allows to aphid transmission of this virus. Materials and methods Production and purification of BWYV Particles of isolate FL1 of wild type (WT) BWYV originating from lettuce were purified from aphid-infected Montia perfoliata plants [8], according to the method developed by van den Heuvel et al. [60]. The concentration of purified particles was determined by reading the optical density at 260 nm and using an extinction coefficient of 8.6. Purified particles were stored in citrate buffer [0.1 M sodium citrate, pH 6.0] at −80 ◦ C until use. Maintenance of insects A luteovirus-free colony of Myzus persicae (clone Colmar, Mp-Col) was maintained on Capsicum annuum plants and was reared in a growth chamber at 20 ◦ C as previously described [47]. Production of BWYV-specific monoclonal antibodies A six weeks old female BALB/c mouse (obtained from Dr. P. Pothier, Laboratoire d’Immunotechnologies, Facult´e de M´edecine, Universit´e de Bourgogne, Dijon) was injected intraperitoneally with 50 µg equivalent proteins of purified particles of BWYV-WT in complete Freund’s adjuvant. The animal received four additional injections at 2 weeks interval of the particles in incomplete Freund’s adjuvant. One month after the last injection, the mouse received a final injection of particles as described by Seddas et al. [53]. Cell fusion and screening of hybridoma secreting antibodies reacting with BWYV particles were carried out according to Seddas et al. [52] using a triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) for screening. The antigen preparation used for screening was healthy or BWYV-infected N. clevelandii plant extract (0.2 g · ml−1 of extraction buffer). Specificity of these Mabs was tested using purified BWYV virions (1 µg · ml−1 ) that were either not modified after purification or were treated with 5 mM sodium metaperiodate, 0.75 U PNGase-F, 0.75 U α-D-galactosidase or 0.75 U O-glycosydase DS. Monoclonal antibodies (Mabs) obtained from mouse ascitic fluid were purified using a protein A affinity column [52]. Virus transmission by aphids All aphid transmission experiments were done at 20 ◦ C as described [47]. Third or fourth instar nymphs or adults were given a 24 hr acquisition access period (AAP) with or without different concentrations of lectins and 50 µg · ml−1 of a purified BWYV suspension through a stretched

