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J. Biol. Chem., Vol. 277, Issue 1, 135-140, January 4, 2002

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The Capsid Protein of a Plant Single-stranded RNA Virus Is Modified by O-Linked N-Acetylglucosamine^*

M. Rosario Fernández-Fernández, Emilio Camafeita, Pedro Bonay^§, Enrique Méndez, Juan Pablo Albar, and Juan A. García^¶

From the  Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, and ^§ Centro de Biología Molecular "Severo Ochoa," Campus de la Universidad Autónoma de Madrid, 28049 Cantoblanco, Madrid, Spain

Received for publication, July 20, 2001, and in revised form, October 16, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plum pox virus (PPV) is a member of the Potyvirus genus of plant viruses. Labeling with UDP-[³H]galactose and galactosyltransferase indicated that the capsid protein (CP) of PPV is a glycoprotein with N-acetylglucosamine terminal residues. Mass spectrometry analysis of different PPV isolates and mutants revealed O-linked N-acetylglucosamination, a modification barely studied in plant proteins, of serine and/or threonine residues near the amino end of PPV CP. CP of PPV virions is also modified by serine and threonine phosphorylation, as shown by Western blot analysis with anti-phosphoserine and anti-phosphothreonine antibodies. Thus, "yin-yang" glycosylation and phosphorylation may play an important role in the regulation of the different functions in which the potyviral CP is involved.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

O-Linked N-acetylglucosamine (O-GlcNAc) is a simple monosaccharide modification that is abundant on serine or threonine residues of nuclear and cytoplasmic eukaryotic proteins (1, 2). Although many proteins of animal origin have been found to carry this modification, including transcription factors, cytoskeletal proteins, nuclear pore proteins, viral proteins, oncogene products, and tumor suppressors, very little is known about its incidence in the plant world (3). O-GlcNAc has been suggested to be reciprocal to phosphorylation (2), and some studies carried out on different proteins have shown that O-GlcNAc and O-phosphate alternatively occupy the same or adjacent sites. This supports the hypothesis that the saccharide could somehow be blocking phosphorylation (4).

Structural proteins from several animal viruses such as the cytomegalovirus basic phosphoprotein (5), the adenovirus fiber protein (6), and the baculovirus gp41 protein (7), as well as the non-structural NS26 protein from a rotavirus (8), have been described to be modified by O-GlcNAc, but no function for these modifications has been envisaged.

Plum pox virus (PPV)¹ is a member of the Potyvirus genus of plant viruses. PPV is the causal agent of sharka, a very devastating disease affecting stone fruit crops of the Prunus genus. The potyvirus genome consists of a single-stranded messenger-polarity RNA molecule of about 10 kb, with a VPg protein at its 5' end and a poly(A) tail at its 3' end. This genome is translated into a large polyprotein that is further processed by three virus-encoded proteases (9, 10). The genome is encapsidated by ~2000 units of a single type of capsid protein (CP), encoded at the 3' end of the genome (11) (Fig. 1).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Sequence comparison of the N-terminal amino acids of several PPV isolates and mutants. Foreign sequences included in the chimeric viruses are in bold letters, and dashes indicate the amino acids deleted by the NAT mutation. Schematic representations of the potyvirus genome and of the three regions of the potyvirus CP are shown above the sequences.

The potyvirus CP is involved in cell to cell and long distance movement inside the plant. In particular, the N- and C-terminal parts of the CP are known to be involved in systemic movement (12, 13). Moreover, the N-terminal region of the potyvirus CP, which is extremely variable among different virus species, holds an Asp-Ala-Gly (DAG) amino acid triad that is essential for aphid transmission (14, 15).

