Rotavirus NS26 is modified by addition of single O-linked residues of N-acetylglucosamine

VIROLOGY 182, 8-16 (1991)

Rotavirus NS26 Is Modified by Addition of Single O-Linked Residues of IV-Acetylglucosamine

SILVIA ADRIANA GONZALEZ’ AND OSCAR R. BURRONE*

lnstituto de lnvestigaciones Bioquimicas, Fundaci6n Campomar-CONICET and Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Avenida Patricias Argentinas 435, 1405, Buenos Aires, Argentina

Received November 6, 1990; accepted January 8, 199 1

We studied the post-translational modification of NS26, the protein product of rotavirus gene 11 segment. Based on the presence of a putative N-glycosylation site and the high content of serine and threonine residues in gene 11 amino acid sequence we investigated whether NS26 is modified by carbohydrate addition. Specific antibodies raised against the gene 11 product expressed in Escherichia co/i recognized in infected cells two polypeptides with apparent molecular weight of 26,000 (26-kDa polypeptide) and 28,000 (28-kDa polypeptide). Pulse-chase experiments demonstrated that the 26-kDa product was processed to the 28-kDa polypeptide. Both polypeptides were metabolically labeled with [3H]glucosamine, indicating the presence of a carbohydrate moiety on the protein. NS26 was found to be resistant to endo-,!%fV-acetylglucosaminidase H and endo-@Vacetylglucosaminidase Flpeptide:N-glycosidase F treatment, but sensitive to removal by alkali-induced p-elimination, suggesting that the saccharide chain was attached to the protein via an 0-glycosidic linkage. Chromatographic analysis of total acid hydrolysates of [3H]glucosamine-labeled NS26- bound carbohydrate indicated the presence of N-acetylglucosamine. In addition, mild alkaline treatment of NS26 in the presence of NaB3H, identified the O-linked carbohydrate moiety as N-acetylglucosamine. Taken together, these data demonstrate that NS26 is processed to a 28-kDa polypeptide by addition of O-linked monosaccharide residues of N-acetylglucosamine. 0 1991 Academic Press, Inc.

INTRODUCTION

Rotaviruses, members of the Reoviridae family, are the major etiologic agents of infantile gastroenteritis in many mammalian species including man (reviewed by Estes et al., 1983; Kapikian and Chanock, 1985). Their genome, composed of 11 segments of double- stranded (ds) RNA, is surrounded by two icosahedral layers (shells) of proteins. The inner shell consists of a core of the dsRNA genome and the structural proteins VP1 , VP2, and VP3 surrounded by the major inner shell protein VP6, whereas the outer shell is composed of VP7 and VP4. By contrast, the nonstructural proteins NS53, NS34, NS35, NS26, and NS28 are only present in infected cells. Of all the rotavirus proteins, five of them have been shown to bear post-translational modifications. VP2 and VP6 appear to be myristilated (Clark and Desselberger, 1988) whereas both VP7 and NS28 contain N-linked high-mannose oligosaccharide residues, a finding consistent with their exclusive rough endoplasmic reticulum localization (Kabcenell and At- kinson, 1985). Interestingly, NS26, the protein product

’ To whom correspondence and requests for reprints should be addressed at University of Brussels (ULB), Department of Molecular Biology, Rue des Chevaux 67, 1640 Rhode-Saint-Genese. Belgium. Fax: 32-2-650 99 99.

’ Present address: International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-3401 2 Trieste. Italy.

0042.6822/91 $3.00 Copynght 0 1991 by Academic Press, Inc

All rights of reproduction I” any form reserved.

of genome segment 11, exhibits an apparent molecular mass of 28,000 (28 kDa) which differs from that of the primary translation product obtained in cell-free translation systems (26 kDa), suggesting that it may undergo a post-translational modification (Ericson et al., 1982; Welch et a/., 1989). Although it has recently been shown that NS26 is a phosphoprotein (Welch et al., 1989), removal of the phosphate group did not change the electrophoretic mobility of the protein, thus remaining unknown the nature of the main modification responsible for the observed molecular weight shift. The deduced amino acid sequence of the major open reading frame of gene 11 shows a potential N-glycosylation site (Asn-Xaa-Ser/Thr) at residue 20 and a high content of the amino acids serine (20%) and threonine (5%) (Gonzalez et al., 1989; Gonzalez and Burrone, 1989) which suggests two possible modes of modification, N-linked and O-linked glycosylation, respectively. In this paper we demonstrate that NS26 bears O-linked N-acetylglucosamine (0-GlcNAc) monosaccharide residues, a novel carbohydrate-peptide linkage which has been shown to be present on proteins localized to the cytoplasmic and nucleoplasmic com- partments of the cell (for a review, see Hart et a/., 1988, 1989). In addition, we show that 0-glycosylation ac- counts for the observed molecular weight of 28 kDa for NS26. To our knowledge, NS26 is the second example of an 0-GlcNAc-bearing glycoprotein to be described in viruses.