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parafilm membrane. Lectins and virus particles were prepared in 20% (w/v) sucrose in the artificial diet MP148 [27]. After AAP, aphids were transferred onto virus-free M. perfoliata seedlings to assess their capacity to transmit the virus. Eight aphids were placed on each test plant for a 4-day inoculation access period (IAP). The plants were tested 4 weeks later for BWYV infection using the TAS-ELISA protocol. Deglycosylated or periodate-oxidated virions were used in parallel experiments to assess the influence of the glucidic residues on aphid transmission. SDS-PAGE of BWYV capsid proteins Two µg of BWYV capsid proteins in 25 µl were processed for solubilization: after addition of an equivalent volume of Laemmli buffer, the proteins were solubilized for 10 min at 100 ◦ C [32] before being processed for SDS-PAGE, as described by Dozolme et al. [16]. SDS-PAGE was done according to the method of Laemmli [32]. The slab gels were 1 mm thick and consisted of a 14% (w/v) acrylamide resolving gel and a 4% (w/v) acrylamide stacking gel with 10 wells for samples. Polypeptides in the resolving gels were either fixed in 30% (v/v) ethanol, 5% (v/v) acetic acid for silver staining [7] or transferred onto Immobilon-PTM or nitrocellulose membranes (Millipore). Western blot experiments After separation of BWYV capsid proteins by SDS-PAGE, the proteins were transferred onto Immobilon-PTM membrane using a Millipore semi-dry transfer apparatus (80 V, 2.5 mA · cm−2 for 40 min at room temperature) according to the manufacturer’s instructions. Nonspecific protein binding sites were blocked with 2% (w/v) Tween 20 in Tris buffer saline (TBS) [200 mM NaCl, 20 mM Tris-HCl, pH 7, 6] for 15 min at room temperature. The membrane was then incubated for 16 hr at 20 ◦ C with anti-BWYV monoclonal antibody 7C2. These antibodies were used at 10 µg · ml−1 in TBST [TBS containing 0.05% (w/v) Tween 20] and were incubated at room temperature for 1 hr. After 5 washings with TBST, the immune complexes were revealed by 2 hr incubation with alkaline phosphatase labelled goat antimouse polyclonal antibodies [53]. Glycoprotein detection and lectin binding After separation of BWYV capsid proteins by SDS-PAGE and transfer onto nitrocellulose membrane, glycoproteins were detected by a colorimetric method using periodic acid/Schiff detection [55] or the Bio-Rad detection kit according to the manufacturer’s instructions. After transfer onto Immobilon-PTM (Millipore) membranes, the carbohydrates on BWYV capsid proteins were identified using 10 µg · ml−1 of different biotinylated lectins [i.e. from Bandeireae simplicifolia (BSI) and Maclura pomifera (MPA) that interact specifically with α-D-galactosyl and N-acetyl-α-D-galactosaminyl residues, Canavalia ensiformis (ConA) or Lens culinaris (LcH) that have an affinity for terminal α-D-mannosyl and α-D glucosyl residues, Dolichos biflorus (DBA) that interact with α-N-acetyl-galactosamine oligomers, Maackia amurensis (MAA) that interact with sialic acid-containing glycoconjugates, Sambucus nigra (SNA) that have an affinity for α-Neu-N-acetyl galactosaminyl residues, Solanum tuberosum (STA) that interact with N-acetyl-β-D-glucosamine oligomers, Tetragonolobus purpureas (TPA) that possess an affinity for α-L-fucosyl residues, and Triticum vulgaris (WGA) that interact with N-acetyl-β-D-glucosaminyl residues and N-acetyl-β-D-glucosamine oligomers] [16]. Negative control experiments were run in parallel using deglycosylated fetuin (Bio-Rad) instead of BWYV capsid proteins in order to verify the specificity of lectin binding.

Glycosylation of BWYV is implicated in aphid transmission

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Deglycosylation and oxidation of BWYV particles Aliquots of purified virions (20 µg) were pelleted and deglycosylated using α-D-galactosidase from Sigma or enzymes supplied in deglycosylation or deglycosylation enhancement kits of Bio-Rad. To remove N-linked oligosaccharides from glycoproteins, 20 µg purified BWYV particles were incubated with 10 mU of peptide: N-glycosidase F (PNGase F) for 24 hr at 37 ◦ C in 50 mM sodium phosphate buffer pH 6. A mixture of 40 mU NANase II, 30 mU HEXase I, 10 mU GALase III, 0.75 U α-D-galactosidase, and 10 mU O-glycosidase DS in 50 mM sodium phosphate buffer pH 6 was incubated for 3 hr at 37 ◦ C to enhance deglycosylation and release O-linked oligosaccharides from glycoproteins. A specific deglycosylation was carried out using α-D-galactosidase at different concentrations (0, 0.25, 0.50, 0.75, 1 U) in 50 mM sodium phosphate buffer pH 6 for 3 hr at 37 ◦ C. Different concentrations of sodium metaperiodate (0, 0.1, 0.2, 0.5, 1, 2, 5, and 10 mM), diluted in 50 mM sodium acetate buffer pH 4.5 and incubated 2 hr at 37 ◦ C were used to modify the glucidic moieties of virions. Deglycosylated and oxidated virions were dialysed 3 times against 0.1 M sodium citrate buffer pH 6.0, then adjusted to 20% (w/v) sucrose and 50 µg · ml−1 of virions before being used for BWYV aphid transmission experiments. Deglycosylated virion proteins were analyzed by SDS-PAGE and Western-blot experiments using Mab 7C2 and by ELISA using the three selected Mabs. Northern-blot RNA phenol extraction was carried out using 2 µg of BWYV particles that were untreated, in presence of 500 µg · ml−1 lectin from Maclura pomifera (MPA), Bandeireae simplicifolia (BSI), Lens culinaris (LcH), Canavalia ensiformis (Con A), or treated with 5 mM sodium metaperiodate, 0.75 U PNGase-F, 0.75 U α-D-galactosidase or 0.75 U O-glycosydase DS [10]. After separation in denaturing agarose gel electrophoresis, RNA was electro-transferred onto nylon membrane (Amersham). Northern-blot experiments were carried out using a digoxigenin-UTP-labeled DNA probe (Roche Diagnostics) complementary to the 3 -terminal 185 residues of the viral RNA [48].

