Analysis of lettuce necrotic yellows virus structural proteins with monoclonal antibodies and concanavalin A

VIROLOGY

166,486-494

(1988)

Analysis of Lettuce Necrotic Yellows Virus Structural with Monoclonal Antibodies and Concanavalin

Proteins A

RALF G. DIETZGEN’ AND R. I. B. FRANCKI Department

of Plant Pathology,

Waite Agricultural

Research Institute, The University of Adelaide,

Glen Osmond, 5064, South Australia

Received March 7, 1988; accepted May 17, 1988 Three major structural proteins of lettuce necrotic yellows virus (LNW) were identified by discontinuous polyacrylamide gel electrophoresis (PAGE) to have M, - 78,000 (G), 57,000 (N), and 19,000 (M). Unreduced G and M proteins had faster mobilities in PAGE indicating the presence of disulfide bonds. The G protein was shown to be glycosylated with a complex network of oligosaccharides containing /3-N-acetylchitobiose N-linked to asparagine residues of the protein. Up to 17 additional minor bands were also detected in silver-stained electrophoretograms. In Western immunoblots, 9 of these (M, - 27,000-220,000) were recognized by a monoclonal antibody to the N protein and another 6 (Mr - 58,000-180,000) with a monoclonal antibody to the G protein, indicating that they were degradation products or aggregates of these two viral proteins. Two minor silver-stained bands failed to react with either of the monoclonal antibodies, but were recognized by polyclonal anti-LNW serum and are probably the L (M, - 190,000) and NS (M, - 38.000) viral proteins. 0 1gsSAcademic Press, Inc.

INTRODUCTION Lettuce necrotic yellows virus (LNYV) is a member of the family Rhabdoviridae (Matthews, 1982) and infects a number of plant species and its aphid vectors (Francki et a/., 1988). The virus appears to contain five structural proteins L, G, N, NS, and M (Dale and Peters, 1981) and a model of the virus particle has been proposed by Francki and Randles (1980). However, when proteins from highly purified virus preparations are subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and stained with silver nitrate, numerous minor polypeptide components are detected in addition to the three major proteins G, N, and M (Dietzgen and Francki, 1987b). Monoclonal antibodies (MCAs) (Kiihler and Milstein, 1975) have proven to be valuable tools for the structural analysis of plant viruses (Halk and DeBoer, 1985) and the study of complex mixtures of antigens contained in the particles of animal rhabdoviruses (Ogden et al., 1986). In this paper, we describe experiments in which we have used monoclonal antibodies to the G and N proteins of LNYV and concanavalin A (Con A) to determine the nature and origin of the LNYV proteins detected by PAGE.

LNYV isolated from garlic with similar biological properties to the S.E.3 isolate (Stubbs and Grogan, 1963). The virus was purified by celite-filtration, differential centrifugation, and column chromatography on calcium phosphate gel as described by Francki et al. (1988).

Polyacrylamide

Proteins were suspended in 62.5 ml\/l Tris-HCI, pH 6.8, 39/o SDS, 5% 2-mercaptoethanol, 10% glycerol, and 0.01% bromphenol blue (electrophoresis sample buffer) and separated by electrophoresis on slab gels using the SDS discontinuous buffer system described by Laemmli (1970). The separated proteins were visualized by staining with silver nitrate (Wendrychowski et a/., 1986). Production and purification antibodies (MCAs)

’ To whom requests for reprints should be addressed.

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of monoclonal

BALB/c mice were immunized with sucrose densitygradient purified virus preparations. Three intraperitoneal injections of LNYV treated with 0.5% Triton X-l 00 in Freund’s adjuvant were given. For the last booster, undissociated LNYV was injected intravenously 3 to 4 days before the cell fusion. Antibody titers were monitored by an indirect ELISA. The spleen cells of mice having titers of greater than 1: 100,000 were fused with P3-X63-Ag8.653 myeloma cells (Kearney et a/., 1979) using the cell fusion procedure described by Lane et a/. (1 986). One milliliter of a sterile-filtered 50% polyeth-

MATERIALS AND METHODS Virus propagation and purification Nicotiana glutinosa plants were grown under glasshouse conditions and inoculated mechanically with