8

ROTAVIRUS 0-GLYCOSYLATION

MATERIALS AND METHODS

Cells and viruses

MA1 04 cells were grown as monolayers in Dul- becco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 pg/ml streptomycin (all from Gibco Laboratories, Grand Island, NY). Porcine rotavirus OSU strain was propagated and grown in MA1 04 cells as previously described (Estes et a/., 1979).

Expression of gene 11 protein product in Escherichia co/i

A full-length cDNA clone corresponding to OSU genome segment 11 (Gonzalez and Burrone, 1989) was used to express the protein encoded by its longest open reading frame in a bacterial system (pUR 288 plasmid) which produces fusion proteins with @-galac- tosidase (P-Gal) (Ruther and MUller-Hill, 1983). The cDNA clone was linearized with EcoRI, and after filling in its ends with Klenow fragment of DNA polymerase 1 (Biolabs Inc.), it was ligated to phosphotylated BamHl linkers (Biolabs Inc.). The cDNA insert was excised from pUC19 plasmid by digestion with BarnHI and the purified 670-bp insert was ligated to BarnHI-digested and dephosphorylated pUR288 vector with T4 DNA li- gase (Biolabs Inc.). Plasmids bearing inserts in the desired transcriptional orientation were identified by Sacl digestion. The correct construct, designated pUR288: 1 1, was verified by dideoxynucleotide sequencing of the alkali-denatured supercoiled plasmid (Hattori and Sakaki, 1986).

Synthesis of NS26 protein in E. co/i

E. co/i JMlOl cells harboring the pUR288:l 1 plasmid were grown to an OD,,, = 0.5-0.65 at 37” in Luria broth medium supplemented with 100 pg ampicillin/ml and the expression of the fusion protein was induced with 2 mM isopropyl fl-p-thiogalactopyranoside (IPTG; Bethesda Research Laboratories, Gaithersburg, MD) for 90 min. Bacteria were lysed and disrupted by boiling in 2 ml of 1 X Laemmli’s sample buffer (Laemmli, 1970) and a fraction of the lysate (5 ~1) was analyzed by sodium dodecyl sulfate (SDS)-7.5% polyacrylamide gel electrophoresis and staining with Coomassie bril- liant blue R250. The fusion protein P-Gal:NS26 was purified from a preparative SDS-7.5% polyacrylamide gel by electroelution in 25 ml\/l Tris (pH 8.3), 192 m/l/l glycine, and 0.1% SDS buffer. The yield of fusion protein was quantitated by Bradford method (Bradford, 1976) and further used as antigen for immunization of guinea pigs.

Immunization schedule

Two rotavirus-seronegative guinea pigs were inocu- lated with 200 pg of the purified ,!?-Gal:NS26 fusion protein three times. The first inoculation was intramuscu- lar with complete Freund’s adjuvant and the subse- quent injections were intradermic with incomplete adjuvant at 15 and 30 days. Guinea pigs were bled by heart puncture 7 days after the last immunization and the presence of antibodies to NS26 was verified by reaction of the serum with nitrocellulose filters containing IPTG-induced and noninduced bacterial lysates harboring the pUR288: 1 1 plasmid, and rotavirus-infected MA1 04 cell lysates (corresponding to approximately 1 O5 cells). Antigen-antibody reaction wasdevel- oped by reaction with 1251-labeled protein A followed by autoradiography.

Western blot analysis

Lysates of infected and mock-infected MA1 04 cells (corresponding to lo5 cells) were resolved on a 10% SDS-polyacrylamide gel and electroblotted onto a nitrocellulose filter after published procedures (Burnette, 1981). Filters were reacted with NS26 antiserum followed by incubation with ‘251-labeled protein A and subjected to autoradiography (Burnette, 1981).

Radiolabeling of rotavirus-infected cells

Confluent monolayers of MA104 cells in 35 mm di- ameter petri dishes were infected at high multiplicity (20-30 PFU of trypsin-activated OSU rotavirus per cell) as described elsewhere (Ericson et al., 1982). At 3-4 h postinfection the medium was changed to methionine- deficient DMEM containing 50 KCi of [35S]methionine (800 to 1200 Ci/mmol; New England Nuclear Corp., Boston, MA) and 5 pg actinomycin D (AD; Sigma Chem- ical Co., St. Louis, MO) per milliliter. For pulse-chase experiments, the cells were labeled for 10 min in methionine-deficient DMEM containing 100 &i of [35S]methionine and 5 pg AD/ml. The labeling medium was removed and replaced with DMEM supplemented with 5 pg AD and 0.3 mg cold methionine per milliliter and incubated for various lengths of time. Alternatively, for labeling the carbohydrate moiety of viral glycoproteins, cells were incubated in glucose-deficient DMEM medium containing 0.1 mg glucose/ml for 1 hr beforevirus infection. After viral adsorption, cells were washed twice with Dulbecco’s phosphate-buffered saline (PBS) and incubated in glucose-free DMEM containing 5 pg AD/ml. At 4.5 hr postinfection cells were pulsed for 2 hr in glucose-free DMEM containing 100 &i [1,6- 3H]glucosamine (52 Ci/mmol; New England Nuclear)

