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Immunochemical identification of rubella virus hemagglutinin
VIROLOGY
126. 194-203 (1983)
lmmunochemical
Identification
M. NEAL Departmats
of Neurology, Hygiene,
WAXHAM
of Rubella JERRY
AND
Virus Hemagglutinin
S. WOLINSKY’
Immunology and Infectious Di.seases, Schools of Medicine, Public Health Th.e Johns Hopkins University. Baltimore, Maryland 21205
and
Received August 13, 1982; accepted December 8, 1982
Purified rubella virus contains three major structural polypeptides whose apparent molecular weights are 62,000, a 47,000-54,000 complex, and 38,000 when analyzed by polyacrylamide gel electrophoresis. Both the 62,000 and 47,OOC-54,000 dalton polypeptides are glycosylated, but they vary in their relative [‘Hlglucosamine and [‘Hlmannose content. Limited-digest peptide maps confirm that each polypeptide is distinct and that the 47,00054,000 dalton complex is a series of three glycopolypeptides with extensive similarities. Under nonreducing conditions, both the 62,000 dalton glycopolypeptide and the 47,00054,000 dalton complex exist as monomers and also as disulfide-linked complexes. A complex of 105,000 daltons is a dimer of the 62,000 dalton glyeopolypeptide and a second, at 95,000 daltons, is a mixed disulfide-bonded dimer of the 62,000 dalton glycopolypeptide and 47,000-54,000 dalton complex. The 38,000 dalton polypeptide migrated exclusively as a dimer of 78,000 daltons when unreduced. Two monoclonal antibodies which inhibit the hemagglutinin function of the virus were shown to be directed against the 62,000 dalton glycopolypeptide. This glycoprotein is therefore responsible at least in part for the hemagglutinin function of rubella virus.
INTRODUCTION
Rubella virus has recently been classified as the sole member of the genus Rubivirus in the family Togaviridae (Fenner, 1976). A characteristic feature of the togaviruses is their lipid envelope which is acquired by budding through host cell membranes, modified by the insertion of virus-specified glycoproteins (Brown, 1980). The importance of these viral glycoproteins in mediating biological functions has been well documented for several representative members of the togavirus family (Simons et al, 1980). However, little is known about the relationships between the biological functions of rubella virus and specific viral proteins. Several groups have described the structural proteins of rubella virus (Vaheri and ’ Address correspondence and reprint requests to Jerry S. Wolinsky, M. D., Department of Neurology, The Johns Hopkins University, Adolph Meyer Building 6-181D, 600 N. Wolfe Street, Baltimore, Md. 21205. 0042-6822/83 $3.00 Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved.
Hovi, 1972; Bardelleti et aZ.,1975; Ho-Terry and Cohen, 1980; Payment et al., 1975; Liebhaber and Gross, 1972). Yet, as recently reviewed (Van Alstyne, 1981), there continue to be discrepancies in the literature concerning the number and molecular weights of the viral proteins. Most workers agree that rubella virus contains three major structural proteins, One is nonglycosylated, cosediments with the infectious viral RNA, and is the presumed capsid protein. The other two are glycoproteins found in the lipid envelope. One of the major biological properties of rubella virus is its well-documented ability to agglutinate red blood cells (Stewart et al., 1967; Hobbins et ab, 1967). Subfractionation experiments have shown that spikes consisting of the two glycoproteins can be isolated from virions and these spikes retain the hemagglutination conditions under appropriate activity (Trudel et aZ.,1980). Furthermore, a recent study of electrophoretic and biological alterations produced by proteolytic treat194
RUBELLA
HEMAGGLUTININ
ment of virions with trypsin indicated that the larger of the two glycoproteins may mediate the hemagglutinin function (HoTerry and Cohen, 1980). In this report we have characterized the structural polypeptides of rubella virus by electrophoresis, and we have used an immunochemical approach coupled with biological inhibition assays to associate the hemagglutinin function with the larger of the two glycoproteins. MATERIALS
AND
METHODS
Cells and viruses. Vero cells obtained from the American Type Culture Collection were routinely cultured in Eagle’s minimum essential medium (MEM) supplemented with vitamins, nonessential amino acids, 10% heat-inactivated fetal bovine serum (GIBCO Laboratories, Grand Island, N. Y.), and 10 pug/ml of gentamycin (Schering Corp., Kenilworth, N. J.). The Therien strain of rubella virus was plaque purified twice and stocks were expanded in Vero cells, using a multiplicity of infection (m.0.i.) of 0.01 plaque-forming units (PFU) per cell. Stock virus used for these studies had a titer of 106.3PFU/ml. Growth and puri&cation
of rubella virus.
Vero cells were grown in 850~cm2 roller bottles (Corning Glasswares, Corning, N. Y.) and infected when subconfluent at an m.o.i. of 0.1 PFU per cell. Infected culture medium was collected at 48 hr postinfection, clarified, and stored at 4” and the cultures were replenished with fresh medium, Harvests were then repeated daily for four additional days. The pooled virus harvest was concentrated approximately loo-fold using constant flow-countercurrent dialysis over a 100,000 dalton membrane (Millipore, Bedford, Mass.) as previously described (Bellini, 1979). The virus was then purified by banding in two successive discontinuous 25%/50% (w/w) sucrose gradients in NET (0.1 MNaCl, 0.001 M EDTA, 0.01 M Tris, pH 7.4) by centrifugation at 95,000g for 2 hr in a Sorvall AH-627 rotor. Material sedimenting at the 25% /50% sucrose interface was collected, dialyzed against NET, and stored at -70”. Preparation of radiolabeled virus. Sub-
195
confluent Vero cell monolayers in 75-cm2 flasks (Corning) were infected at an m.o.i. of 1 PFU per cell. After 48 hr the medium was replaced with MEM completely deficient in either leucine or methionine and the cells were incubated for 1 hr at 37”. The medium was then replaced with the appropriate deficient medium containing [3H]leucine (60 Ci/mmol, Amersham, Arlington Hts., Ill.) or r5S]methionine (1300 Ci/mmol, Amersham) and supplemented with 5% MEM. [3H]Glucosamine-labeled virus was grown identically in MEM with fructose substituted for glucose as a source. [3H]Glucosamine carbohydrate (Amersham) had a specific activity of 30 Ci/mmol. Virus was labelled with [3H]mannose (30 Ci/mmol) by adding the isotope directly to MEM. After an additional 24 hr incubation at 37” the radiolabeled supernatant fluids were collected, clarified, and virus was pelletted at 95,000 g for 1 hr. The pellet was resuspended and banded on a 25%/50% sucrose gradient as described above. The purified virus was repelleted and suspended in lysate buffer [0.025MTris-HCl, pH 7.6,0.5 MNaCl, 0.001 M EDTA containing 1% Zwittergent 3-14 (Calbiochem-Berhring, La Jolla, Calif.), 0.5% sodium deoxycholic acid (Sigma Chemical Co., St. Louis, MO.), 0.1% sodium dodecyl sulfate (SDS: BDH Chemicals Ltd., Poole, England), and tosylamide-Z-phenyethyl chloromethyl ketone at 10 pg/ml (Sigma)] and stored at -20”. Immunoprecipitation and SDS-polyacrylamide gel electrophoresis (PAGE).