Results Monoclonal antibodies against BWYV Mabs directed against BWYV were produced using an enriched fraction of BWYV particles obtained from infected M. perfoliata plants. About 80% of the hybridomas secreted antibodies which reacted more strongly with healthy tobacco plants than with infected ones. Only three stable clones specific for BWYV were obtained. Mabs 2E4 and 4D1 are IgG1 subclass whereas Mab 7C2 is a IgG2a subclass; all Mabs possess a λ light chain. The three Mabs detected virions after periodate oxidation (5 mM) and deglycosylation treatments (0.75 U PNGase-F, 0.75 U α-D-galactosidase or 0.75 U O-glycosydase DS) in ELISA (Table 1) and Mab 7C2 also detected the deglycosylated forms of the CP and RT proteins in Western-blot experiments (Fig. 2, lane 4b), suggesting that the three Mabs are directed against a peptidic epitope. Mabs 2E4 and 4D1 did not react with the CP or the RT viral proteins in Western-blots and are considered to be directed against conformational epitopes whereas Mab 7C2 recognized CP and RT viral proteins of BWYV in Western-blots

BWYV-infected tobacco 2.90 ± 0.020 3.15 ± 0.015 3.05 ± 0.020

Healthy tobacco

0.030 ± 0.005 0.020 ± 0.010 0.030 ± 0.005

Name

2E4 4D1 7C2

3.30 ± 0.030 3.45 ± 0.045 3.40 ± 0.035

Untreated BWYV virions 3.20 ± 0.020 3.30 ± 0.010 3.40 ± 0.015

Na periodate treated BWYV virions 3.10 ± 0.005 3.40 ± 0.025 3.20 ± 0.010

PNGase-F treated BWYV virions

3.40 ± 0.010 3.05 ± 0.020 3.40 ± 0.010

α-D-galactosidase BWYV virions

3.25 ± 0.020 3.20 ± 0.020 3.20 ± 0.020

O-glycosydase DS treated BWYV virions

Table 1. Monoclonal antibodies against BWYV particles. Monoclonal antibodies were raised against purified BWYV particles. See Materials and methods for details. TAS-ELISA was carried out using healthy or BWYV-infected N. clevelandii tobacco plants, purified BWYV particles (0.1 µg per well) untreated after purification or treated with 5 mM sodium metaperiodate (Na periodate), 0.75 U PNGase-F, 0.75 U α-D-galactosidase or 0.75 U O-glycosydase DS as described in Material and methods. ELISA results (optical densities read at 405 nm) are means of four independent experiments

972 P. Seddas and S. Boissinot

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Fig. 1a, b. Detection of glycoproteins after transfer of SDS-PAGE profile of BWYV capsid proteins. a: Detection of CP and RT proteins of BWYV using 10 µg · ml−1 7C2 monoclonal antibody (1), the Schiff colorimetric reaction (2), and the glycosylation detection kit of Bio-Rad (3). The equivalent of 2 µg protein of purified BWYV particles was deposited in each lane. CP and RT are indicated on the left. b: Detection of CP and RT proteins of BWYV using the lectins from Bandeireae simplicifolia (BSI), Canavalia ensiformis (ConA), Dolichos biflorus (DBA), Lens culinaris (LcH), Maackia amurensis (MAA), Maclura pomifera (MPA), Sambucus nigra (SNA), Solanum tuberosum (STA), Tetragonolobus purpureas (TPA) and Triticum vulgaris (WGA). CP and RT are indicated on the left. Equivalent of 2 µg protein of purified BWYV particles was deposited in each lane