0042-6822/88

gel electrophoresis

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ylene glycol (Merck 4000) solution was added over a 45-set period at 37” to a mixture of 5 X 1O7 myeloma and 1 x 1O8spleen cells and then diluted dropwise with RPMI 1640 medium supplemented with 15010 fetal calf serum. The fusion mixture was plated into 24-well tissue culture plates containing peritoneal exudate feeder cells. Culture supernatants from growing hybridomas were screened for LNYV-specific antibodies by indirect ELISA and Western immunoblot assays of purified LNYV preparations and extracts from uninfected N. glutinosa leaves. Hybridoma clones obtained by two cycles of limiting dilution cloning were grown in vitro and in viva. Antibodies were precipitated from the culture supernatant with 50% ammonium sulfate (Jonak, 1980). These enriched antibody preparations and the clarified ascitic fluid, which had been absorbed with silicon dioxide (Sigma) to remove lipids (Neoh et a/., 1986), were further purified by affinity chromatography on protein A-agarose (MAPS II kit, Bio-Rad, Richmond, CA) according to the manufacturer’s protocol. Eluted antibody preparations were concentrated by placing dialysis tubing in solid polyethylene glycol and subsequent dialysis against 20 mM Tris-HCI, pH 7.8, containing 20 mM NaCl at 4”. The antibody concentration was determined spectrophotometrically with A280 “,,, 1.4 = 1 mg/ml. In the case of less than 1 mg/ml, bovine serum albumin was added to 1 mg/ml. The affinity-purified MCAs were stored in aliquots at -20”. Characterization

of monoclonal

antibodies

The isotype of MCAs was determined by an indirect ELISA using affinity-purified goat anti-mouse subclassspecific antibodies (Haemmerling and Haemmerling, 1981). lmmunodiffusion tests with detergent-disrupted LNYV were conducted in 0.75% agar as described by Randles and Carver (197 1). Polyclonal

antisera

Rabbit antiserum to LNYV prepared by McLean et a/. (1971) was used. Mouse antisera to LNYV elicited by detergent-dissociated virus were collected on the day the animals were sacrificed for preparation of monoclonal antibodies. ELBA An indirect ELISA was used to detect polyclonal rabbit and mouse antibodies and for the initial screening of some hybridoma supernatants. The buffers described by Clark and Adams (1977) were used throughout and all incubations were at 25”. Polystyrene microtiter plates (Nunc, Denmark) were coated with purified

VIRUS PROTEINS

virus preparations

487

or clarified extracts of uninfected

N.

glutinosa leaves. After blocking residual binding sites on the plastic by incubation with 1o/obovine serum albumin (BSA) in 0.9% NaCI, a dilution series of the antibody samples was added. Then a 1: 1000 dilution of affinity-purified anti-rabbit or anti-mouse IgG that had been labeled with alkaline phosphatase (Sigma, Miles) was incubated in the plate. Both the sample and conjugate buffers contained heparin at a final concentration of 5 units/ml (Dietzgen and Francki, 1987a). After incubation with 1 mg/ml p-nitrophenylphosphate, the A 4o5nmwas measured with an ELISA reader (Bio-Rad). Preimmune serum of rabbits and mice and myeloma cell supernatant served as negative controls. Western immunoblotting Proteins were transferred electrophoretically from SDS-polyacr-ylamide gels to nitrocellulose membranes (0.2 pm pore size, Schleicher & Schuell, Germany) at 60-100 V for 4-5 hr in 25 mM Tris, 192 mM glycine, 209/o methanol (Towbin et al., 1979). Fluorescent labeled protein markers (Law and Lingwood, 1985) also electrophoresed in gels, were used to monitor the transfer and to localize nitrocellulose membrane sections to be cut for probing with different antibody preparations. Protein molecular weight markers were stained with india ink (Glenney, 1986) or colloidal gold (Bio-Rad) after the transfer. BSA, non-fat dry milk and gelatin at 3% (w/v) were compared for their efficiency of blocking free binding sites on the nitrocellulose membrane without interfering with the specific antibody binding. BSA was found to be optimal for both monoclonal and polyclonal antibodies that reacted with LNYV structural proteins; milk and gelatin led to an overall reduction in specific signals. After a brief wash in rinse buffer (10 mM Tris-HCI, pH 7.4, 150 mM NaCI, 1 mM EDTA) containing 0.1% Triton X-l 00 (RBT), residual binding sites on the nitrocellulose membrane were blocked by incubation for 2 hr at 25” in rinse buffer containing 3% BSA and 1O/O normal goat serum. Antibody preparations, diluted in RBT containing 1o/oBSA, 5 units/ml heparin, and 0.02% sodium azide, were agitated with the membrane for 2 hr at room temperature in sealed plastic bags. Heparin was included in the immunoblot sample buffer to avoid charge-related nonspecific reactions (Dietzgen and Francki, 1987a). Following washes with four changes of RBT, the proteins were incubated for 2 hr at room temperature with alkaline phosphatase-conjugated affinity-purified anti-mouse or anti-rabbit IgG (Sigma or Miles) diluted 1 :lOOO in sample buffer. After being