The CP from a potyvirus, the potato virus A (PVA), has been described recently to be phosphorylated in serine and threonine residues (16), and this modification appears to down-regulate the RNA binding function of PVA CP. In this paper we explain that PPV CP, in addition to being phosphorylated, is modified by O-linked N-acetylglucosamination. At least one of the glycosylated residues lies at the N-terminal region of the protein, namely at a 15-amino acid sequence that is deleted in some natural non-aphid-transmissible (NAT) PPV mutants (17, 18). To our knowledge this is the first extensive characterization of this modification in a plant protein.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Virus Infection and Purification-- PPV strains R and PS and the chimeras PPV-CPV (19) and PPV-MCMV (20) were used in these experiments. Viruses were propagated in Nicotiana clevelandii plants. To establish systemic infection, young plants were mechanically inoculated by rubbing three leaves per plant with crude sap from PPV-infected leaves (1 g of leaf tissue in 2 ml of 5 mM sodium phosphate, pH 7.2), using Carborundum as an abrasive. The virus was purified according to Laín et al. (21).

Galactosyltransferase Assay-- Eight µg of purified PPV virions were labeled in a 100-µl reaction mixture with uridine diphospho[6-³H]galactose (UDP-galactose) (5-20 Ci/mmol) (Amersham Biosciences, Inc.) and bovine milk galactosyltransferase (galactosyltransferase) (Sigma) according to the method described by Roquemore et al. (2). As positive and negative controls, a protein mixture containing 10 µg of chymotrypsinogen A, ovalbumin, serum albumin, and aldolase (Roche Molecular Biochemicals) was labeled in the same conditions. UDP-galactose labeling of galactosyltransferase in the absence of other added proteins was also carried out as an additional control. After the labeling reaction the samples were loaded in a Sephadex G-50 column to separate labeled proteins from unincorporated UDP-galactose.

PPV CP was immunoprecipitated with a mixture of protein A-Sepharose (Sigma) and polyclonal serum against PPV (or a control preimmune serum) in a buffer containing 150 mM NaCl, 10 mM Tris-HCl, pH 7.8, 1% Triton X-100, and 0.1% SDS.

Labeled products (before or after immunoprecipitation) were separated in a 12.5% SDS-polyacrylamide gel and detected by fluorography, after treatment of the gel with Amplify (Amersham Biosciences, Inc.).

Western Blot Analysis-- Two different amounts of purified virions of wild-type PPV-R and PPV-PS and chimeric PPV-CPV in addition to a negative control, bovine serum albumin (Roche Molecular Biochemicals), were separated by SDS-PAGE in triplicate. One gel was stained with Coomassie Blue, and the two other ones were transferred to nitrocellulose membranes. The membranes were saturated with 2% bovine serum albumin and subjected to immunodetection. Two different monoclonal antibodies, anti-phosphoserine and anti-phosphothreonine (Calbiochem), were used as primary antibodies at a concentration of 0.1 µg/ml. The secondary antibody was a peroxidase-conjugated goat anti-mouse purchased from Jackson ImmunoResearch Laboratories (1:1500). The peroxidase reaction was developed with the ECL^TM kit (Amersham Biosciences, Inc.).

Matrix-assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) Analysis of PPV CP Digested with Proteases-- Ten µg of purified PPV-R, PPV-CPV, and PPV-MCMV virions were digested for 5-15 min at room temperature with 1.7 ng of trypsin (Promega) in a buffer containing 25 mM Tris-HCl, pH 8, and 1 mM EDTA in a reaction volume of 10 µl. Alternatively, 10 µg of purified virions were first digested for 5 h at room temperature with 1 µg of protease V8 (Sigma) in 10 µl of 10 mM pH 8, then 5 ng of trypsin were added to 5 µl of the digestion mixture, and the reaction was left at room temperature for 30 min.

For MALDI-TOF analysis 0.5-µl aliquots of the reaction mixtures were deposited onto the stainless steel MALDI probe and allowed to dry at room temperature. Then 0.5 µl of matrix solution (saturated -cyano-4-hydroxycinnamic acid in 33% aqueous acetonitrile and 0.1% trifluoroacetic acid) were added and again allowed to dry at room temperature. The spectra were acquired in a MALDI-TOF Bruker Reflex^TM II equipped with visualization optics and a nitrogen laser (337 nm). Linear mode was used, with an acceleration voltage of 28.5 and 1.5 kV in the linear detector, accumulating 100 spectra corresponding to the same number of laser shots in conditions of threshold irradiance. The spectrometer was externally calibrated using the singly and doubly charged signals from bovine insulin.