10 GONZiiLEZ AND BURRONE

and 5 Kg AD/ml. Labeling was stopped by washing the dishes with cold PBS and the cells were harvested and lysed at 4’ in 250 ~1 of TNN lysis buffer (100 mMTris- HCI [pH 8.01, 250 mM NaCI, 0.59/o Nonidet-P40, 1 mM phenylmethylsulfonyl fluoride [PMSF]). The lysates were spun at 12,000 g for 2 min and the postnuclear supernatants were stored at -20” for immunoprecipitation analysis.

lmmunoprecipitations

[35S]Methionine- and [3H]glucosamine-labeled viral lysates (100 ~1) were subjected to immunoprecipitation by incubation with 1 ~1 NS26 antiserum at 4” during 2 hr. As control, lysates were immunoprecipitated with 1 ~1 preimmune guinea pig serum. A 30-~1 sample of protein A-Sepharose CL-4B beads (Pharmacia, Uppsala, Sweden; 10% (w/v) in TNN buffer) was added and further incubated for 30-60 min at 4”. Pelleted beads were washed batchwise and consecutively four times with buffer containing 100 mn/l Tris-HCI (pH 8.0) 500 mM LiCI, 1% Nonidet-P40, 1 rnM PMSF, and 0.1% bovine serum albumin, prior to release of antibody-antigen complexes by boiling in 1 X Laemmli’s sample buffer and analysis on SDS-l 2% polyacrylamide gels. Visualization of radiolabeled proteins was enhanced by fluorography (Bonner and Laskey, 1974) followed by exposure at -70” with preflashed X-ray film (Kodak XAR-5 film) for 16-24 hr (for 35S-labeled proteins) and for 1 O-l 5 days (for 3H-labeled proteins).

Endoglycosidases treatment of viral proteins

For endoglycosidases treatment, infected cell lysates were immunoprecipitated essentially as described above, except that immunocomplexes were eluted from protein A-Sepharose beads by boiling in 100-l 50 ~1 of buffer 10 mM Tris-HCI (pH 7.5) 0.059/o SDS, 1 mhll PMSF. Endo-P-N-acetylglucosaminidase H (endo H; Sigma) digestions were performed in 50 mn/l triethanolamine acetate buffer (pH 5.5) and 0.2 U/ml enzyme for 6 hr at 37” (Trimble and Maley, 1984). Endo-fi-A/-acetylglucosaminidase F (endo F; Boehrin- ger Mannheim, Biochemicals) digestions were carried out in 100 mM phosphate buffer (pH 6.3) with 5 U/ml enzyme, under conditions favoring the enzyme’s peptide:/+glycosidase F activity (PNGase F) (Freeze and Varki, 1986). Controls of 35S-labeled cell lysates (5-l 0 ~1) incubated under the same conditions without endo H or endo F/PNGase F were included. The reactions were stopped by addition of an equal volume of 2X electrophoresis sample buffer and boiling for 3 min.

P-Elimination assay

NS26 immunocomplexes were eluted from protein A-Sepharose beads in 100-l 50 ~1 of a solution containing 7 M urea, 1 mM PMSF. NaOH to a final concen- tration of 0.1 M was added to one half and incubated at 23” during 18 hr. An aliquot of 35S-labeled infected cell lysate (5-l 0 ~1) was incubated under the same conditions as control of mild alkali treatment.

Strong acid hydrolysis of [3H]glucosamine-labeled NS26

[3H]Glucosamine-labeled NS26 was immunoprecipitated from OSU-infected cell lysates as described above and eluted from protein A-Sepharose beads by boiling 2 min in 10 mll/lTris-HCI (pH 7.5) 0.05% SDS, and 1 mh/l PMSF. The purity of the glycoprotein was checked by SDS-polycrylamide gel electrophoresis of an aliquot of the eluted material and comparing its mobility with that of molecular weight calibration standards. The procedures described below are adapted from Cummings et a/. (1983). The sample was hydro- lyzed in 300 ~1 of 4 N HCI for 4 hr at loo”, and then the acid was removed by evaporation under reduced pressure. The sugars were reacetylated by treatment with acetic anhydride as described by Baenziger and Korn- feld (1974) and desalted over an Amberlite mixed bed resin. The eluate containing the reacetylated monosac- charides was dried by evaporation under reduced pressure.

Paper chromatography

Descending paper chromatography of sugars was performed for 30 hr on Whatman No. 1 filter paper. Solvent systems used were solvent A, ethyl acetate:pyridine:water (8:2: 1) for separation of reduced N-acetylated sugars, and solvent B, l-bu- tanol:pyridine:water (6:4:3). N-Acetylglucosamine and N-acetylgalactosamine were separated by chromatography in solvent system B on borate-impregnated paper (Cardini and Leloir, 1957). Standard N-[3H]acetylglucosamine and N-acetylgalactosamine were pre- pared by acetylation of the amino sugars (Baenziger and Kornfeld, 1974). The sugar alcohols of N-acetylglucosamine and N-acetylgalactosamine were pre- pared by NaB3H, reduction (Li et al., 1978). The other standards were silver stained (Li et al., 1978). In the case of radiolabeled sugars, lanes were marked into 0.5-cm sections and excised after chromatography for radioactivity quantitation by liquid scintillation counting.