Immunoadsorbents were prepared by incubating 25 ~1 of the appropriate antibody with 20 ~1 of a 1:l slurry of protein A Sepharose CL-4B (Pharmacia, Piscataway, N. J.) in 0.01 M phosphate buffer pH 7.4 at 4” for 1 hr. Unadsorbed antibody was removed by washing the immunoadsorbents with lysate buffer. Lysate buffer-disrupted radiolabeled antigen was then incubated with the immunoadsorbent for 2 hr at 4”. Unbound antigen was removed by exhaustively washing the immunoadsorbents with lysate buffer. Antigen-antibody complexes were dissociated prior to electrophoresis by the addition of SDS sample buffer with or without a reducing
196
WAXHAM
AND
agent, and heating at 100” for 5 min. Polyacrylamide gel electrophoresis was performed using 10% acrylamide (acrylamide:bisacrylamide:30:0.8) resolving gels, and the discontinuous buffer system of Laemmli (1970), and radiolabeled protein bands were visualized by fluorography (Bonner and Laskey, 1974). Electrophoresis was for 3 hr at 25 mA per gel. Relative molecular weights were determined by comparison of migration with the following standards, visualized after staining with 0.5% Coomassie Blue G-250:phosphorylase B (94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100), @lactoglobulin (18,400), and lactalbumin (14,400), (Pharmacia). Two-dimensional SDS-PAGE. Two-dimensional SDS-PAGE was performed essentially as detailed by Smith and Hightower (1981). Briefly, reducing agent was omitted from electrophoresis in the first dimension, following which the gel lane was excised, equilibrated with sample buffer containing 5% 2-mercaptoethanol (Fisher Scientific, Silver Spring, Md), placed horizontally onto a second SDS gel, and electrophoresed as before. Both resolving gels contained 10% acrylamide. Peptide mapping. Peptide maps were generated from gel slices containing individual polypeptides by either partial cleavage with cyanogen bromide (Pierce Chemical Co., Rockford, Ill.) as described by Nikodem and Fresco (1979) or by limited proteolysis as described by Cleveland et al. (1979) using 0.5 pg of Staphylococcus aureus V8 protease (Miles Laboratories, Inc., Elkhart, Ind.) for each sample well. Limited proteolysis with S. aureus V8 protease of an entire lane of viral polypeptides separated by SDS-PAGE was also performed according to the method of Bordier and Crettol-Jarvinen (1979). Reduction and carboxymethylation of protein. Radiolabeled virus solubilized in
lysate buffer was reduced by the addition of 2-mercaptoethanol to a final concentration of 0.15 Mand incubated in a stoppered tube at 37” for 6 hr. The reduced proteins were then carboxymethylated by the addition of 30 pg of iodoacetamide (Fisher)
WOLINSKY
in lysate buffer and incubated at 15” for 45 min. Remaining iodoacetamide was reacted with excess 2-mercaptoethanol and the reduced carboxymethylated proteins were exhaustively dialyzed against lysate buffer for use as targets for immunoprecipitation. Hemagglutination
inhibition
assay.
Hemagglutination inhibition (HAI) assays were performed in V bottom microtiter plates (Dynatech Laboratories Inc., Alexandria, Va.) using l-day-old chick red blood cells as indicators (Liebhaber, 1970). All antisera were the IgG fraction from protein A Sepharose CL-4B chromatography (Server et al., 1982). Generation
of rubella-specijc
ant&n&es.
Four-week-old BALB/c mice were sensitized by intraperitoneal injection of 50 pg of purified virus. An additional 10 pg of virus was injected intravenously 5 days before donor spleens were harvested. The immune donor spleen cells were separated and fused with P3X63-Ag 8.653 myeloma cells essentially as described by Server et al. (1982). Anti-rubella-secreting hybrids were detected by an enzyme-linked immunoadsorbent assay (Wolinsky et al., 1982) and single cell clones selected by limit dilution. Hyperimmune rabbit anti-rubella antibodies and the cerebrospinal fluid from a patient with progressive rubella panencephalitis served as polyvalent antisera. These reagents have been characterized previously (Wolinsky et al, 1982). RESULTS
Characterization of the Polypeptides rified Virus
of Pu-
Polypeptides of virions labeled with [35S]methionine or rH]leucine were analyzed using SDS-PAGE. Similarly processed unlabeled virus samples were composed exclusively of partially disrupted and intact virions as determined by electron microscopic analysis of negatively stained preparations (data not shown). The electrophoretic patterns further demonstrate the purity of these viral preparations and show the three major structural polypeptides of rubella virus (Fig. 1). These have relative molecular weights of 62,000, a se-
RUBELLA
a
b
197
HEMAGGLUTININ
C
105kL 95kN
d
e
P78
w62 SIP54 9P47 ~38
FIG. 1. Identification of rubella virus structural polypeptides. Metabolically labeled rubella virus was disrupted under reducing conditions (lanes a-d) or nonreducing conditions (lane e) and analyzed on 10% SDS-polyacrylamide gels. Virus was labeled with [3H]leucine (lane a), [aH]mannose (lane b), [%lglueosamine (lane c), or [%]methionine in (lanes d and e). Variations in migration patterns are due to the length of the respective electrophoresis times, and do not represent qualitative differences in relative mobilities.
ries of related bands from 47,000-54,000, which by limited peptide mapping (see below) are essentially identical, and 38,000. Two minor components were also consistently detected with approximate molecular weights of 95,000 and 105,000. Selective labeling of virus with [3Hlglucosamine or [3H]mannose demonstrated that the two proteins of 62,000 daltons and the 47,00054,000 dalton complex are glycosylated as are the two minor high molecular weight species (Fig. 1, lanes b and c). Comparative analysis of densitometric tracings of Fig. 1, lanes b and c, shows that there are differences in the amounts of mannose and glucosamine incorporated into the two major glycoproteins. The 62,000 dalton glycopolypeptide contains much more mannose than glucosamine, while the 47,000-54,000 dalton complex contains more glucosamine than mannose. These two types of carbohydrate patterns are indicative of oligosaccharide side chains of the simple and complex types, respectively (Kornfeld and Kornfeld, 1980). A different electrophoretic pattern of [%]methionine-labeled polypeptides was obtained when reducing agent was omitted from the sample buffer (Fig. 1, lane e). The 38,000 dalton protein was no longer detectable and a new band was apparent at 78,000 daltons. In addition, there was a
distinct shift in the relative proportion of label from the 62,000 dalton and 47,00054,000 dalton complex regions to higher molecular weight components at 105,000 and 95,000. The 62,000 dalton glycopolypeptide migrated slightly faster under nonreducing conditions, while the 47,00054,000complex migrated similarly whether unreduced or reduced. Two-Dimensional SDS-PAGE The differences in electrophoretic patterns between unreduced and reduced rubella virus polypeptides suggested that multichain disulfide-linked polypeptides exist in the virion. These complexes and their components were specifically identified using SDS-PAGE in two dimensions, the first in the absence of reducing agent, followed by reduction and reelectrophoresis in a second dimension (Fig. 2). The two major glycopolypeptides are present as unlinked monomers, as shown by their migration near the diagonal. The 62,000 dalton glycopolypeptide migrated slightly above the diagonal, indicating a faster electrophoretic mobility when unreduced, as previously noted. This result is consistent with a more compact polypeptide structure in the absence of reducing agent, due to the presence of intrachain disulfide bonds.