(Fig. 1a, lane 1) and ELISA (Table 1), suggesting that it is directed against a linear epitope. The CP and RT proteins of BWYV are glycosylated Using prediction server programs developed by the Center for Biological Sequence Analysis (Technical University of Denmark, http://www.cbs.dtu.dk), we identified different potential N-glycosylation sites in the capsid proteins of BWYV. The CP protein possesses 3 potential N-glycosylation sites whereas the RT protein possesses five potential N-glycosylation sites, two of which are located in the conserved region of RTD (Fig. 3). Ser211, Ser215, Ser217, Thr221, and Ser592 are potential O-glycosylation sites in the RT protein domain. Except for Ser592, these potential O-glycosylation sites are located in the conserved region of RTD, between two proline residue (Fig. 3). The presence of sugar residues in the CP and/or the RT proteins was demonstrated by Schiff colorimetric reaction (Fig. 1a, lane 2) as well as by a glycosylation detection kit (Fig. 1a, lane 3).

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Fig. 2. Silver staining (1a–4a) or Western-blot using 10 µg · ml−1 7C2 monoclonal antibody (1b–4b) of SDS-PAGE profile of BWYV capsid proteins. BWYV capsid proteins (2 µg in each lane) were separated by SDS-PAGE after deglycosylation of the virions using PGNase F (2a and 2b) or the deglycosylation enhancement kit of Bio-Rad combined with 0.75 U α-D galactosidase (4a and 4b). Unmodified BWYV capsid proteins were run as control incubated in the corresponding buffers without addition of glycosydase (1a, 3a, 1b and 3b). Relative molecular mass (MMr) are indicated on the left

Fig. 3. Potential N- and O-glycosylation sites in the amino acid sequence of BWYV CP and RT coat proteins. CP protein encoding by ORF3 is indicated between square brackets. RT protein is encoding by ORF3 and ORF5. RT domain (RTD) encoding by ORF5 is in italics (ORF5). Conserved RTD in luteoviruses is delimited by brackets. Potential N-glycosylation sites are indicated in bold and underlined whereas serine and threonine residues that are potentialy O-glycosylation sites are indicated in italics, bold and underlined

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Deglycosylation experiments were carried out to determine the relative MW of the glucidic moiety of the capsid proteins. Using the N-linked oligosaccharide deglycosylation protocol (Fig. 2, lanes 2a and 2b), no difference in the relative MW was observed for CP (25 kDa) and for RT (55 kDa) compared to the non deglycosylated control (Fig. 2, lanes 1a and 1b). When deglycosylation was performed with the enhanced deglycosylation protocol combined with 0.75 U α-D-galactosidase, which included O-linked oligosaccharide deglycosylation (Fig. 2, lanes 4a and 4b), a decrease of the relative MW of the RT protein was observed (52 kDa) compared to non-deglycosylated protein (Fig. 2, lanes 3a and 3b); however, no significant difference was observed in the relative MW of the deglycosylated CP protein. In order to identify which sugars are present in the CP and RT proteins, we tested their ability to bind various lectins (Fig. 1b). Seven lectins (BSI, ConA, DBA, LcH, MPA, TPA and WGA) reacted with the CP and RT proteins whereas no binding was observed between BWYV CP and RT proteins and MAA, SNA or STA. The specificity of the different lectins tested suggests that α-L-fucosyl residues (TPA), α-N-acetyl-galactosamine oligomers (DBA), α-D-galactosyl and N-acetyl-α-D-galactosaminyl residues (BSI, MPA) as well as N-acetyl-β-Dglucosaminyl residues (WGA) are present on the two BWYV capsid proteins. Other sugars such as α-D-glucose or α-D-mannose (ConA, LcH) could also be present on the terminal glucidic core extremity on the BWYV capsid protein; however, the glucidic cores present on CP and RT do not contain sialic acid (MAA), α-Neu-N-acetyl galactosaminyl residues (SNA), nor N-acetyl-β-D-glucosamine oligomers (STA). These lectin binding reactions were specific because no binding was observed using deglycosylated fetuin instead of BWYV capsid proteins (data not shown). Glucidic groups of CP and RT proteins are implicated in aphid transmission of BWYV When virions were treated with sodium metaperiodate which is known to open the aromatic circle of carbohydrates, they lost the ability to be transmitted by aphids Table 2. Aphid transmission experiments after treatment of BWYV particles. Aphid transmission experiments were carried out after metaperiodate oxidation of purified BWYV particles (left) or after deglycosylation experiments (right). Results represent the percentage of plants that have been found by ELISA to be BWYV-infected 4 weeks after aphid transmission of virus. Results are means of four independent experiments using 8 plants each time Sodium metaperiodate 10 mM 5 mM 2 mM 1 mM 0.1 mM