ROTAVIRUS 0-GLYCOSYLATION 11

3H labeling of saccharides

After NS26 immunoprecipitation from OSU-infected cell lysates, the viral protein was eluted from protein A-Sepharose beads and precipitated in 8 vol acetone to remove salts and detergent and resuspended in 25 ~1 of 0.2 M sodium carbonate, pH 1 1.6. P-Elimination under reducing conditions was carried out by addition of 1 vol of 0.2 N NaOH containing 50 mCi of NaB3H, (7.6 mCi/mmol; NEN) and incubation at 23” during 18 hr. The sample was brought to 4” and neutralized with 1 M acetic acid. The 3H-labeled saccharides were passed over a Dowex 5OW-X8H+ column and eluted with water. Borate was removed by repeated evaporation with 0.1 M glacial acetic acid in methanol at 40”. The 3H-labeled saccharides were resuspended in 400 ~1 distilled water. One-half of the sample was subjected to strong acid hydrolysis and analyzed together with the nontreated sample by paper chromatography in buffer system B.

RESULTS

Characterization of an antiserum to gene 11 protein product

Porcine OSU rotavirus gene 1 1 product was expressed in E, co/i as a fusion protein with P-galactosi- dase and further used to raise monospecific polyclonal antibodies to NS26 in guinea pigs. As a first step in the characterization of gene 1 1 product, immunoprecipita- tions of [35S]methionine-labeled protein extracts of OSU-infected MA104 cells at 4 hr postinfection were carried out using the antiLNS26 serum. Figure 1 A (lane 5) shows that two polypeptides with apparent molecular weights of 26 kDa and 28 kDa were detected. These products are specific to gene 1 1, since preimmune guinea pig serum did not immunoprecipitate any of these species (Fig. 1 A, lane 4) and no product was detected when proteins from mock-infected MA1 04 cells were immunoprecipitated with anti-NS26 serum (Fig. 1 A, lane 3). The same molecular weight polypeptides were detected when OSU-infected MA1 04 lysates were analyzed by Western blot (not shown), con- firming the specificity of the detected bands in infected cells. To determine whether the two detected bands corresponded to different products of genome segment 11 or processed polypeptides of NS26, pulse- chase experiments were performed. OSU-infected cells were labeled with [35S]methionine during 10 min and then incubated in the absence of radioactive precursor during 50 min. Each cellular lysate was immunoprecipitated with NS26 antiserum and analyzed by polyacrylamide gel electrophoresis and autoradiogra-

phy. The pulse-chase experiments clearly demonstrated that the 26-kDa polypeptide is processed to the larger molecular weight (28 kDa) polypeptide (Fig. 1 B). Similar results have been obtained by Welch et al. (1989) using an antiserum against the gene 1 1 protein of simian rotavirus SAl 1 synthesized in baculovirus expression system, albeit some lower molecular weight bands (20, 22 kDa, and smaller) were also observed. By contrast, we consistently detected in pulse-chase experiments only a band of 28 kDa, which strongly suggests that the main product of the longest open reading frame of gene 11 is a 28-kDa polypeptide.

NS26 post-translational modification

The results described above indicated that NS26 exhibits a post-translational modification. Based on the presence of a putative N-glycosylation site in gene 11 sequence (Gonzalez et al., 1989) we first investigated the possibility of N-glycosylation on NS26 by means of endoglycosidases digestion analysis. The enzymes used were endo H and endo F which cleave high-mannose and complex N-linked oligosaccharides from proteins, respectively. The 26- and 28-kDa polypeptides were immunoprecipitated from a [35S]methionine- labeled total protein extract and subjected to endoglycosidases digestion. Both polypeptides were resistant to endoglycosidases treatment (Fig. 2, lanes 2 and 3), demonstrating the absence of either high-mannose or complex N-linked oligosaccharides on NS26. The enzymes activity on the viral lysate from which NS26 products were immunoprecipitated was verified by the sensitivity of the two well-characterized viral N-glycoproteins, VP7 and NS28, to both endo H and endo F/PNGase F. This is shown in the digestion of 35S-labeled total viral protein extract (Fig. 2, lanes 4 and 6 versus lanes 5 and 7). The fact that the amino acid sequence of gene 11 product is rich in serines and threonines prompted us to next analyze whether it contained O-linked saccharides. The immunoprecipitated 35S-labeled NS26 polypeptides were subjected to P-elimination under mild alkaline conditions which re- leases O-linked carbohydrates. Interestingly, the 26- and 28-kDa bands were both sensitive to alkaline treatment, yielding a single species migrating slightly faster than the 26-kDa polypeptide (Fig. 3, lane 2 versus lane 1, and lane 4 versus lane 3). That the total viral protein profile was not altered by incubation under the same conditions as NS26 (Fig. 3, lane 5 compared to lane 6) rules out the possibility of nonspecific degradation of the 26- and 28-kDa polypeptides. This result indicated that NS26 is processed into the 28-kDa polypeptide by 0-glycosylation. To further define the nature of its modification, OSU-infected cells were metabolically labeled