198
WAXHAM
AND
The heterogeneity of the 47,000~54,000 dalton complex is again shown when analyzed in this manner. These polypeptides migrated as a diagonal group of at least two and more likely three species. However, they migrated similarly with or without disruption of disulfide bonds, which indicates a lack of extensive intrachain disulfide bonds. The 38,000 dalton polypeptide appears as a spot directly below the 78,000dalton unreduced species far off the diagonal, and identifies the 78,000 dalton species as a disulfide-linked dimer of the 38,000 dalton polypeptide. A distinct shift of radioactivity to the high-molecular-weight components at 95,000 and 105,000 daltons when reducing agent was omitted from single dimension SDS-PAGE was noted above. Analysis of these two species in two-dimensional SDS-PAGE showed that both consisted of disulfidelinked complexes of the two major glycoproteins. The 105,000 dalton species gave rise to a spot that migrated with the 62,000 dalton polypeptide and a spot at 105,000 daltons which must be a form of that polypeptide that is not altered by the reducing treatment. Therefore, the 105,000 dalton species apparently represents a dimeric form of the 62,000 dalton glycopolypeptide. The 95,000 dalton species, upon reduction, dissociated into the 62,000 dalton polypeptide and a heterogeneous spot which migrated in the same manner as the 47,00054,000 dalton complex. Thus the 95,000 dalton species apparently represents a mixed disulfide-linked dimer of the 62,000 dalton glycopolypeptide and the 47,000-54,000 dalton glycopolypeptide complex. Cyanogen Bromide and Limited Lliges tion Maps
Proteolytic
The number of unique polypeptides of rubella virus was determined by generating peptide maps for each species. Cyanogen bromide (CNBr) peptides are shown in Fig. 3. Each major polypeptide separated by SDS-PAGE was partially cleaved with CNBr and the resulting fragments were separated in a 15% SDS-polyacrylamide gel. The 62,000 dalton glycopolypeptide (lanes a and b) and 38,000 dalton
WOLINSKY
nonreduced -
38
@a \
FIG. 2. Two-dimensional SDS-PAGE. [“S]Methionine-labeled rubella virus was disrupted and electrophoresed under nonreducing conditions (top panel). A second, identically prepared gel lane was re-equilibrated in SDS sample buffer containing reducing agent, and re-electrophoresed perpendicularly into a second SDS-polyacrylamide gel (middle panel). Both gels contained 10% acrylamide and were fluorographed as described. The lower panel diagrammatically identifies the polypeptides shown in the middle fluorograph above. The monomeric forms of the glycopolypeptides are represented by the filled outlines near the diagonal and the oligomeric forms are represented by the hollow outlines below the diagonal. The reduced form of the capsid protein which also falls below the diagonal is represented by the hatched outline. The molecular weights for the reduced polypeptides are noted on the left in kilodaltons.
polypeptide (lanes c and d) yielded different peptides that were also distinct from those generated from the glycopolypeptides in the 47,000-54,000 dalton complex. That complex was subdivided into three bands and the peptide maps generated
RUBELLA
ab
HEMAGGLUTININ
cd
199
efghij
FIG. 3. Cyanogen bromide (CNBr) peptide mapping. The major polypeptides or rubella virus were first separated by SDS-PAGE identified by staining with Coomassie blue and then excised and digested with CNBr (lanes a, c, e, g, and i) in acidic acetonitrile. Parallel slices were incubated under identical conditions which lacked CNBr (lanes b, d, f, h, and j) to monitor acid hydrolysis. Lanes a and b are the 62,000 dalton glycopeptide. Lanes c and d are the 38,000 dalton polypeptide and e-j represent the three regions of the 47,000-54,000 dalton complex decreasing in molecular weight from right to left, the arrowhead indicates the peptide referred to in the text. Peptides were analyzed on 15% SDS-polyacrylamide gels
from each of these bands were similar (lanes e, g, and i). Only one peptide is seen to differ in each of these three lanes (arrowhead), and it is possibly the peptide that imparts the heterogeneous banding pattern to this protein complex in SDSpolyacrylamide gels. Whether this peptide differs because of a variation in glycosylation or is generated by some type of aberrant cleavage cannot be determined from these results. These observations were further supported by limited proteolytic maps generated with S. aureue V8 protease (Fig. 4). Using this enzyme, which has a different amino acid specificity from CNBr and with a different amino acid label ([YS]methionine) in the polypeptides, unique maps were again generated for both the 62,000 dalton glycopolypeptide and 38,000 dalton polypeptide. The components of the 47,00054,000 dalton complex were again shown to be three related glycopolypeptides similar to each other but different from the other species.
Determination of the Activity and Specificity of the Mono&ma1 Antibodies The biological activity of the monoclonal antibodies was determined by standard hemagglutination inhibition assay. At a starting protein concentration of about 1 mg/ml, both of the monoclonal antibodies used in these experiments consistently inhibited hemagglutination at dilutions of 1280 to 2560. To identify the viral polypeptide possessing the hemagglutinin function, the monoclonal antibodies were used to immunoprecipitate [35S]methionine-labeled polypeptides from disrupted virus particles. Both of the monoclonal antibodies used in these studies precipitated the 62,000 dalton glycopolypeptide (Fig. 5, lanes c and d). The small amount of the 47,000-54,000 dalton glycopolypeptide seen in immunoprecipitates of virions disrupted in the presence of reducing agent was not present in immunoprecipitates of virions that were disrupted without the reducing agent.
200
WAXHAM
gp6:
AND
gp58p47 p3S
WOLINSKY
a
b
c
d
WC. 4. Peptide mapping of $S]methionine-labeled rubella virus proteins. Two identical samples were electrophoresed under reducing conditions on 10% SDS-polyacrylamide gels as described. One gel lane was excised, transferred to a 15% acrylamide gel, overlayed with 20 pg of S. aureus V8 protease in sample buffer, and electrophoresed at 5 mA overnight. The second gel lane was held in a methanol-acetic acid solution until the digest gel was complete, then both were processed for fluorography. The one-dimensional pattern of the sample is shown at the top of the left panel; electrophoresis was from left to right. The four gel lanes on the right represent digests of the individually excised glycopolypeptides, each digested in the second dimension with 0.5 pg of S aureus V8 protease. Lane a is the 62,000 dalton glycopolypeptide, lanes b, c, and d are the three regions of the 4’7,000-54,000 dalton complex, decreasing in molecular weight from right to left.