The post-source decay (PSD) MALDI spectra were measured on a Bruker Reflex^TM III MALDI-TOF mass spectrometer equipped with the SCOUT^TM source in positive ion reflector mode using delayed extraction (22). Spectra were recorded in 14 segments, each successive segment representing a 20% reduction in reflector voltage. The precursor ion was selected by FAST^TM deflecting pulses. About 200 shots were averaged per segment, and the segments were pasted, calibrated, and smoothed under the computer control of Bruker XTOF 5.0.3 software. Data analysis was performed using Bruker BioTools 2.0 software.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PPV Virions Are Labeled by Galactosyltransferase-- A common and sensitive method frequently used in the detection of O-linked N-acetylglucosamination of proteins is to covalently label the protein-bound sugar with UDP-[³H]galactose and galactosyltransferase (2). Thus, to gain a first insight into the possibility that the PPV CP could be modified by O-GlcNAc residues, PPV-R virions purified from N. clevelandii plants were subjected to that treatment. Two different lots of purified virions were treated in order to ensure that the results are representative of the general PPV population rather than specific features of a batch of purified virions. The fluorography of the reaction products, separated by SDS-PAGE, revealed the presence of proteins labeled with [³H]galactose in the PPV samples, with a main band having the electrophoretic mobility expected for PPV-R CP (Fig. 2, lanes 1 and 2). Ovalbumin, a well known glycoprotein, was strongly labeled, whereas other proteins included in the control reactions remained unlabeled (Fig. 2, lane 3). Although it was not detected in the fluorography shown in Fig. 2 (lane 4), measurement by liquid scintillation of the incorporated radioactivity demonstrated a weak autolabeling of the galactosyltransferase (data not shown).

View larger version (61K): [in this window] [in a new window]   Fig. 2.   [3H]Galactose labeling of PPV-purified virions. Two different batches of purified PPV virions (lanes 1, 5, and 6 and lanes 2, 7, and 8) were enzymatically labeled with galactosyltransferase and UDP-[3H]galactose. A mixture of marker proteins containing chymotrypsinogen A, ovalbumin, serum albumin, and aldolase (lanes 3 and 9) was also subjected to the labeling reaction. The reaction products were loaded on the gel and subjected to SDS-PAGE directly (lanes 1-4) or after immunoprecipitation (6 times the amount directly loaded) with anti-PPV (lanes 6, 8, 9, and 10) or preimmune (lanes 5 and 7) sera. Total (lane 4) or immunoprecipitated with anti-PPV serum (lane 10) products of a labeling reaction lacking substrate were also analyzed. The gel was treated with Amplify (Amersham Biosciences, Inc.) and autoradiographed. The sizes of prestained broad range molecular weight markers (Bio-Rad) run in the lane labeled Mr are indicated at the left side of the panel.

The [³H]galactose labeling of PPV-CP by the galactosyltransferase treatment was confirmed by immunoprecipitation. A protein with the size expected for PPV-R CP was immunoprecipitated by a polyclonal antibody against PPV from the products of the labeling reactions of PPV virions (Fig. 2, lanes 6 and 8). No labeled bands were detected when these samples were immunoprecipitated with a preimmune serum (Fig. 2, lanes 5 and 7) or when the labeled products of the control reactions were immunoprecipitated with the anti-PPV serum (Fig. 2, lanes 9 and 10).