12 GONZALEZ AND BURRONE

A B P c p c

anti-NS26 - - + +

FIG. 1. Detection of gene 11 product in infected cells by immunoprecipitation. A. Mock-infected and OSU-infected MA104 cells were pulse-labeled with 50 &i [35S]methionine/ml for 60 min at 5 hr postinfection and then immunoprecipitated with either preimmune or anti-NS26 serum. Lanes: 1, mock-infected cell lysate before the addition of antiserum; 2, OSU-infected cell lysate before the addition of antiserum; 3, mock-infected cell lysate immunoprecipitated with anti-NS26 serum; 4 and 5, OSU-infected cell lysate immunoprecipitated with preimmune guinea pig serum (lane 4) or anti-NS26 serum (lane 5). Samples were analyzed by electrophoresis on a SDS-12% polyacrylamide gel. B. Processing of NS26. OSU-infected MA1 04 cells were pulsed with [35S]methionine for 10 min (P), then chased for 50 min in the absence of radioactive precursor (C), and NS26 polypeptides were analyzed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis as described above. Minor bands of approximately 98. and 41.kDa (A, lane 5; B, lane C+) correspond to nonspecific binding of the most abundant viral proteins to the protein A-Sepharose beads, Numbers indicate molecular weights in thousands. Exposure was for 18 hr.

with [3H]glucosamine (GlcN) at 4 hr postinfection for 2 hr. As expected, both 26- and 28kDa protein products were labeled with [3H]GlcN (Fig. 4, lane 1) and the 3H label was released upon P-elimination (Fig. 4, lane 2) demonstrating that NS26 is indeed a glycoprotein whose carbohydrate moiety is attached via O-linkages to serine or threonine (or both). It is worth mentioning that GlcN was incorporated into the 26-kDa species in a mild base labile O-linkage, which indicates that this polypeptide is not the primary translation product of NS26. Therefore, the 26- and 28-kDa products detected in infected cells are both post-translationally modified forms of the same polypeptide. Taken together, the p-elimination analysis and the 3H-labeling data show that NS26 is an 0-glycoprotein of 28 kDa and that 0-glycosylation is the modification responsible for its processing.

Analysis of NS26-bound carbohydrate

There are two distinct pathways for O-linked oligosaccharide biosynthesis. One of them occurs within

the Golgi apparatus and is initiated by the addition of N-acetylgalactosamine (GalNAc) residues to the hy- droxyl groups of threonine and serine. Biosynthesis proceeds then through the sequential addition of carbohydrate residues by glycosyltransferases located in the Golgi apparatus, yielding structurally diverse oligosaccharide chains. By contrast, the other pathway in- volves the 0-glycosidic linkage to protein of single monosaccharide residues of N-acetylglucosamine (O- GlcNAc). This modification has been found to be present on cytosolic and nuclear glycoproteins (Hart et al., 1988, 1989).

In order to identify the O-linked carbohydrate structure attached to NS26, OSU-infected MA104 cells were grown in the presence of [3H]GlcN for 2 hr and NS26 was isolated by immunoprecipitation as described above. Preliminary experiments using [35S]- methionine labeling had established the optimum time of NS26 synthesis under our conditions of infection to be about 4-6 hr postinfection. This time period was chosen for the carbohydrate-labeling experiments


12 3456 7 C En&H -+-+-- -

En&F --t--+ -

- 98

#@I& -41

dim -37

FIG. 2. Endoglycosidases treatment of NS26. 35S-labeled proteins from OSU-infected cells were immunoprecipitated with NS26 antiserum and digested wrth endo H or endo F as indicated (lanes l-3), and analyzed by electrophoresis on a 12% SDS-polyacrylamide gel. Total infected cell lysate incubated under the same conditions either in the absence (lanes 5 and 7) or the presence (lanes 4 and 6) of endoglycosidases was used as control of enzyme activity. C: control of 35S-labeled viral proteins which was not subjected to incubation. Numbers indicate molecular weights in thousands.

since a large amount of NS.26 would be available for labeling and its detection would be optimized. Since GlcN is a precursor which is incorporated into glycoproteins as A/-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), and sialic acid, a pulse of [3H]GlcN could result in the addition of either [3H]- GalNAc, the first sugar to be added to O-linked chains processed in the Golgi apparatus, or O-linked [3H]GlcNAc. The purified [3H]GlcN-labeled NS26 was treated with 4 N HCI for 4 hr at 100” to hydrolyze its oligosaccharide moiety. This treatment released a single radiolabeled sugar that comigrated with authentic GlcNAc on paper chromatography (Fig. 5) indicating that GlcNAc was most likely the only sugar attached to NS26. To confirm this, NS26 was P-eliminated under reducing conditions using NaB3H, to label the sugar involved in the O-linkage with the peptide backbone. This material was analyzed by paper chromatography (Fig. 6A) together with a fraction which had been further subjected to strong acid hydrolysis (Fig. 6B). The fact that no oligosaccharide chain was detected by direct analysis of the 3H-labeled P-elimination product (Fig. 6A) and that N-acetylglucosaminitol, which origi- nates in the P-elimination reaction by the reduction of GlcNAc, was the only labeled product obtained in both cases (Figs. 6A and 6B) clearly demonstrates that GlcNAc is O-linked to NS26 as monosaccharide structures.