This suggested that the 47,000~54,000dalton complex was coimmunoprecipitated as the 62,000 dalton:47,000-54,000 dalton disulfide-linked complex described above. Further proof for the specificity of the monoclonal antibodies for the 62,000 dalton glycopolypeptide was obtained by reducing and alkylating virus proteins before immunoprecipitation. The monoclonal antibodies specifically precipitated the 62,000 dalton glycopolypeptide (Fig. 6, lanes c and d). A small amount of the 38,000 dalton polypeptide was shown to nonspecifically precipitate even with Protein ASepharose alone (Figs. 5 and 6, lane e). Polyvalent antisera from a hyperimmunized rabbit and cerebrospinal fluid from a case of progressive rubella panencephalitis previously shown to recognize all of the structural proteins of the virus precipitated all of the structural proteins before or after reduction and alkylation, showing that the chemically modified proteins retained their antigenicity (Figs. 4 and 5, lanes a and b).
DISCUSSION
These studies were designed to identify the structural proteins of rubella virus and to begin to define the functional characteristics of the individual polypeptides using an immunochemical approach. Metabolically labeled, purified rubella virus when disrupted under reducing conditions and electrophoresed in SDS-PAGE is composed of three major and two minor structural components. The major components include a 62,000 dalton glycopolypeptide, a complex of three glycopolypeptides migrating at a molecular weight of 47,000~54,000, and a nonglycosylated 38,000 dalton polypeptide. By analogy with current nomenclature for other members of the togavirus family, these three species correspond to El, E2, and C, respectively. Similar profiles have been reported by Vaheri and Hovi (1972) and Payment et al. (1975) with minor differences in apparent molecular weights probably reflecting differences in the separation techniques used.
RUBELLA
a
201
HEMAGGLUTININ
b
C
d
FIG. 5. Immunological identification of rubella virus proteins. Lysates of [3SS]methionine-labeled virions were immunoprecipitated with cerebrospinal fluid from a patient with progressive rubella panencephalitis (lane a), from rabbit hyperimmunized with purified rubella virus (lane b), monoclonal antibody (lanes c and d) or protein A-Sepharose CL-4B al,ne to monitor nonspecific precipitation products (lane e), and analyzed on 10% SDS-polyacrylamide gels.
They do, however, differ substantially from results reported by Van Alstyne et al (1981) who used a different purification procedure from that used in this report and by the other investigators. The studies of Vaheri and Hovi (1972) and Payment et al. (1975) have shown that the nonglycosylated C polypeptide is not envelope associated but does cosediment with viral RNA, supporting its designation as the core protein C. Two-dimensional SDS-PAGE has now demonstrated that this capsid protein C exists as a disulfide-linked dimer in native form which migrates as a single species of 78,000 daltons under nonreducing conditions. Vaheri and Hovi (1972) also suggested that E2, their VPII, has a broad migration pattern due to large amounts of glycosylation. We were able to resolve the highly glycosylated E2 into a group of three species. The peptide maps of these species generated by cyanogen bromide cleavage appeared to differ only by a single peptide, which may represent the peptide that imparts heterogeneous migration to E2. Whether this peptide differs only in its extent of glycosylation or represents altered forms of the polypeptide backbone remains to be determined. In addition to the three major structural
polypeptides, we consistently noted two minor high-molecular-weight components of 105,000 and 95,000 daltons. When analyzed by two-dimensional (nonreducing and reducing) SDS-PAGE, the larger of the two was determined to be a dimer of El, and the second was shown to be a heterodimer of El and E2. Ho-Terry and Cohen (1980) recently reported a single glycoprotein which they called A that migrated closely to the combined molecular weights of their two major glycoproteins and seemed susceptible to reduction. They proposed that A might be a disulfide-bonded dimer of El and E2, but they made no attempt to characterize it further. Delineation of whether the homo- and heterodimers are present in the virion or are artifacts induced in the preparation of the proteins for SDS-PAGE will require further investigation. However, the presence of three distinct forms of El (El, El-El, El-E2) could well prove to be important in modulating the function of this protein. We were able to select two monoclonal antibodies which inhibit hemagglutination of rubella virus to very high titer. Immunoprecipitation patterns of radiolabeled viral proteins and analysis by SDSPAGE showed that both minor high-molecular-weight proteins and the 62,000
202
WAXHAM
a
AND WOLINSKY
b
C
d
e
w62 9P54 9P47 ~38
FIG. 6. Immunological identification of reduced and carboxymethylated rubella virus proteins. Lysates of [“Slmethionine-labeled rubella virus were immunoprecipitated with cerebrospinal fluid from a patient with progressive rubella panencephalitis (lane a), hyperimmune rabbit anti-rubella antibody (lane b), monoclonal antibody (lanes c and d), or protein A-Sepharose alone (lane e) and analyzed on 10% SDS-polyacrylamide gels.
dalton protein were recognized by these antibodies and by definition share the same antigenic site. This is because each of the proteins contains the El glycoprotein in either the dimeric (El-El), heterodimeric (El-E2), or monomeric form (El). This interpretation was supported by the demonstration that following conditions which disrupted disulfide linked proteins only the El protein was specifically immunoprecipitated. These results show that the El protein of rubella virus must include determinants involved in hemagglutinin function, which is consistent with both HoTerry and Cohen’s preliminary digestion studies with rubella virus (1980) and the finding that monoclonal antibodies generated against the El glycoprotein of Sindbis virus, a togavirus of the alphavirus subgroup, also inhibit hemagglutination (Chanas et aL, 1982). In summary, our studies show that rubella virus contains three major structural proteins, El, E2, and C, and that the hemagglutinin function is associated with the El glycoprotein. ACKNOWLEDGMENTS The authors thank Drs. David C. Merz and Alfred C. Server for critical comments during the course of
these studies and Ms. Linda Kelly for preparation of the manuscript. This work was aided by the United Cerebral Palsy Research and Educational Foundation and a Research Career Development Award, NS00443 (JSW) from the National Institutes of Neurological and Communicative Disorders and Stroke. REFERENCES BELLINI, W. J., TRUDGETT,A., and MCFARLIN, D. E. (1979). Purification of measles virus with preservation of infectivity and antigenicity. J. Gen Viral. 43, 633-639.
BONNER,W. M., and LASKEY, R. A. (1974). A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83-88.
BORDIER,C., and CRETTOL-JARVINEN, A. (1979). Peptide mapping of heterogeneous protein samples. J. Biol. Chm.
254, 2565-2567.