0% 0% 0% 4.16% ± 5.55 8.33% ± 5.55

0 (buffer control)

95.83% ± 5.55

1U 0.75 U 0.5 U 0.25 U 0.10 U 0.01 U 0 (buffer control)

N-glycosidase F

O-glycosidase DS

α-D-galactosidase

0% 0% 0% 4.16% ± 5.55 0% 0% 95.83% ± 5.55

100% 100% 100% 95.83% ± 5.55 95.83% ± 5.55 100% 100%

0% 4.16% ± 5.55 76.66% ± 7.22 76.66% ± 7.22 95.83% ± 5.55 100% 100%

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(Table 2), suggesting that carbohydrates are important and must be in native form for aphid transmission to occur. Treatment of the virions with different concentrations of PNGase F which cleaves all common asparagine-linked glycan chains from glycoproteins inhibits the aphid transmission of the virus whatever the concentration of enzyme used (Table 2), suggesting that N-linked oligosaccharides are necessary for aphid transmission of BWYV. In contrast, aphid transmission of BWYV was not inhibited after O-glycosidase DS treatment of virus particles (Table 2). Moreover, specific deglycosylation of the virus particles using different concentrations of αD-galactosidase, which cleaves terminal α-linked galactose groups showed that aphid transmission was totally suppressed with 0.75 U enzyme and only partialy suppressed when 0.25 U or 0.5 U enzyme was used (Table 2). In order to identify which sugars in the CP or RT proteins may be implicated in aphid transmission of BWYV, we carried out lectin competition tests by adding lectins to BWYV particles during the 24 hr AAP (Table 3). Whereas WGA, TPA, DBA and ConA did not affect aphid transmission at any of the lectin concentrations used, MPA, BSI, LcH inhibited aphid transmission at 250 µg/ml, 500 µg/ml and 250 µg/ml, respectively (Table 3). Integrity and stability of BWYV particles In order to verify that the various treatments (addition of lectins, deglycosylation or metaperiodate oxidation) did not degrade BWYV virions, the presence of genomic viral RNA was assessed using Northern-blot experiments (Fig. 4). The Table 3. Lectin-competition in BWYV Aphid transmission experiments. Aphid transmission experiments were carried out using 50 µg · ml−1 of purified BWYV and different lectin concentrations during a 24 hr AAP as described in Material and Methods. BWYV-infection was monitored using TAS-ELISA and 7C2 monoclonal antibody. Results come from three independent experiments (different lots of purified BWYV particles, successive independent aphid transmission experiments) and represent the percentage of BWYV-infected plants relatively to aphid-inoculated plants, 4 weeks after aphid transmission of virus. Results are means of three independent experiments using 8 plants each time Lectin concentration 2 mg · ml−1 1 mg · ml−1 750 µg · ml−1 680 µg · ml−1 600 µg · ml−1 500 µg · ml−1 250 µg · ml−1 100 µg · ml−1 50 µg · ml−1 25 µg · ml−1 0 µg · ml−1 (control)