488

DIETZGEN AND FRANCKI

washed in four changes of RBT the bound antibody was detected with a substrate solution containing either Fast Red TR (O’Connor and Ashman, 1982) or nitroblue tetrazolium/phenazine methosulfate/5-bromo4-chloro-3-indolyl phosphate (Ey and Ashman, 1986). The former substrate was used in Figs. 2, 3, and 4. The latter substrate was used from there on, since its reaction product was easier to photograph and led to an increased sensitivity of the assay. The specificity of MCAs for protein or carbohydrate antigenic determinants was determined by treatment of blots with periodic acid to remove oligosaccharides as described by Woodward et al. (1985). For screening of hybridoma culture supernatants, nitrocellulose membranes containing proteins electroblotted from preparative polyacrylamide SDS gels were cut into 0.4-cm-wide strips and processed as described above. All washes and incubation steps were done in a slot incubator. Culture supernatants to be assayed were diluted 1: 1 with sample buffer. Affinity

purification

of monospecific

antibodies

Antibodies were eluted from nitrocellulose membranes as described by Rybicki (1986). LNYV proteins, separated by preparative PAGE, were electroblotted to nitrocellulose membrane, blocked with BSA, and incubated with 1:20 diluted hybridoma culture supernatant of MCA-N or polyclonal antiserum to LNYV (1:2000) for 2 hr at 25”. Following extensive washing, the membrane was air-dried and aligned with india ink-stained side tracks. Strips corresponding to the N protein and its assumed degradation products were cut and the respective bound antibodies were eluted with 1 .O ml of 0.1 M glycine-HCI, pH 2.8, containing 0.15 M NaCI. After 5 min the strips were removed and the solution was neutralized with 2 M Trizma base. The antibody preparations were diluted 1:5 in sample buffer and assayed by immunoblotting as described above. Controls included antibodies eluted from the G protein band and from nitrocellulose regions not containing viral proteins. Glycoprotein

analysis

Glycosylated proteins were detected by affinoblotting as described by Faye and Chrispeels (1985). Proteins were transferred electrophoretically from SDSpolyacrylamide gels to nitrocellulose membrane and fixed for 5 min with acetic acid/isopropanol/water (10: 25:65, v/v). Following the blocking of residual binding sites with gelatin, the membrane was first reacted with 20 pg/ml of concanavalin A (Con A) and then with 30 rglml of horseradish peroxidase. Bound peroxidase