BARDELETTI,G., KESSLER,N., and AYMARD-HENRY, M. (1975). Morphology, biochemical analysis and neuraminidase activity of rubella virus. Arch Virol 49, 175-186. BROWN,D. T. (1980). The assembly of alphaviruses. In “The Togaviruses” (R. W. Schlesinger, ed.), pp. 473-501. Academic Press, New York. CHANAS, A. C., GOULD, E. A., CLEGG,J. C. S., and VARMA, M. G. R. (1982). Monoclonal antibodies to Sindbis virus glycoprotein El can neutralize, enhance infectivity, and independently inhibit haemagglutination or haemolysis. J. Gen. Viral. 58,3746.
RUBELLA
HEMAGGLUTININ
CLEVELAND,D. W., FISCHER,S. G., KIRSCHNER,M. W., and LAEMMLI, U. K. (1977). Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem 252, 1102-1106. FENNER, F. (1976). Classification and nomenclature 7. of viruses. Intervirology HOBBINS,T. E., STEWART,G. L., PARKMAN, P. D., HOPPS,H. E., and MEYER, H. M., JR. (1967). Biophysical and biochemical properties of rubella virus hemagglutinin. Fed Proc 26,421. HO-TERRY,L., and COHEN,A. (1980). Degradation of rubella virus envelope components. Arch ViroL 65, 1-13. KORNFELD,R., and KORNFELD,S. (1980). Structure of glycoproteins and their oligosaccharide units. In “The Biochemistry of Glycoproteins and Proteoglycans” (W. J. Lennarz, ed.), pp. l-34. Plenum, New York. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680-685. LIEBHABER, H. (1970). Measurement of rubella antibody by hemagglutination inhibition. Variables affecting rubella hemagglutination. J. Immunol 194, 818825. LIEBHABER,H., and GROSS,P. A. (1972). The structural proteins or rubella virus. Virology 47, 684693.
NIKODEM,V., and FRESCO,J. R. (1979). Protein fingerprinting by SDS-gel electrophoresis after par-
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tial fragmentation with CNBr. Anal Biochem. 97, 382-386.
SERVER,A. C., MERZ,D. C., WAXHAM,M. N., and WOLINSKY,J. S. (1982). Differentiation of mumps virus strains with monoclonal antibody to the HN glycoprotein. InLfect.Immun 35,179-186. SIMONS,K., GAROFF,H., and HELENIUS,A. (1980). Alphavirus proteins. In “The Togaviruses” (R. W. Schlesinger, ed.), pp. 317-333. Academic Press, New York. SMITH, G. W., and HIGHTOWER,L. E. (1981). Identification of the P proteins and other disulfide-linked and phosphorylated proteins of Newcastle Disease virus. J. ViroL 37, 256-267. STEWART,G. L., PARKMAN,P. D., HOPPS,H. E., DoucLAS, R. D., HAMILTON,J. P., and MEYER, H. M., JR. (1967). Rubella virus hemagglutination-inhibition test. New EngL J. Med 276,554-557. TRUDEL, M., RAVAORINORO,M., and PAYMENT, P. (1980). Reconstitution of rubella hemagglutinin on liposomes. Can J. MicrobioL 26,899-904. VAHERI, A., and HOVI, T. (1972). Structural proteins and subunits of rubella virus. J. ViroL 9, 10-16. VAN ALSTYNE,D., KRYSTAL,G., KETTYLS,G. D., and BOHN,E. M. (1981). The purification of rubella virus (RV) and determination of its polypeptide composition. Virologl/ 108, 491-498. WOLINSKY, J. S., WAXHAM, M. N., HESS,J. L., TOWNSEND,J. J., and BARINGER,J. R. (1982). Immunochemical features of a case of progressive rubella panencephalitis. Clin Exp. ImmunoL 48, 359-366.
126. 194-203 (1983)
lmmunochemical
Identification
M. NEAL Departmats
of Neurology, Hygiene,
WAXHAM
of Rubella JERRY
AND
Virus Hemagglutinin
S. WOLINSKY’
Immunology and Infectious Di.seases, Schools of Medicine, Public Health Th.e Johns Hopkins University. Baltimore, Maryland 21205
and
Received August 13, 1982; accepted December 8, 1982
Purified rubella virus contains three major structural polypeptides whose apparent molecular weights are 62,000, a 47,000-54,000 complex, and 38,000 when analyzed by polyacrylamide gel electrophoresis. Both the 62,000 and 47,OOC-54,000 dalton polypeptides are glycosylated, but they vary in their relative [‘Hlglucosamine and [‘Hlmannose content. Limited-digest peptide maps confirm that each polypeptide is distinct and that the 47,00054,000 dalton complex is a series of three glycopolypeptides with extensive similarities. Under nonreducing conditions, both the 62,000 dalton glycopolypeptide and the 47,00054,000 dalton complex exist as monomers and also as disulfide-linked complexes. A complex of 105,000 daltons is a dimer of the 62,000 dalton glyeopolypeptide and a second, at 95,000 daltons, is a mixed disulfide-bonded dimer of the 62,000 dalton glycopolypeptide and 47,000-54,000 dalton complex. The 38,000 dalton polypeptide migrated exclusively as a dimer of 78,000 daltons when unreduced. Two monoclonal antibodies which inhibit the hemagglutinin function of the virus were shown to be directed against the 62,000 dalton glycopolypeptide. This glycoprotein is therefore responsible at least in part for the hemagglutinin function of rubella virus.
INTRODUCTION
Rubella virus has recently been classified as the sole member of the genus Rubivirus in the family Togaviridae (Fenner, 1976). A characteristic feature of the togaviruses is their lipid envelope which is acquired by budding through host cell membranes, modified by the insertion of virus-specified glycoproteins (Brown, 1980). The importance of these viral glycoproteins in mediating biological functions has been well documented for several representative members of the togavirus family (Simons et al, 1980). However, little is known about the relationships between the biological functions of rubella virus and specific viral proteins. Several groups have described the structural proteins of rubella virus (Vaheri and ’ Address correspondence and reprint requests to Jerry S. Wolinsky, M. D., Department of Neurology, The Johns Hopkins University, Adolph Meyer Building 6-181D, 600 N. Wolfe Street, Baltimore, Md. 21205. 0042-6822/83 $3.00 Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved.