MPA

WGA

TPA

0 0

100

95.8 ± 5.5

BSI

DBA 95.8 ± 5.5 95.8 ± 5.5

LcH

ConA

0 0

100

0 0 72.7 ± 7 95.8 ± 5.5

95.8 ± 5.5 95.8 ± 5.5 91.7 ± 5.5 95.8 ± 5.5

95.8 ± 5.5

96.2 ± 4.9

0 0 4.2 ± 5.5 91.7 ± 5.5

95.8 ± 5.5 100 95.8 ± 5.5 95.8 ± 5.5

91.7 ± 5.5 95.8 ± 5.5 95.8 ± 5.5 95.8 ± 5.5

4.2 ± 5.5 91.7 ± 5.5 96.2 ± 4.9 95.8 ± 5.5

95.8 ± 5.5

95.8 ± 5.5

100

96.2 ± 4.9

0

96.2 ± 4.9 95.8 ± 5.5 100 95.8 ± 5.5 100 96.2 ± 4.9

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Fig. 4. Control of BWYV integrity particles by Northern-blot. Genomic RNA (gRNA) was extracted from 2 µg of untreated or modified BWYV particles, separated by denaturing agarose electrophoresis, then detected by Northern-blot using a digoxigenin-UTP-labeled DNA probe complementary to the 3 -terminal 185 residues of the viral genomic RNA. BWYV particles were incubated in presence of 500 µg · ml−1 lectin from Maclura pomifera (MPA), Bandeireae simplicifolia (BSI), Lens culinaris (LcH) or were treated with 5 mM sodium metaperiodate (Na Periodate), 0.75 U PNGase-F (PNGase F) or 0.75 U α-D-galactosidase (Galactosidase). Positive controls were carried out using untreated virions (Control) or virions incubated with 500 µg · ml−1 lectin from Canavalia ensiformis (Con A) or 0.75 U O-glycosidase (O-Glycosidase)

same amounts of BWYV RNA were detected in all extracts (Fig. 4) indicating that BWYV virions were not degraded. These results were also supported by serological testing of modified virions. ELISA responses obtained with the three monoclonal antibodies (2E4, 4D1 and 7C2) after treatment of virions with 5 mM sodium metaperiodate, 0.75 U PNGase-F, 0.75 U α-D-galactosidase or 0.75 U O-glycosydase DS (Table 1) were the same as for the untreated virions, indicating that the various treatments did not cause some unspecific deleterious effect on the capsids. Discussion Lectins with specificity for different oligosaccharides and various glycosidases, in conjunction with SDS-PAGE and Western-blotting, have been used successfully to determine the type of oligosaccharides found in the glycoproteins of several viruses [15, 26, 44, 58]. Our results suggest that the two BWYV structural proteins (CP and RT) are N-glycosylated and contain α-L-fucose, N-acetyl-αD-galactosamine, N-acetyl-β-D-glucosamine, and sugars among α-D-glucose or α-D-mannose, and probably α-D-galactose. These results are supported by the fact that the N-terminal extremity of RT protein possesses the same 203 amino acid sequence as CP protein [39]. This extensive range of sugars is usual for proteins glycosylated in plants [34, 36]. Despite the fact that the same sugars could be present on CP and RT, results show that the level of glycosylation of CP is lower than of RT. This agrees with glycosylation site analysis of RT and CP which showed that only RT protein contained potential O-glycosylation sites and possesses more potential N-glycosylation sites than CP. The carbohydrate moiety in O-glycosylated proteins is large enough on RT protein so that its removal with a combination of NANase II, HEXase I, GALase III, α-D-galactosidase and O-Glycosidase DS leads to an increase in mobility of RT protein. Whereas