was visualized by adding 4-chloro-1 -naphthol or a luminescent substrate (Laing, 1986). The affinity for concanavalin A was demonstrated by using ovalbumin as a control glycoprotein and by inclusion of 15 mM methylcr’o-mannopyranoside in all buffers. The identity of the oligosaccharides was determined by hydrolysis with glycosidases of different specificities. Blocked membranes were treated at 37” for 48 hr with jackbean a-mannosidase, ,&AI-acetylglucosaminidase (Sigma), with buffers and concentrations as specified by Faye and Chrispeels (1985), and endo-P-N-acetylglucosaminidase F (New England Nuclear) (Thotakura and Bahl, 1987) at 10 units of activity in 100 mM NaH,PO,, 50 mM EDTA, pH 6.1 (Elder and Alexander, 1982). Duplicate blots were incubated in the respective buffers without enzyme. RESULTS Protein analysis of highly purified LNYV preparations LNYV preparations purified by sucrose density gradient centrifugation were essentially free of host plant proteins because they reacted strongly in ELISA using a mouse anti-LNYV polyclonal serum which failed to react when tested against extracts from uninfected N. glutinosa plants (data not shown). PAGE of all fractions from such gradients revealed that most of the protein was present in three fractions (Figs. 1A and B) which, by electron microscopy, were shown to contain, almost exclusively, intact virus particles (data not shown). The three fractions contained a complex pattern of protein bands that was readily visualized by silver staining (Fig. 1 B). Three major protein bands were identified by their electrophoretic mobilities as the G, N, and M proteins (Dale and Peters, 1981) but numerous minor bands were also resolved. Electron microscopy of LNYV preparations treated with Nonidet-P40 at a concentration of 1% to remove the envelope (Randles and Francki, 1972) revealed only long nucleocapsid strands (data not shown). PAGE of density gradient fractions showed that the N protein sedimented into the gradient whereas much of the G protein and some of the M protein remained at the top (Figs. 1C and D). However, some G and M protein was also present in all the fractions. The N protein sedimented with the nucleocapsids as reported by Randles and Francki (1972) but substantial amounts of both G and M proteins were also present. The significance of this remains to be determined. Immunological analysis of LNYV structural proteins N and G Two monoclonal antibodies, one specific to the N protein (MCA-N) and another to the G protein

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489

FIG. 1. Protein analysis of LNW subjected to sucrose density gradient centrifugation. A preparation of LNW, purified by column chromatography and concentrated by centrifugation, was divided into two equal aliquots. One aliquot was subjected to centrifugation into a 1O-40% (w/v) sucrose density gradient for 20 min at 36,000 rpm in a Spinco SW41 rotor (A and B). To the other aliquot, Nonidet-P40 was added to 1% (v/v) before centrifugation under the conditions described above, except for 45 min (C and D). Both gradients were fractionated and scanned at 254 nm (A and C). To each fraction (600 ~1) was added 66 ~1 of 1OX Laemmli sample buffer and after heating at 95” for 5 min, 40-~1 aliquots were subjected to electrophoresis in 10% polyacrylamide and the gels were stained with silver (B and D). Positions to which the major virus proteins G. kl, and M migrated are shown to the left of(B).

(MCA-G), were selected for the analysis of LNYV structural proteins. MCA-N was shown to belong to the IgG, and MCA-G to the IgG, subclasses. Both reacted specifically with LNYV in indirect ELISA (data not shown) but neither precipitated the virus in agar immunodiffusion tests. The MCAs recognized their respective proteins and several minor ones when used to probe dissociated LNYV preparations in Western immunoblots (Fig. 2, lanes 1). However, only the N and G proteins were recognized in protein extracts from LNYV-infected N. glutinosa leaves (Fig. 2, lanes 2) and no signals were detected in similar preparations from uninfected N. glutinosa (Fig. 2, lanes 3). This suggested that the minor bands recognized by the MCAs were degradation products of the N and G proteins, respectively. PAGE analysis of proteins from LNYV and virus nucleocapsid preparations revealed a complex silver staining pattern (Fig. 3A) that was similar to that of immunoblots probed with mouse polyclonal antiserum to LNYV (data not shown). lmmunoblotting with MCA-N

(Fig. 3B), however, revealed the presence of one major polypeptide (M, - 57,000) and up to 6 minor, faster migrating bands (M, - 54,000, 52,000, 50,000, 48,000, 29,000, and 27,000). In addition, three bands migrating slowly, with estimated M, of 110,000, 160,000, and 220,000 were detected. None of these bands was detected when mouse preimmune serum was used for probing or primary antibodies were omitted, indicating that they were not the result of nonspecific binding of IgG or enzyme conjugate (data not shown). It is concluded that the minor proteins migrating faster than the N protein were degradation products, and those migrating slower were dimeric, trimerit, and tetrameric aggregates of the N protein. Further evidence that the minor proteins were derived from the N protein was obtained from the results of experiments with monospecific monoclonal and polyclonal antibodies purified from nitrocellulose-immobilized protein bands (data not shown). Antibodies eluted from either the intact N protein (n/r, - 57,000) or the assumed major degradation products of M, - 54,000