Hovi, 1972; Bardelleti et aZ.,1975; Ho-Terry and Cohen, 1980; Payment et al., 1975; Liebhaber and Gross, 1972). Yet, as recently reviewed (Van Alstyne, 1981), there continue to be discrepancies in the literature concerning the number and molecular weights of the viral proteins. Most workers agree that rubella virus contains three major structural proteins, One is nonglycosylated, cosediments with the infectious viral RNA, and is the presumed capsid protein. The other two are glycoproteins found in the lipid envelope. One of the major biological properties of rubella virus is its well-documented ability to agglutinate red blood cells (Stewart et al., 1967; Hobbins et ab, 1967). Subfractionation experiments have shown that spikes consisting of the two glycoproteins can be isolated from virions and these spikes retain the hemagglutination conditions under appropriate activity (Trudel et aZ.,1980). Furthermore, a recent study of electrophoretic and biological alterations produced by proteolytic treat194
RUBELLA
HEMAGGLUTININ
ment of virions with trypsin indicated that the larger of the two glycoproteins may mediate the hemagglutinin function (HoTerry and Cohen, 1980). In this report we have characterized the structural polypeptides of rubella virus by electrophoresis, and we have used an immunochemical approach coupled with biological inhibition assays to associate the hemagglutinin function with the larger of the two glycoproteins. MATERIALS
AND
METHODS
Cells and viruses. Vero cells obtained from the American Type Culture Collection were routinely cultured in Eagle’s minimum essential medium (MEM) supplemented with vitamins, nonessential amino acids, 10% heat-inactivated fetal bovine serum (GIBCO Laboratories, Grand Island, N. Y.), and 10 pug/ml of gentamycin (Schering Corp., Kenilworth, N. J.). The Therien strain of rubella virus was plaque purified twice and stocks were expanded in Vero cells, using a multiplicity of infection (m.0.i.) of 0.01 plaque-forming units (PFU) per cell. Stock virus used for these studies had a titer of 106.3PFU/ml. Growth and puri&cation
of rubella virus.
Vero cells were grown in 850~cm2 roller bottles (Corning Glasswares, Corning, N. Y.) and infected when subconfluent at an m.o.i. of 0.1 PFU per cell. Infected culture medium was collected at 48 hr postinfection, clarified, and stored at 4” and the cultures were replenished with fresh medium, Harvests were then repeated daily for four additional days. The pooled virus harvest was concentrated approximately loo-fold using constant flow-countercurrent dialysis over a 100,000 dalton membrane (Millipore, Bedford, Mass.) as previously described (Bellini, 1979). The virus was then purified by banding in two successive discontinuous 25%/50% (w/w) sucrose gradients in NET (0.1 MNaCl, 0.001 M EDTA, 0.01 M Tris, pH 7.4) by centrifugation at 95,000g for 2 hr in a Sorvall AH-627 rotor. Material sedimenting at the 25% /50% sucrose interface was collected, dialyzed against NET, and stored at -70”. Preparation of radiolabeled virus. Sub-
195
confluent Vero cell monolayers in 75-cm2 flasks (Corning) were infected at an m.o.i. of 1 PFU per cell. After 48 hr the medium was replaced with MEM completely deficient in either leucine or methionine and the cells were incubated for 1 hr at 37”. The medium was then replaced with the appropriate deficient medium containing [3H]leucine (60 Ci/mmol, Amersham, Arlington Hts., Ill.) or r5S]methionine (1300 Ci/mmol, Amersham) and supplemented with 5% MEM. [3H]Glucosamine-labeled virus was grown identically in MEM with fructose substituted for glucose as a source. [3H]Glucosamine carbohydrate (Amersham) had a specific activity of 30 Ci/mmol. Virus was labelled with [3H]mannose (30 Ci/mmol) by adding the isotope directly to MEM. After an additional 24 hr incubation at 37” the radiolabeled supernatant fluids were collected, clarified, and virus was pelletted at 95,000 g for 1 hr. The pellet was resuspended and banded on a 25%/50% sucrose gradient as described above. The purified virus was repelleted and suspended in lysate buffer [0.025MTris-HCl, pH 7.6,0.5 MNaCl, 0.001 M EDTA containing 1% Zwittergent 3-14 (Calbiochem-Berhring, La Jolla, Calif.), 0.5% sodium deoxycholic acid (Sigma Chemical Co., St. Louis, MO.), 0.1% sodium dodecyl sulfate (SDS: BDH Chemicals Ltd., Poole, England), and tosylamide-Z-phenyethyl chloromethyl ketone at 10 pg/ml (Sigma)] and stored at -20”. Immunoprecipitation and SDS-polyacrylamide gel electrophoresis (PAGE).
Immunoadsorbents were prepared by incubating 25 ~1 of the appropriate antibody with 20 ~1 of a 1:l slurry of protein A Sepharose CL-4B (Pharmacia, Piscataway, N. J.) in 0.01 M phosphate buffer pH 7.4 at 4” for 1 hr. Unadsorbed antibody was removed by washing the immunoadsorbents with lysate buffer. Lysate buffer-disrupted radiolabeled antigen was then incubated with the immunoadsorbent for 2 hr at 4”. Unbound antigen was removed by exhaustively washing the immunoadsorbents with lysate buffer. Antigen-antibody complexes were dissociated prior to electrophoresis by the addition of SDS sample buffer with or without a reducing
196
WAXHAM
AND
agent, and heating at 100” for 5 min. Polyacrylamide gel electrophoresis was performed using 10% acrylamide (acrylamide:bisacrylamide:30:0.8) resolving gels, and the discontinuous buffer system of Laemmli (1970), and radiolabeled protein bands were visualized by fluorography (Bonner and Laskey, 1974). Electrophoresis was for 3 hr at 25 mA per gel. Relative molecular weights were determined by comparison of migration with the following standards, visualized after staining with 0.5% Coomassie Blue G-250:phosphorylase B (94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100), @lactoglobulin (18,400), and lactalbumin (14,400), (Pharmacia). Two-dimensional SDS-PAGE. Two-dimensional SDS-PAGE was performed essentially as detailed by Smith and Hightower (1981). Briefly, reducing agent was omitted from electrophoresis in the first dimension, following which the gel lane was excised, equilibrated with sample buffer containing 5% 2-mercaptoethanol (Fisher Scientific, Silver Spring, Md), placed horizontally onto a second SDS gel, and electrophoresed as before. Both resolving gels contained 10% acrylamide. Peptide mapping. Peptide maps were generated from gel slices containing individual polypeptides by either partial cleavage with cyanogen bromide (Pierce Chemical Co., Rockford, Ill.) as described by Nikodem and Fresco (1979) or by limited proteolysis as described by Cleveland et al. (1979) using 0.5 pg of Staphylococcus aureus V8 protease (Miles Laboratories, Inc., Elkhart, Ind.) for each sample well. Limited proteolysis with S. aureus V8 protease of an entire lane of viral polypeptides separated by SDS-PAGE was also performed according to the method of Bordier and Crettol-Jarvinen (1979). Reduction and carboxymethylation of protein. Radiolabeled virus solubilized in
lysate buffer was reduced by the addition of 2-mercaptoethanol to a final concentration of 0.15 Mand incubated in a stoppered tube at 37” for 6 hr. The reduced proteins were then carboxymethylated by the addition of 30 pg of iodoacetamide (Fisher)
WOLINSKY
in lysate buffer and incubated at 15” for 45 min. Remaining iodoacetamide was reacted with excess 2-mercaptoethanol and the reduced carboxymethylated proteins were exhaustively dialyzed against lysate buffer for use as targets for immunoprecipitation. Hemagglutination
inhibition
assay.