Furthermore, we investigated the possibility that gene 11 product might bear O-linked mannose-containing saccharide structures in addition to 0-GlcNAc since O-linked mannosyl residues have been shown to exist on certain cytoplasmic proteins (Finne et al., 1979; Margolis et a/., 1979; Krusius et a/., 1986). OSU- infected cells were labeled with [2-3H]mannose and the protein lysate was immunoprecipitated with NS26 antiserum. No signal was obtained even after pro- longed exposure of the gel (data not shown), which confirms that 0-GlcNAc is the only modification responsible for NS26 processing.

DISCUSSION

In the present paper we addressed the question of the post-translational modification of rotavirus gene 1 1 product. Specific antibodies raised against the polypeptide encoded by the major open reading frame of gene 1 1 recognized in protein extracts of infected cells two polypeptides with apparent molecular weight of 26,000 and 28,000. The data presented here demonstrate that the primary product of gene 11 is processed to the 28-kDa polypeptide by addition of single O-

A 12 34 B 5 6

026

d26

Anllib - 020

FIG. 3. Alkali-induced cleavage of NS26. OSU-infected MA104 cells were pulsed with [%S]methionine or pulse-labeled and then chased as described in Fig. 1 and NS26 polypeptides were recov- ered by immunoprecipitation of cell lysates. Lanes: 1 and 2, immunoprecipitated polypeptides from pulse-labeled cell lysate which were incubated at 23” for 18 hr in the absence (lane 1) or presence (lane 2) of 0.1 N NaOH; 3 and 4, immunoprecipitated polypeptldes from pulse-chase labeled cell lysate which were incubated under the same conditions as above in the absence (lane 3) or the presence (lane 4) of alkali: 5 and 6. %S-labeled viral protein lysate also rncu- bated under the same conditions either in the absence (lane 5) or the presence of alkali (lane 6) was used as control. The arrow heads indicate the position of the P-elimination product. Numbers indicate molecular weights in thousands.

14 GONZALEZ AND BURRONE

FIG. 4. Metabolic labeling of NS26 with [3H]glucosamine. At 4 hr postinfection OSU-infected MA104 cells were labeled for 2 hr in glucose-free MEM containing 100 yCi [3H]glucosamine/ml. Protein extracts were immunoprecipitated with NS26 antiserum and analyzed directly (lane 1) or after@-elimination treatment under mild alkaline conditions (lane 2) by electrophoresis on a SDS-l 0% polyact-y- amide gel. Numbers to the left of the figure indicate the positions of molecular weight standards given in thousands. Exposure was for 15 days.

linked residues of GlcNAc. The presence of O-linked saccharides on NS26 was first suggested by its resistance to endo H and endo F/PNGase F treatment (which hydrolyze N-linked oligosaccharides) and its sensitivity to p-elimination. Further evidence that O- GlcNAc is added to NS26 was obtained from thefollow- ing results: (i) the 26- and 28-kDa polypeptides were metabolically labeled with [3H]GlcN; (ii) NS26-asso- ciated 3H-label disappeared upon P-elimination, indicating that the 3H-labeled carbohydrate is attached via O-linkages to the protein; (iii) [3H]GlcNAc was the single sugar released upon total acid hydrolysis of the NS26-bound carbohydrate; (iv) N-acetylglucosaminitol was the only product released upon P-elimination under reducing conditions of gene 11 product.

Furthermore, the gene 11 protein shows a potential N-glycosylation site which is conserved in the pre-

dieted product of all the known sequences of gene 11. However, this target site for N-glycosylation seems to be inaccessible to the endoplasmic reticulum N-glycosylation machinery, which is consistent with the cytoplasmic localization of NS26 (Welch eta/., 1989). Thus, NS26 provides another example of a protein in which the sole presence of a consensus N-glycosylation site is not sufficient for N-glycosylation.

It is important to point out that the 26-kDa polypeptide detected in infected cells is not the primary product of gene 11 since it was 3H-labeled and sensitive to ,&elimination, which suggests that 0-GlcNAc residues are likely to be added to NS26 in the cytoplasm within the first minutes of its synthesis, as has been observed for the addition of 0-GlcNAc to the p62 nuclear pore protein (Davis and Blobel, 1987). In addition, the saccharide moiety released upon P-elimination of the 26- and 28-kDa polypeptides gave a single peak by chromatographic analyses, indicating that both forms repre- sent post-translationally modified versions of the same polypeptide which only differ in their degree of O-glycosylation.