490

DIETZGEN

-G

N-

FIG. 2. Specificity of MCAs in immunoblots. Purified LNYV (lanes 1) and leaf extracts from LNW-infected (lanes 2) and uninfected (lanes 3) Nicofiana glutinosa leaves were resolved by 10% SDSPAGE and electroblotted to nitrocellulose membrane. (A) was reacted with MCA-N and (B) with MCA-G. Each MCA was affinity purified and used at 1 rg/ml.

and 29,000, reacted with both the intact N protein and the minor proteins. The degradation of the N protein in LNYV preparations was reduced when 1 mM of the protease inhibitor /V-p-tosyl-L-lysine chloromethyl ketone was added to the extraction buffer, but not with 1 mlVI phenylmethylsulfonyl fluoride, 10 ml\/l dithiothreitol, or 20 mM iodoacetic acid. Several bands with M, less than 27,000 contained in nucleocapsid preparations (Fig. 3A, lane 6) were not revealed by immunoblotting with MCA-N (Fig. 3B). These polypeptides may be either degradation products of other structural proteins or N protein degradation products which do not contain the MCA-N epitope. lmmunoblots of LNYV and virus envelope preparations probed with MCA-G (Fig. 4B) revealed the G protein (I@ - 78,000). In addition, the antibody recognized two faster (Mr - 71,000 and 58,000) and four slower migrating (rJr, - 94,000, 135,000, 170,000, and 180,000) components. Like the G protein, all these minor components were shown to be glycosylated by their ability to bind Con A (Fig. 4C), but none was recognized by either MCA-N or immunoglobulins from preimmune mouse serum (data not shown). The faster migrating components may be degradation products of the G protein and the slower migrating components may be aggregates of the G protein and various degradation products, or possibly anomalously migrating components that vary in the extent of glycosylation. The migration of LNYV N protein in PAGE was unaffected by exposure to the reducing agent 2-mercaptoethanol, whereas the mobilities of both the G and M

AND FRANCKI

proteins were decreased by reduction (Figs. 5A and B). The apparent I’@ of the G protein was calculated to be 67,000 when determined under nonreducing conditions as opposed to 78,000 after reduction. Similarly, the M, of the M protein was calculated to be 18,000 before and 19,000 after reduction, respectively. This indicates that both the G and M protein conformation is stabilized by disulfide bonds whereas the N protein is not. Both reduced and nonreduced G protein bound MCA-G and Con A (Figs. 5C and D). This indicates that reduction did not change the epitope specific to the monoclonal antibody and that the protein was glycosylated before and after reduction. MCA-G appears to be specific for an epitope on the protein moiety because it showed similar reactivity in immunoblots treated with periodic acid prior to probing with the antibody and control blots (Figs. 5E and F). However, it may be possible that the carbohydrate was not removed by this treatment (H. Zola, personal communication).

Glycosylation of LNYV G protein Con A binds specifically to a variety of carbohydrates, including N-acetylglucosamine, mannose, glu-

4p220 4pmo

4 p110 +

1

P57 z4 45P

4p29 4~27

123456123456 FIG. 3. Analysis of polypeptides derived from LNYV N protein by immunoblotting with MCA-N. Increasing amounts of dissociated LNW in ratios of 1:5: 15:40 (lanes l-4) and of isolated viral nucleocapsids in ratios of 1:20 (lanes 5 and 6) were resolved on 10% SDSpolyacrylamide gels and either stained with silver (A) or electroblotted and probed with 1 pg/ml of MCA-N (B). Estimated M, (X 103) of N protein derived polypeptides (p) are indicated by arrowheads on the right. The mobilities of M, markers (X 103) and the major LNYV proteins G, N, and M are indicated on the left. The star on the right indicates a faint band in (B) representing the G protein, stained with india ink before the immunoblot.