Hemagglutination inhibition (HAI) assays were performed in V bottom microtiter plates (Dynatech Laboratories Inc., Alexandria, Va.) using l-day-old chick red blood cells as indicators (Liebhaber, 1970). All antisera were the IgG fraction from protein A Sepharose CL-4B chromatography (Server et al., 1982). Generation
of rubella-specijc
ant&n&es.
Four-week-old BALB/c mice were sensitized by intraperitoneal injection of 50 pg of purified virus. An additional 10 pg of virus was injected intravenously 5 days before donor spleens were harvested. The immune donor spleen cells were separated and fused with P3X63-Ag 8.653 myeloma cells essentially as described by Server et al. (1982). Anti-rubella-secreting hybrids were detected by an enzyme-linked immunoadsorbent assay (Wolinsky et al., 1982) and single cell clones selected by limit dilution. Hyperimmune rabbit anti-rubella antibodies and the cerebrospinal fluid from a patient with progressive rubella panencephalitis served as polyvalent antisera. These reagents have been characterized previously (Wolinsky et al, 1982). RESULTS
Characterization of the Polypeptides rified Virus
of Pu-
Polypeptides of virions labeled with [35S]methionine or rH]leucine were analyzed using SDS-PAGE. Similarly processed unlabeled virus samples were composed exclusively of partially disrupted and intact virions as determined by electron microscopic analysis of negatively stained preparations (data not shown). The electrophoretic patterns further demonstrate the purity of these viral preparations and show the three major structural polypeptides of rubella virus (Fig. 1). These have relative molecular weights of 62,000, a se-
RUBELLA
a
b
197
HEMAGGLUTININ
C
105kL 95kN
d
e
P78
w62 SIP54 9P47 ~38
FIG. 1. Identification of rubella virus structural polypeptides. Metabolically labeled rubella virus was disrupted under reducing conditions (lanes a-d) or nonreducing conditions (lane e) and analyzed on 10% SDS-polyacrylamide gels. Virus was labeled with [3H]leucine (lane a), [aH]mannose (lane b), [%lglueosamine (lane c), or [%]methionine in (lanes d and e). Variations in migration patterns are due to the length of the respective electrophoresis times, and do not represent qualitative differences in relative mobilities.
ries of related bands from 47,000-54,000, which by limited peptide mapping (see below) are essentially identical, and 38,000. Two minor components were also consistently detected with approximate molecular weights of 95,000 and 105,000. Selective labeling of virus with [3Hlglucosamine or [3H]mannose demonstrated that the two proteins of 62,000 daltons and the 47,00054,000 dalton complex are glycosylated as are the two minor high molecular weight species (Fig. 1, lanes b and c). Comparative analysis of densitometric tracings of Fig. 1, lanes b and c, shows that there are differences in the amounts of mannose and glucosamine incorporated into the two major glycoproteins. The 62,000 dalton glycopolypeptide contains much more mannose than glucosamine, while the 47,000-54,000 dalton complex contains more glucosamine than mannose. These two types of carbohydrate patterns are indicative of oligosaccharide side chains of the simple and complex types, respectively (Kornfeld and Kornfeld, 1980). A different electrophoretic pattern of [%]methionine-labeled polypeptides was obtained when reducing agent was omitted from the sample buffer (Fig. 1, lane e). The 38,000 dalton protein was no longer detectable and a new band was apparent at 78,000 daltons. In addition, there was a
distinct shift in the relative proportion of label from the 62,000 dalton and 47,00054,000 dalton complex regions to higher molecular weight components at 105,000 and 95,000. The 62,000 dalton glycopolypeptide migrated slightly faster under nonreducing conditions, while the 47,00054,000complex migrated similarly whether unreduced or reduced. Two-Dimensional SDS-PAGE The differences in electrophoretic patterns between unreduced and reduced rubella virus polypeptides suggested that multichain disulfide-linked polypeptides exist in the virion. These complexes and their components were specifically identified using SDS-PAGE in two dimensions, the first in the absence of reducing agent, followed by reduction and reelectrophoresis in a second dimension (Fig. 2). The two major glycopolypeptides are present as unlinked monomers, as shown by their migration near the diagonal. The 62,000 dalton glycopolypeptide migrated slightly above the diagonal, indicating a faster electrophoretic mobility when unreduced, as previously noted. This result is consistent with a more compact polypeptide structure in the absence of reducing agent, due to the presence of intrachain disulfide bonds.
198
WAXHAM
AND
The heterogeneity of the 47,000~54,000 dalton complex is again shown when analyzed in this manner. These polypeptides migrated as a diagonal group of at least two and more likely three species. However, they migrated similarly with or without disruption of disulfide bonds, which indicates a lack of extensive intrachain disulfide bonds. The 38,000 dalton polypeptide appears as a spot directly below the 78,000dalton unreduced species far off the diagonal, and identifies the 78,000 dalton species as a disulfide-linked dimer of the 38,000 dalton polypeptide. A distinct shift of radioactivity to the high-molecular-weight components at 95,000 and 105,000 daltons when reducing agent was omitted from single dimension SDS-PAGE was noted above. Analysis of these two species in two-dimensional SDS-PAGE showed that both consisted of disulfidelinked complexes of the two major glycoproteins. The 105,000 dalton species gave rise to a spot that migrated with the 62,000 dalton polypeptide and a spot at 105,000 daltons which must be a form of that polypeptide that is not altered by the reducing treatment. Therefore, the 105,000 dalton species apparently represents a dimeric form of the 62,000 dalton glycopolypeptide. The 95,000 dalton species, upon reduction, dissociated into the 62,000 dalton polypeptide and a heterogeneous spot which migrated in the same manner as the 47,00054,000 dalton complex. Thus the 95,000 dalton species apparently represents a mixed disulfide-linked dimer of the 62,000 dalton glycopolypeptide and the 47,000-54,000 dalton glycopolypeptide complex. Cyanogen Bromide and Limited Lliges tion Maps
Proteolytic
The number of unique polypeptides of rubella virus was determined by generating peptide maps for each species. Cyanogen bromide (CNBr) peptides are shown in Fig. 3. Each major polypeptide separated by SDS-PAGE was partially cleaved with CNBr and the resulting fragments were separated in a 15% SDS-polyacrylamide gel. The 62,000 dalton glycopolypeptide (lanes a and b) and 38,000 dalton
WOLINSKY
nonreduced -
38
@a \
FIG. 2. Two-dimensional SDS-PAGE. [“S]Methionine-labeled rubella virus was disrupted and electrophoresed under nonreducing conditions (top panel). A second, identically prepared gel lane was re-equilibrated in SDS sample buffer containing reducing agent, and re-electrophoresed perpendicularly into a second SDS-polyacrylamide gel (middle panel). Both gels contained 10% acrylamide and were fluorographed as described. The lower panel diagrammatically identifies the polypeptides shown in the middle fluorograph above. The monomeric forms of the glycopolypeptides are represented by the filled outlines near the diagonal and the oligomeric forms are represented by the hollow outlines below the diagonal. The reduced form of the capsid protein which also falls below the diagonal is represented by the hatched outline. The molecular weights for the reduced polypeptides are noted on the left in kilodaltons.