MALDI-TOF Analysis Suggests That the N-terminal Region of PPV-CP Is Modified with O-GlcNAc Residues-- The labeling of PPV-CP with galactosyltransferase demonstrates that this protein is modified with sugars that have terminal residues of N-acetylglucosamine, but it gives no indication of the nature of the saccharide architecture linked to the polypeptide bond. To obtain more information about the sugars linked to PPV-CP, purified virions of the PPV-R isolate were subjected to a mild trypsin digestion, and the resulting peptides were analyzed by MALDI-TOF (Fig. 3). This protease treatment is expected to mainly affect the N- and C-terminal regions of the protein, which are known to be exposed at the surface of the virions. In agreement with this, a peptide was detected with the size (4075 Da) of a tryptic peptide spanning amino acids 1 to 39 of the CP of PPV-R (no cleavage appears to take place efficiently at arginine 4). Two other major peptide mass signals appeared in the spectrum at m/z = 4278 and 4481. These two peptides differ from the 4075 peptide in 203 and 406 Da, as expected for a single and double modification with O-GlcNAc. The assumption that the 4278 and 4881 peptides are variants of the 4075 peptide linked to one or two residues of O-GlcNAc was further supported by the fact that the three main peaks were accompanied by small satellite peaks 42 Da apart. The three minor peptides could be the result of N-acetylation of the unmodified and the glycosylated forms of the N-terminal fragment of CP, which is in accordance with the previously reported blockade of the N-terminal ends of several PPV proteins (23).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   MALDI-TOF analysis of PPV virions digested with proteinases. Purified virions were digested with trypsin for 5 min (PPV-R and PPV-CPV) or 15 min (PPV-MCMV) (panel A) or with V8 for 5 h and trypsin for 30 min (panel B) and subjected to MALDI-TOF analysis. The mass (in daltons) assigned to the different peaks is indicated above them. The N-terminal region of each virion CP is shown using the amino acid one-letter code. Sequences deleted by the NAT mutation and replaced by foreign sequences in PPV-CPV and PPV-MCMV are underlined. The arrows indicate the position where trypsin and V8 cleave according to the spectra results.

The 10 to 39 Sequence of PPV CP Is O-GlcNAc-modified-- Two different approaches were followed to confirm the O-GlcNAc modification of the N-terminal segment of PPV-CP: tryptic peptide analysis of mutated CPs and PSD analysis of the peptides presumably derived from the N-terminal region of the protein.

If the allocation of the 4075 peptide to the 1-39 amino acid sequence was correct, this peptide could not be the target for N-glycosylation, because it does not include any asparagine residue. On the contrary, it includes two serine and two threonine residues, which might be the target for O-glycosylation. PPV-CPV and PPV-MCMV are two PPV-R chimeras in which the 15 amino acids that are deleted in the NAT PPV mutants (17, 18) are replaced by sequences coding 15 amino acids of the VP2 protein of canine parvovirus (19) and 9 amino acids of murine cytomegalovirus pp89 phosphoprotein (20), respectively. Potential targets of trypsin digestion and O-GlcNAc modification differ in these chimeras from those of PPV-R. Two additional arginine residues are present in the 1-39 sequence of the PPV-CPV CP, which appear to be recognized by the trypsin treatment. Cleavage at arginine 26 gives rise to a 2910-Da peptide, which was observed in the spectrum of Fig. 3A, whereas the 2511-Da peptide, derived from cleavage at arginine 23, could only be detected at longer incubation times (data not shown). A peak corresponding to a 4276-Da peptide that results from trypsin cleavage at arginine 39 was detected at short digestion times (Fig. 3A). However, no peaks attributable to O-GlcNAc modifications of this peptide could be detected, as expected from the absence of serine and threonine residues in the 1-39 sequence of the PPV-CPV CP sequence. The additional peak at m/z = 2757 displayed in the spectrum of Fig. 3A, could be attributed to a partially digested peptide from the C-terminal region of PPV CP and disappeared at longer incubation times (data not shown).

The MALDI-TOF spectrum of the tryptic peptides of PPV-MCMV CP revealed the presence of a peak at the position expected for the 4341-Da peptide resulting from trypsin cleavage at arginine 39 (Fig. 3A). Moreover, a peak corresponding to the acetylated form of this peptide (4383 Da) was also detected, and it was more prominent than those from the other PPV CP samples (Fig. 3A). Despite the fact that serine 16 of PPV CP is conserved in PPV-MCMV CP and that this chimeric protein contains a new threonine residue, no peaks that could correspond to peptides modified with O-GlcNAc residues were detected in the spectrum (Fig. 3A). This result appears to indicate that the MCMV-derived threonine 24 is not susceptible to O-GlcNAc modification and that either serine 16 is not glycosylated in wild-type PPV CP or its new context in PPV-MCMV CP prevents its glycosylation.