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116 97 G66N-

29-

FIG. 4. Analysis of polypeptides derived from LNW G protein by immunoblotting with MCA-G and affinoblotting with Con A. Protein from dissociated LNW was resolved on 8% SDS-polyacrylamide gels and stained with silver (A), electroblotted. and probed with 1 pg/ ml of MCA-G (B) or probed with Con A (C). Protein markers (myosin, M, = 205 X 1 03; P-galactosidase, M, = 116 X 103; phosphorylase b, M, = 97 X 103; bovine serum albumin, M, = 66 X 103; and carbonic anhydrase, Mr = 29 X 103) are shown in lane 1. Dissociated LNW at relative loadings in a ratio of 1: 10 are shown in lanes 2 and 3, respectively, and in ratios of 1:5:15 in lanes 4-6, respectively. A preparation of dissociated viral envelopes (obtained from the top of a density gradient such as that shown in Fig. 1C) at relative loadings in a ratio of 1:6 are shown in lanes 7 and 8, respectively, and ovalbumin (1 pg/lane) in lanes 9. Estimated M, (Xl 03) of G protein-derived polypeptides (p) are indicated by arrowheads on the right. The mobilities of M, markers (Xl 03) and the major LNW proteins G, N, and M are indicated on the left.

case, and sorbose (Faye and Chrispeels, 1985) and denatured glycoproteins are more susceptible to hydrolysis by exo- and endoglycosidases than glycoproteins in their native conformation (Thotakura and Bahl, 1987). Hence, the susceptibility of LNYV G protein to glycosidases was tested on LNYV proteins immobilized on nitrocellulose byelectroblotting from PAGES, followed by probing with Con A. Endo-@II-acetylglucosaminidase F from Flavobacterium meningosepticum (Endo F) (Elder and Alexander, 1982) almost completely deglycosylated LNYV G protein (Fig. 6, lanes 1 and 2) and the ovalbumin used as a control (Fig. 6, lanes 3). However, the G protein proved resistant to exo-@V-acetylglucosaminidase and jackbean cY-mannosidase which, however, digested the control ovalbumin (data not shown). From these specificities we conclude that the oligosaccharides of LNYV G protein consist of a complex network containing /V-acetylchitobiose N-linked to asparagine residues of the protein backbone. A search for the L and NS proteins of LNYV In a search for the L and NS proteins of LNYV, we examined the proteins of highly purified virus prepara-

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VIRUS PROTEINS

tions following electroblotting from polyacrylamide gels to nitrocellulose. In these experiments we used LNYV preparations purified from N. ghtinosa that had been infected for only 1O-l 1 days. Such preparations appeared to contain smaller amounts of N and G protein degradation products than virus isolated from plants infected for longer periods. Electroblots were cut into strips and some were stained with colloidal gold to detect all the proteins in the virus preparations (Fig. 7, lane 3). Other blot strips were probed with a variety of antibody preparations to identify specific protein bands. None of the proteins reacted with a mouse preimmune serum (Fig. 7, lane l), but all the gold stained bands were recognized by a mouse polyclonal antiserum to LNYV (Fig. 7, lane 2) which was shown not to react with proteins extracted from leaves of N. glutinosa (data not shown). The N protein and a few minor components were recognized by MCA-N (Fig. 7, lane 4) and similarly the G protein and few other minor components were recognized by MCA-G (Fig. 7, lane 5). From Fig. 7 it can be seen that at least two proteins were detected by both gold-staining and the polyclonal antibodies, but not by MCA-N or MCA-G. One of these proteins had the electrophoretic mobility correspondand another to M, ing to M, - 180,000-190,000

Gr%i N-

%-

r-

12

12

12

12

12

12

FIG. 5. Influence of reducing conditions on the structural proteins of LNW. A virus preparation was divided into two aliquots; one was dissolved in Laemmli sample buffer and heated for 5 min at 95” (lane 1) and the other was dissolved in sample buffer without 2-mercaptoethanol, and kept at 4” (lane 2). The samples were resolved in 8% SDS-polyacrylamide gels and stained with silver (A). The remaining gel was electroblotted to nitrocellulose membrane and stained with colloidal gold (B), affinoblotted with Con A(C), affinoblotted with Con A in the presence of methyl-Luo-mannopyranoside, and probed with 1 /*g/ml MCA-G (E and F). Nitrocellulose in (E) was pretreated with periodic acid to remove carbohydrate. G, N, and M on the left indicate the positions of the three major viral proteins in the reduced (r) and nonreduced (nr) forms.