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RT protein is predicted to have a molecular weight of 74 kDa, its migration in SDS-PAGE measured with total proteins from BWYV-infected protoplasts indicated a size of about 80 kDa [9], the difference likely being due to glycosylation. In the present work, the RT protein extracted from purified BWYV was cleaved during virus purification, leading to a C-terminally truncated form of about 55 kDa [3, 8, 9, 64] or 52 kDa after enhanced O-deglycosylation treatment (this work). Several amino acid residues of CP protein have been implicated in aphid transmission [9, 47], and a model for the three-dimensional structure of the BWYV S domain of the CP subunit has been proposed based on the model of PLRV [57]. Among the three potential N-glycosylation sites on the BWYV coat protein, Asn50 is not located in a secondary structure element whereas Asn162 and Asn181 are implicated in an α-helix and a β-sheet respectively [10, 57]. For this reason, we hypothesize that Asn50 could be the amino acid that is N-glycosylated on the CP of BWYV and on the N-terminal domain of the RT protein. Five potential O-glycosylation sites have been identified in the conserved RTD found in all luteoviruses. Four of them are located just downstream of the CP cistronsuppressible termination codon in an alternating proline residue sequence of 15 amino acids [39]. Moreover, Ser211, Ser215, Ser217 and Thr221 are surrounded by Pro +3 and −1, an environment that strongly favours glycosylation [14]. Glycosylation is common in envelope proteins of animal viruses [28] but has only rarely been described in plant viruses [6, 15, 20, 44, 58]. In the case of PPV, it has been shown that serine and/or threonine residues can be glycosylated or phosphorylated. This “ying-yang” glycosylation and phosphorylation may play an important role in regulating the different functions of potyviral coat proteins [20]. Oligosaccharides have a broad range of functions in glycoproteins, ranging from stabilization of the native conformation, resistance to proteolytic degradation, intracellular targetting to subcellular compartments and stabilization of protein–protein interactions [28, 29, 63]. The oligosaccharides have been suggested to be important for maintaining the structure of virus particles. It has been proposed that O-GlcNAcylation could protect the surface-exposed N-terminus of PPV coat protein from degradation, although no significant differences in infectivity or virus accumulation in herbaceous hosts have been observed between O-GlcNAc modified PPV mutants and wild-type PPV [20]. Recently, it has been reported that the surface-located and glycosylated N-terminal CP segments of intact PVX virions induce the formation around virions of a columnar-type shell of bound water molecules, which could play a major role in maintaining the surface structure [6]. In the case of TSWV, the envelope glycoproteins are known to play a critical role in the ability of thrips vectors to acquire the virus [5], and it is plausible that the N-linked oligosaccharides present on TSWV glycoproteins play a key role during the initial steps of virus-vector thrips interactions [44]. In the case of BSMV and CPMV, the glycosylation of their CP proteins has been proposed to be a determinant of seed-transmissibility [45]. In the present work, we have shown that CP is N-glycosylated whereas RT proteins of BWYV are

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likely to be O- and N-glycosylated. Aphid transmission of the virus is inhibited after treatments such as oxidation of the glucidic cores by sodium metaperiodate or deglycosylation of BWYV particles by PNGase F or α-D-galactosidase. The fact that PGNase F treatment abolished virus transmissibility supports the idea that a particular sugar, such as α-D-galactose, N-linked to the viral CP and RT proteins is necessary for promoting virus transmission, even if the N-linked glucidic core is so small that a decrease in the two capsid proteins migration cannot be detected in our conditions. The presence of this particular sugar N-linked to CP protein could explain that a RT-deleted mutant can also be aphidtransmissible [47, 53]. In contrast, O-glycosidase DS treatment alone does not inhibit aphid transmission, nor produce a decrease in molecular weight of CP and RT (data not shown) whereas a decrease of the relative molecular weight of the RT protein was only observed using the enhanced deglycosylation protocol that combines NANase II, HEXase I, GALase III, α-D-galactosidase and O-glycosidase DS treatments. O-glycosidase DS only releases unsubstituted disaccharide Gal(β1,3)GalNAc(α1) attached to serine or threonine. Modifications of the Ser/Thr linked Gal(b1,3)GalNAc core such as galactose substitutions inhibit O-glycosidase cleavage and complete deglycosylation of such modified cores requires additional enzymes such as α-D-galactosidase, NANase II, HEXase I, and GALase III. Our results demonstrate that α-D-galactose is probably present in the glucidic cores of the two capsid proteins and that α-D-galactosidase treatment does not produce a sufficient decrease in molecular weight of CP and RT to be detectable in SDS-PAGE (data not shown). We have also shown that aphid transmission of BWYV on membranes was inhibited after α-D-galactosidase treatment of BWYV particles or when lectins specific for α-D-galactose were used. Moreover, the non transmission of BWYV in these experiments cannot be attributed to a degradation of virus particles. Thus, we propose that the glucidic cores linked to the CP and RT proteins are essential for aphid transmission of BWYV, and that α-D-galactose residues, located in CP and/or RT protein that is incorporated into the capsid via its CP moiety with the RT domain protruding from the virion surface [8], play a fundamental role in the interaction between virion and aphid. However, it is not clear if the glucidic cores play a direct role in the entry of the virions in epithelial cells of the aphid, as has been described when human immunodeficiency virus (HIV) penetrates human cells [4], or if the glucidic cores play an indirect role by protecting BWYV particles from degradation in the insect body by interacting with symbionin, the major protein synthetized by the endosymbiont Buchnera aphidicola, which is known to prevent virion degradation [62]. Nevertheless, we cannot rule out the possibility that disturbance of glycosylation could affect directly the infectivity of the virions rather than their aphid transmissibility. Several lectins, such as ConA or lectin from Griffonia simplicifolia, are toxic to mammalian cells and their toxicity is due to their binding to cell-surface sugars of cells, which leads to them being taken up by the cell itself where they inhibit protein synthesis by interfering with peptide chain elongation on polyribosomes [37]. Lectins from plants present an insecticidal activity most probably through