The 1-39 peptide is too large to be suitable for an accurate MALDI-PSD analysis. For this reason, purified PPV-R virions were subjected to controlled double protease digestion with V8 and trypsin. V8 mainly cleaves at the carboxylic sites of glutamic acid residues, although in some conditions it also cleaves at aspartic acid residues. MALDI-TOF analysis revealed the release of three peptides with masses corresponding to a peptide of the N-terminal region of PPV-R CP spanning amino acids 10-39 (2974 Da) and to singly (3177 Da) and doubly (3380 Da) O-GlcNAc-modified forms of that peptide (Fig. 3B). These three peptides were subjected to MALDI-PSD analysis (Fig. 4 and data not shown). The fragmentation spectrum of the smaller peptide (2974 Da) was in very good agreement with the 10-39 sequence (83% of the expected b and y ions could be assigned, in contrast with the 10% ion assignment obtained for a random sequence of the same size). The spectrum of the middle size peptide (3177 Da) conformed to the 10-39 sequence carrying a single O-GlcNAc modification. The proximity of the serine and threonine residues precluded an accurate identification of the modified residues, although the two threonine residues appeared to be the most likely candidates (78 and 72% of b/y ions assigned for threonines 24 and 19, respectively). Similarly, the fragmentation spectrum of the largest peptide (3380 Da) supported its identification as the 10-39 peptide with two O-GlcNAc modifications, although it could not ascertain whether single amino acids carried two O-GlcNAc molecules or a couple of amino acids were singly modified and did not identify the modified residues (b/y ion assignments among 50 and 63%).

View larger version (50K): [in this window] [in a new window]   Fig. 4.   MALDI-PSD analysis of PPV virions. Some of the major backbone fragmentations in linear peptides according to Roepstorff and Folhmann (28) (panel A). PSD MALDI spectra from peptides at m/z = 2974 (top), 3177 (middle), and 3381 (bottom). Identified "y" ions considering a +203-Da modification located in the residues highlighted (Thr-15 for m/z = 3177 and Thr-10 and Thr-15 for m/z = 3381) are displayed (panel B).

PPV CP Is Modified by Phosphorylation in Serine and Threonine Residues-- O-GlcNAc modification appears to affect mainly, if not exclusively, proteins that are also modified by phosphorylation (24). In addition, phosphorylation at serine or threonine residues has been demonstrated for the CP of the potyvirus PVA. To assess whether PPV CP was also phosphorylated, purified virions of different PPV isolates were subjected to Western blot analysis with antibodies specific for phosphothreonine or phosphoserine residues. The CPs of all three PPV isolates (PPV-PS and PPV-R, which belong to the strains M and D, respectively, and the PPV-R-derived chimeric virus PPV-CPV) were recognized by both the anti-phosphoserine (Fig. 5B) and the anti-phosphothreonine (Fig. 5C) antibodies. Densitometric analysis revealed that the relative intensities of the PPV-PS anti-phosphoserine and anti-phosphothreonine immunoreactions were 2-3 times higher than those of PPV-R and PPV-CPV.