NS26 is the second example of an O-GlcNAc-bearing glycoprotein to be described in viruses. Interest- ingly, the first viral glycoprotein found to be modified by 0-GlcNAc was the 149-kDa virion basic phosphoprotein of human cytomegalovirus (a nuclear virus) which is located between the capsid and the viral envelope (Benko et al., 1988) whereas NS26 is found in the cytoplasmic compartment of rotavirus-infected cells (a

3

ii I 100

; 50

n ” i0 20 3b 40

cm

FIG. 5. Descending paper chromatogram of the strong acid hydrolysates of [3H]glucosamine-labeled NS26 oligosaccharides. OSU-infected MA1 04 cells were metabolically labeled with [3H]glucosamine as described in Fig. 4. NS26 polypeptides were immunoprecipitated with NS26 antiserum and subjected to strong acid hydrolysis (4 N HCI, 4 hr at 100’). The samples were then dried and analyzed by descending paper chromatography on borate-impregnated paper (solvent B). Arrows indicate the migration of standards: 1, galactose; 2, sialic acid; 3, N-acetylgalactosamine; 4, N-acetylglucosamine.


1 2

c I

A

600

: 600 0 x 400 L ” 200

io i0 io io cm

FIG. 6. Migration on paper chromatography of reduced O-linked NS26 carbohydrate before and after strong acid hydrolysis. NS26 polypeptides were immunoprecipitated from OSU-infected cells and the O-linked carbohydrate moiety was released and chemically labeled upon p-elimination in the presence of NaB3H,. The products were analyzed by paper chromatography (solvent A) before (A) and after (B) strong acid hydrolysis. The migration of standards was as Indicated: 1, maltotriose (used as trisaccharide standard); 2, maltose (used as disaccharide standard): 3, N-acetylglucosaminitol; 4, N- acetylgalactosaminitol; 5, N-acetylgalactosamine; 6, N-acetylglucosamlne.

cytoplasmic virus). As to the significance of 0-GlcNAc modification, it has been proposed that it could play a regulatory role, confer resistance to proteolysis, or be involved in the formation or stabilization of multiprotein complexes (Hart et al., 1988). Localization of NS26 to viroplasms (Welch et al., 1989; our unpublished obser- vations), where single-shelled particles are thought to be synthesized, together with the fact that 0-GlcNAc has been shown to be present on RNA polymerase II transcription factors (Jackson and Tjian, 1988), make it tempting to speculate that NS26 could be a compo- nent of the virus transcriptional machinery. It has recently been shown that NS26 is phosphorylated (Welch et a/., 1989). 0-GlcNAc could therefore reversi- bly “block” sites of phosphorylation on NS26, thereby regulating the protein function. Whatever the explana- tion is for the presence of both phosphate and O-Glc- NAc on NS26, the demonstration of both modifications on NS26 provides a direct clue toward understanding its function during virus replication.

ACKNOWLEDGMENTS

We thank Armando J. A. Parodi and Roberto Couso for their helpful suggestions and discussions on O-linked glycoproteins. S.A.G.

thanks Jo& L. Affranchino for his encouragement and valuable com- ments on the manuscript. This work was supported in part by grants from the Secretaria de Ciencia y TBcnica (SECYT), Programa Na- cional de Biotecnologfa, and SAREC (Swedish Agency for Research Cooperation with Developing Countries).

REFERENCES

AQUINO. D. A., MARGOLIS. R. U., and MARGOLIS. R. K. (1984). Immuno- cytochemical localization of a chondroitin sulfate proteoglycan in nervous tissue. I. Adult brain, retina, and peripheral nerve. J. Ce// Biol. 99, 1117-l 129.

BAENZIGER, J., and KORNFECD, S. (1974). Structure of the carbohydrate units of IgA, Immunoglobulin. II. Structure of the O-glycosidi- tally linked oligosaccharide units. /. Biol. Chem. 249, 7270-7281.

BENKO, M. D., HALTIWANGER, R. S., HART, G. W., and GIBSON, W. (1988). Virion basic phosphoprotein from human cytomegalovlrus contains O-linked N-acetylglucosamlne. Proc. Nat/. Acad. Sci. USA 65, 2573-2577.

BONNER, W. M., and LASKEY, R. A. (1974). A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83-88.

BRADFORD, M. M. (1976). A rapid and sensitive method forthe quantitation of mlcrogram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.

BURNE~E, W. N. (1981). “Western blotting”: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nltrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112, 195-203.

CARDINI, C. E., and LELOIR, L. F. (1957). Enzymatic formation of ace- tylgalactosamlne. /. B/o/. Chem. 225, 3 17-324.

CLARK, B., and DESSELBERGER, U. (1988). Myristylation of rotavirus proteins. /. Gen. Viral. 69, 2681-2686.

CUMMINGS, R. D., KORNFELD, S., SCHNEIDER, W. J., HOBGOOD, K. K., TOLLESHAUG, H., BROWN, M. S., and GOLDSTEIN, J. L. (1983). Biosyn- thesis of N- and 0-llnked oligosaccharides of the low density lipo- protern receptor. /. Biol. Chem. 258, 15,261-l 5,273.