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their carbohydrate binding properties [59]; thus protecting plants from predation by larvae [42, 43]. Transgenic plants transformed with Helianthus tuberosus agglutinin or Con A genes have been demonstrated to have deleterious effects on Myzus persicae by decreasing aphid fecundity and retarding larval development [12, 22]. In the present work, aphid transmission was not inhibited by addition of high concentrations (1–2 mg · ml−1 ) of ConA, DBA, TPA, and WGA in the artificial diet during the 24 hr AAP of BWYV. These results suggest that these lectins have no lethal effect on the endocytosis process that occurs in the midgut of the insect vector. The difference observed between our results and those of Rahb´e et al. [46], who showed a toxic effect of ConA for M. persicae, could be due to the shorter period of exposure to ConA (24 hr) in our experiments. Moreover, our electron microscope observations did not reveal significant morphological differences in the structure of the aphid midgut after acquisition of BWYV in absence or presence of the different lectins (data not shown) suggesting that in our conditions, no lethal effects of lectins was present. There is no direct correlation between aphid transmission and the sugar specificity of the lectin because BWYV aphid transmission was totally inhibited with 250 µg · ml−1 LcH and no inhibition was observed for up to 1 mg · ml−1 ConA, although these two lectins recognize specifically the same carbohydrates: α-D-mannose and α-D-glucose. Compared to LcH, ConA could enhance clathrin-dependent endocytosis and facilitate uptake of BWYV as is the case for another lectin isolated from Xerocomus chrysenteron that was shown to greatly enhance bovine serum albumin uptake in M. persicae [21]. Rahb´e et al. [46] have also shown that there is no correlation between insect toxicity and sugar specificity of lectins. In conclusion, our work demonstrates for the first time that in the case of a circulative virus such as BWYV, lectins may alter aphid transmission by influencing the inoculation and/or the acquisition process. It seems plausible, therefore, that transgenic plants expressing lectin-genes may offer a possible way of controlling insect transmitted plant virus diseases [54]. Acknowledgments This work was supported by grants from the Institut National de la Recherche Agronomique, D´epartement Sant´e des Plantes et Environnement (France). The authors are grateful to Dr. A. Seddas for his help in producing BWYV-specific antibodies, to C. Reinbold for assistance in electron microscopy, to Dr. D. Massotte for her help in detecting glycoproteins, to M.-T. Simonis for technical help in aphid-transmission experiments, to Dr. V. Brault for assistance in Northern-blot analysis and to Dr. M. H. V. van Regenmortel for useful discussion and assistance with manuscript preparation.

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