View larger version (63K): [in this window] [in a new window]   Fig. 5.   Immunodetection of PPV CP phosphorylated at serine and threonine residues. Aliquots of ~1 µg (lanes 2, 4, 6, and 8) or 0.5 µg (lanes 3, 5, 7, and 9) of bovine serum albumin (lanes 2 and 3) and of PPV-PS (lanes 4 and 5), PPV-CPV (lanes 6 and 7), and PPV-R (lanes 8 and 9) virions were loaded in three gels and subjected to 12.5% SDS-PAGE. One gel was stained with Coomassie Blue (panel A). The other two gels were subjected to Western blot analysis with anti-phosphoserine (panel B) and anti-phosphothreonine (panel C) antibodies (Calbiochem). Prestained broad range molecular weight markers (Bio-Rad) were used as standards (lane 1).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Since the first description of protein glycosylation at specific serine or threonine residues by single -N-acetylglucosamine (GlcNAc) moieties (25) a large number of cytoplasmic and nuclear proteins of many eukaryotic organisms have been found to carry this modification (24). O-Glycosylation with terminal GlcNAc modification has been described for nuclear pore complex proteins of tobacco, but in contrast with O-GlcNAc modification of animal proteins, the size of the sugar chains of these tobacco proteins corresponded to more than five monosaccharide units, which were not further characterized (26, 27). On the contrary, the size of one of the peptides detected by the MALDI-TOF analysis of PPV CP corresponds to a single GlcNAc modification (Figs. 3 and 4), resembling the typical O-GlcNAc glycosylation of animal proteins. Taking into account that the sequence of the PPV CP peptide carrying the identified modification has two serine and two threonine residues, the doubly glycosylated form detected by the MALDI-TOF analysis is likely to correspond to two single O-GlcNAc modifications rather than to the attachment of two O-GlcNAc molecules to a single threonine or serine residue, although our present data do not allow us to rule out the latter possibility. The MALDI analysis did not precisely identify the O-GlcNAc-modified residues at the N-terminal region of PPV CP, although the fact that no glycosylation was detected in the 1-39 peptide of PPV-MCMV CP (Fig. 3), which conserves Ser-16, suggests that the modification could affect residues Thr-19, Thr-24, and/or Ser-25. In this regard, the YinOYang program² predicts these three residues, but not Ser-16, as putative targets for O-GlcNAc modification (data not shown).

O-GlcNAc modifications of structural and non-structural proteins of animal viruses with double-stranded RNA (8) or DNA (5-7) genomes have been described. However, to our knowledge, PPV CP is the first protein of a plant virus and of a virus with a positive strand RNA genome, for which a typical O-GlcNAcylation has been unambiguously demonstrated. O-GlcNAc-modified proteins appear to have two common features: they are also phosphorylated, and they form reversible multimeric complexes via interactions that are regulated by phosphorylation (1). The CP of potyviruses may exhibit both traits. Protein-protein and protein-RNA interactions have to be established and disturbed in virion assembly and RNA desencapsidation, respectively. Moreover, very recently it has been shown that phosphorylation down-regulates the RNA binding activity of the CP of a potyvirus, PVA (16). It was demonstrated that PVA CP was phosphorylated in the infected plants, but the authors suggested that it was not phosphorylated when packaged into virions, based on the fact that disrupted particles but not intact virions could be phosphorylated in vitro by a plant protein extract. Our immunoreaction experiments (Fig. 5) appear to indicate that PPV CP was phosphorylated at threonine and serine residues when it was assembled in virions. These data are not in conflict with those of Ivanov et al. (16), because phosphorylation of the assembled CP might be only partial, and disassembled CP would, therefore, be susceptible to further phosphorylation. In agreement with this suggestion, the phosphorylation levels of PPV-PS and PPV-R CPs appear to be different. Probably, these differences reflect changes in the global extent of phosphorylation rather than specific features of the phosphorylation patterns, because sequence analysis showed only minor differences between PPV-PS and PPV-R CPs in the likelihood of phosphorylation at serine and threonine residues predicted by the NetPhos 2.0 program (29) or in the total content of these amino acids (data not shown). The fact that the CP of PPV-CPV, which lacks the two serine and two threonine residues present in the first 39 amino acids of the CP of PPV-R, showed a phosphorylation level similar to that of this protein suggests that the contribution of these threonine and serine residues to the phosphorylation status of the CP assembled in virions is rather low. O-GlcNAcylation is known to be reciprocal to phosphorylation in many proteins; thus, O-GlcNAc residues could be protecting the N terminus of the CP, which is surface-exposed (30), against phosphorylation when the CP is assembled in virions. Interestingly, six of the eight serine and threonine residues that the YinOYang program² predicts to be able to suffer reciprocal O-GlcNAc/phosphate modifications are in the N-terminal region of the PPV-R CP (data not shown). In any case, although the simultaneous detection of O-GlcNAc modification and phosphorylation in PPV virions could be due to the presence of a mixture of molecules carrying each modification in an excluding manner, it appears more likely that CP molecules forming PPV particles could be modified with both phosphate and O-GlcNAc residues, as has been proposed for other proteins (24).