DAVIS, L. I., and BLOBEL, G. (1987). Nuclear pore complex contains a family of glycoproteins that includes ~62: Glycosylatfon through a previously unidentified cellular pathway. Proc. Nat/. Acad. Sci. USA. 64, 7552-7556.

ERICSON, B. L., GRAHAM, D. Y., MASON, B. B., and ESTES, M. K. (1982). Identification, synthesis, and modifications of simian rotavirus SAl 1 polypeptides in Infected cells. /. Viral. 42, 825-839.

ESTES, M. K., GRAHAM, D. Y., GERBA, C. P., and SMITH, E. M. (1979). Simian rotavirus SAl 1 replication in cell cultures. /. Viral. 31, 81 O- 815.

ESTES, M. K., PALMER, E. L., and OBIJESKI. J. F. (1983). Rotaviruses: A review. Cur. Top. Microbial. Immunol. 105, 123-l 84.

FINNE, J., KRUSIUS, T., MARGOLIS, R. K., and MARGOLIS, R. U. (1979). Novel mannitol-containing oligosaccharides obtained by mild alkaline borohydride treatment of a chondroitin sulfate proteoglycan from brain. /. Biol. Chem. 254, 10,295-10,300.

FREEZE, H. H., and VARKI, A. (1986). Endo-glycosidase F and peptide N-glycosidase F release the great majority of total cellular N-llnked oligosaccharides: Use In demonstrating that sulfated N-linked oligosaccharides are frequently found in cultured cells. Biochem. Biophys. Res. Commun. 140, 967-973.

GON~LEZ, S. A., and BURRONE, 0. R. (1989). Porcine OSU rotavirus segment 1 1 shows common features with the viral gene of human origin. Nucleic Acids Res. 17, 6402.

GONZALEZ. S., MAITION, N. M., BELLINZONI. R., and BURRONE, 0. R. (1989). Structure of rearranged genome segment 1 1 in two differ-

16 GONtiLEZ AND BURRONE

ent rotavirus strains generated by a similar mechanism. 1. Gen. Viral. 70, 1329-l 336.

HART, G. W., HALTIWANGER, R. S., HOLT, G. D., and KELLY, W. G. (1989). Glycosylation in the nucleus and cytoplasm. Annu. Rev. Biochem. 58, 841-874.

HART, G. W., HOLT, G. D., and HALTIWANGER, R. S. (1988). Nuclear and cytoplasmic glycosylation: Novel saccharide linkages in unex- pected places. Trends Biochem. Sci. 13, 380-384.

HATORI, M., and SAKAKI, Y. (1986). Dideoxy sequencing method using denatured plasmid templates. Anal. Biochem. 152, 232-238.

JACKSON, S. P., and TJIAN, R. (1988). 0-glycosylation of eukaryotic transcription factors: Implications for mechanisms of transcriptional regulation. Cell 55, 125-l 33.

KABCENELL, A. K., and ATKINSON, P. H. (1985). Processing of the rough endoplasmic reticulum membrane glycoproteins of rotavirus SAl 1. J. Cell Biol. 101, 1270-l 280.

KAPIKIAN, A. Z., and CHANOCK, R. M. (1985). Rotaviruses. ln “Virol- ogy” (B. N. Fields, Ed.), pp. 863-906. Raven Press, New York.

KRUSIIJS, T., FINNE, J.. MARGOLIS, R. K., and MARGOLIS. R. U. (1986). Identification of an 0-glycosidic mannose-linked sialylated tetra-

saccharide and keratan sulfate oligosaccharide in the chondroitin sulfate proteoglycan of brain. J. Biol. Chem. 261, 8237-8242.

LAEMMLI, U. K. (1970). Cleavage of structural proteins during assem- bly of the head of bacteriophage T,. Nature (London) 227, 680- 685.

LI, E., TABAS, I., and KORNFELD, S. (1978). The synthesis of complex- type oligosaccharides. I. Structure of the lipid-linked oligosaccharide precursor of the complex-type oligosaccharides of the vesicu- lar stomatitis virus G protein. J. Biol. Chem. 253, 7762-7770.

MARGOLIS, R. K., THOMAS, M. D., CROCKED, C. P., and MARGOLIS, R. U. (1979). Presence of chondroitin sulfate in the neuronal cytoplasm. Proc. Nat/. Acad. Sci. USA 76, 17 1 l-l 7 15.

ROTHER, U., and MOLLER-HILL, B. (1983). Easy identification of cDNA clones. EMBOJ. 2, 1791-1794.

TRIMBLE, R. B., and MALEY, F. (1984). Optimizing hydrolysis of N- linked high-mannose oligosaccharides by endo-fl-N-acetylglucosaminidase H. Anal. Biochem. 141, 515-522.

WELCH, S.-K. W., CRAWFORD, S. E., and ESTES, M. K. (1989). The rotavirus SAll genome segment 11 protein is a nonstructural phosphoprotein. J. Viral. 63, 3974-3982.

Rotavirus NS26 is modified by addition of single O-linked residues of N-acetylglucosamine

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