DIETZGEN AND FRANCKI

492

- p135 -p94 -G

123123

of LNYV G protein-derived oligosacchaFIG. 6. Characterization rides, Aliquots of a dissociated purified preparation of LNYV at relative loading ratios of 1: 10 (lanes 1 and 2, respectively) and 1 rg of ovalbumin (lane 3) were resolved in 6% SDS-polyacrylamide gels, electroblotted to nitrocellulose, and digested with endoglycosidase F (A) or incubated without enzyme (6) prior to probing with Con A. Affinoblotting was done as described under Materials and Methods.

by Dale and Peters (1981), who used the continuous buffer system of Weber and Osborn (1969). Although the carbohydrate moiety of the LNYV G protein was resistant to some exoglycosidases, it was cleaved from the protein by endoglycosidase F. This and the binding to concanavalin A indicate complex oligosaccharides N-linked to asparagine residues of the protein as in the case of the VSV G protein (Pal and Wagner, 1987). The degradation products of LNYV G and N proteins are most probably generated in vitro during virus purification and/or storage at -ZOO, because we have been unable to detect similar components in protein preparations extracted directly from LNYV-infected leaves. Furthermore, the number and amounts of the degradation products varied considerably from virus preparation to preparation. These degradation products were not detected by Dale and Peters (1981) because they stained their gels with Coomassie brilliant blue which

180-

- 38,000. The former protein was found to be associated with the viral nucleocapsid. Neither protein was glycosylated (data not shown). It seems likely that these are the Land NS proteins, respectively.

DISCUSSION Three major structural proteins, the surface glycoprotein G, the envelope matrix protein M, and the nucleocapsid-associated N protein, were readily identified by discontinuous PAGE. lmmunoblotting with monoclonal antibodies to the G and N proteins established that the majority of the minor bands detected by silver staining were either degradation or aggregation products of the G and N proteins. This included the conspicuous polypeptide of M, about 130,000 referred to as HMW by Dale and Peters (1981) which appears to be an aggregate of the G protein. However, two minor polypeptides which stained with silver and were recognized by antibodies in polyclonal antisera to LNYV were not recognized by the monoclonal antibodies. We tentatively conclude that these are the L and NS proteins. As with the M protein, we have as yet been unable to select monoclonal antibodies to these proteins. When available, such antibodies will be very useful for confirming the identification of the M, L, and NS proteins. Our molecular weight estimates of the LNYV major (G, N, and M) and the putative minor proteins (L and NS) are, with minor discrepancies, similar to those reported

118-

84-

58-

-N

3627-

--M

FIG. 7. Tentative detection of LNYV Land NS proteins. Dissociated virus preparations purified by sucrose density gradient centrifugation (see Figs. 1A and B) was resolved in 10% SDS-polyacrylamide gels and electroblotted to nitrocellulose. The preparative blot was cut into strips. Strip 1 was probed with mouse preimmune serum (diluted 1:2000); strip 2 was similarly probed with mouse polyclonal antiserum to LNYV (diluted 1:2000); strip 3 was stained with colloidal gold; strips 4 and 5 were probed with 0.5 rglml of MCA-N and MCA-G, respectively. The positions of the assigned LNYV structural proteins are indicated on the right. Protein molecularweight markers&-macroglobulin, M, = 180 X 103; fl-galactosidase, M, = 1 16 X 103; fructose 6-phosphate kinase, M, = 84 X 103; pyruvate kinase, M, = 58 x 1 03; lactate dehydrogenase, M, = 36.5 X 1 03; and triosephosphate isomerase, M, = 26.6 x 103) are shown on the left. Some proteins which reacted with MCAs reproduced poorly in the photograph.

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is about 50 times less sensitive. Using sensitive protein detection methods, degradation of structural proteins in preparations of other plant rhabdoviruses have been reported; including the N proteins of northern cereal mosaic and wheat rosette stunt viruses (Shirako and Ehara, 1985; Gong and Zheng, 1986) and the M proteins of sowthistle yellow vein virus (Ziemiecki and Peters, 1976; J. L. Dale, personal communication). Proteolytic activity resulting in the digestion of viral proteins has also been detected in purified VSV preparations despite pretreatment with protease inhibitors by Holland et al. (1972).

ACKNOWLEDGMENTS The authors thank Mr. C. Grivell for excellent technical assistance and Mr. D. Talfourd for the maintenance of plants. R. G. Dietzgen was partially supported by a Feodor-Lynen Research Fellowship from the Alexander von Humboldt Foundation. The project was also supported by the Australian Research Grants Scheme.

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