What is the functional relevance of O-GlcNAc modifications for PPV infection? The fact that PPV-NAT, PPV-CPV, and PPV-MCMV (Fig. 1) are viable indicates that O-GlcNAc-modified residues at the 13-27-amino acid segment deleted by the NAT mutation are not essential. No significant differences in infectivity or virus accumulation in herbaceous hosts have been observed between these mutant viruses and wild-type PPV under experimental conditions (data not shown). This does not preclude the possibility that some differences in adaptation to the selective pressure of their natural environment could exist among these viruses. In this regard, the fact that NAT mutants have appeared spontaneously when PPV was propagated in herbaceous plants after transfer from its natural woody host (17, 18) suggests that NAT region, and possibly its phosphorylation and O-GlcNAcylation, could be important for viral fitness, at least in some hosts. Moreover, the possibility that O-GlcNAcylation of PPV CP is restricted to the N-terminal peptide analyzed in this work is quite unlikely, and an appropriate modification of Ser/Thr residues in other regions of the CP might be relevant for virus viability.

An effect of protein phosphorylation on the stability of poliovirus capsids and its consequent involvement in viral uncoating was described some years ago (31). More recently, it has been shown that phosphorylation of PVA CP affects its capacity to interact with RNA, suggesting that this modification might contribute to the control of the amount of genomic RNA available for translation, replication, encapsidation, and movement (16). A similar role has been suggested for the phosphorylation of the CP of PVX (32) and the movement protein of TMV (33), which has been shown to modulate the effect of these proteins on translation of the genomic RNA. It would be expected that O-GlcNAcylation, reciprocal to phosphorylation, could also play a key role in these regulatory mechanisms.

O-GlcNAc modification of the capsid protein is probably not a specific feature of potyviruses. No data are available on this kind of modification in animal viruses with positive strand RNA genome. However, it has been reported that capsid proteins of some positive strand RNA viruses of plants, namely, barley stripe virus, cowpea mosaic virus (34), and PVX (35), could be glycosylated, although the cowpea mosaic virus results have been recently challenged (36). The saccharide chain appeared to be attached to the potato virus X CP via an O-glycosidic linkage, but a more extensive characterization of the CP sugar moieties was not done. Further work is needed to determine the extent and the relevance of O-GlcNAcylation and phosphorylation, as well as the relationships between these two kinds of modifications, in the CPs of RNA viruses.

	ACKNOWLEDGEMENTS

We thank Elvira Domínguez and Juan A. López for technical assistance, Mario Mellado and José Miguel Rodríguez-Frade from the Centro Nacional de Biotecnología Department of Immunology and Oncology for anti-phosphothreonine and anti-phosphoserine antibodies, and Javier Ortego for help in immunoprecipitation experiments.

	FOOTNOTES

^* This work was supported by Comisión Interministerial de Ciencia y Tecnología, Spain Grants BIO98-0769 and BIO2001-1434 and European Union Grant QLK2-1999-00739.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 34-1-5854535; Fax: 34-1-5854506; E-mail: jagarcia@cnb.uam.es.

Published, JBC Papers in Press, October 17, 2001, DOI 10.1074/jbc.M106883200

² R. Gupta, S. Brunak, and J. Hansen, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: PPV, plum pox virus; CP, capsid protein; CPV, canine parvovirus; PVA, potato virus A; NAT, non-aphid-transmissible; MCMV, murine cytomegalovirus; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; PSD, post-source decay.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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