polypeptide (lanes c and d) yielded different peptides that were also distinct from those generated from the glycopolypeptides in the 47,000-54,000 dalton complex. That complex was subdivided into three bands and the peptide maps generated
RUBELLA
ab
HEMAGGLUTININ
cd
199
efghij
FIG. 3. Cyanogen bromide (CNBr) peptide mapping. The major polypeptides or rubella virus were first separated by SDS-PAGE identified by staining with Coomassie blue and then excised and digested with CNBr (lanes a, c, e, g, and i) in acidic acetonitrile. Parallel slices were incubated under identical conditions which lacked CNBr (lanes b, d, f, h, and j) to monitor acid hydrolysis. Lanes a and b are the 62,000 dalton glycopeptide. Lanes c and d are the 38,000 dalton polypeptide and e-j represent the three regions of the 47,000-54,000 dalton complex decreasing in molecular weight from right to left, the arrowhead indicates the peptide referred to in the text. Peptides were analyzed on 15% SDS-polyacrylamide gels
from each of these bands were similar (lanes e, g, and i). Only one peptide is seen to differ in each of these three lanes (arrowhead), and it is possibly the peptide that imparts the heterogeneous banding pattern to this protein complex in SDSpolyacrylamide gels. Whether this peptide differs because of a variation in glycosylation or is generated by some type of aberrant cleavage cannot be determined from these results. These observations were further supported by limited proteolytic maps generated with S. aureue V8 protease (Fig. 4). Using this enzyme, which has a different amino acid specificity from CNBr and with a different amino acid label ([YS]methionine) in the polypeptides, unique maps were again generated for both the 62,000 dalton glycopolypeptide and 38,000 dalton polypeptide. The components of the 47,00054,000 dalton complex were again shown to be three related glycopolypeptides similar to each other but different from the other species.
Determination of the Activity and Specificity of the Mono&ma1 Antibodies The biological activity of the monoclonal antibodies was determined by standard hemagglutination inhibition assay. At a starting protein concentration of about 1 mg/ml, both of the monoclonal antibodies used in these experiments consistently inhibited hemagglutination at dilutions of 1280 to 2560. To identify the viral polypeptide possessing the hemagglutinin function, the monoclonal antibodies were used to immunoprecipitate [35S]methionine-labeled polypeptides from disrupted virus particles. Both of the monoclonal antibodies used in these studies precipitated the 62,000 dalton glycopolypeptide (Fig. 5, lanes c and d). The small amount of the 47,000-54,000 dalton glycopolypeptide seen in immunoprecipitates of virions disrupted in the presence of reducing agent was not present in immunoprecipitates of virions that were disrupted without the reducing agent.
200
WAXHAM
gp6:
AND
gp58p47 p3S
WOLINSKY
a
b
c
d
WC. 4. Peptide mapping of $S]methionine-labeled rubella virus proteins. Two identical samples were electrophoresed under reducing conditions on 10% SDS-polyacrylamide gels as described. One gel lane was excised, transferred to a 15% acrylamide gel, overlayed with 20 pg of S. aureus V8 protease in sample buffer, and electrophoresed at 5 mA overnight. The second gel lane was held in a methanol-acetic acid solution until the digest gel was complete, then both were processed for fluorography. The one-dimensional pattern of the sample is shown at the top of the left panel; electrophoresis was from left to right. The four gel lanes on the right represent digests of the individually excised glycopolypeptides, each digested in the second dimension with 0.5 pg of S aureus V8 protease. Lane a is the 62,000 dalton glycopolypeptide, lanes b, c, and d are the three regions of the 4’7,000-54,000 dalton complex, decreasing in molecular weight from right to left.
This suggested that the 47,000~54,000dalton complex was coimmunoprecipitated as the 62,000 dalton:47,000-54,000 dalton disulfide-linked complex described above. Further proof for the specificity of the monoclonal antibodies for the 62,000 dalton glycopolypeptide was obtained by reducing and alkylating virus proteins before immunoprecipitation. The monoclonal antibodies specifically precipitated the 62,000 dalton glycopolypeptide (Fig. 6, lanes c and d). A small amount of the 38,000 dalton polypeptide was shown to nonspecifically precipitate even with Protein ASepharose alone (Figs. 5 and 6, lane e). Polyvalent antisera from a hyperimmunized rabbit and cerebrospinal fluid from a case of progressive rubella panencephalitis previously shown to recognize all of the structural proteins of the virus precipitated all of the structural proteins before or after reduction and alkylation, showing that the chemically modified proteins retained their antigenicity (Figs. 4 and 5, lanes a and b).
DISCUSSION
These studies were designed to identify the structural proteins of rubella virus and to begin to define the functional characteristics of the individual polypeptides using an immunochemical approach. Metabolically labeled, purified rubella virus when disrupted under reducing conditions and electrophoresed in SDS-PAGE is composed of three major and two minor structural components. The major components include a 62,000 dalton glycopolypeptide, a complex of three glycopolypeptides migrating at a molecular weight of 47,000~54,000, and a nonglycosylated 38,000 dalton polypeptide. By analogy with current nomenclature for other members of the togavirus family, these three species correspond to El, E2, and C, respectively. Similar profiles have been reported by Vaheri and Hovi (1972) and Payment et al. (1975) with minor differences in apparent molecular weights probably reflecting differences in the separation techniques used.
RUBELLA
a
201
HEMAGGLUTININ
b
C
d
FIG. 5. Immunological identification of rubella virus proteins. Lysates of [3SS]methionine-labeled virions were immunoprecipitated with cerebrospinal fluid from a patient with progressive rubella panencephalitis (lane a), from rabbit hyperimmunized with purified rubella virus (lane b), monoclonal antibody (lanes c and d) or protein A-Sepharose CL-4B al,ne to monitor nonspecific precipitation products (lane e), and analyzed on 10% SDS-polyacrylamide gels.
They do, however, differ substantially from results reported by Van Alstyne et al (1981) who used a different purification procedure from that used in this report and by the other investigators. The studies of Vaheri and Hovi (1972) and Payment et al. (1975) have shown that the nonglycosylated C polypeptide is not envelope associated but does cosediment with viral RNA, supporting its designation as the core protein C. Two-dimensional SDS-PAGE has now demonstrated that this capsid protein C exists as a disulfide-linked dimer in native form which migrates as a single species of 78,000 daltons under nonreducing conditions. Vaheri and Hovi (1972) also suggested that E2, their VPII, has a broad migration pattern due to large amounts of glycosylation. We were able to resolve the highly glycosylated E2 into a group of three species. The peptide maps of these species generated by cyanogen bromide cleavage appeared to differ only by a single peptide, which may represent the peptide that imparts heterogeneous migration to E2. Whether this peptide differs only in its extent of glycosylation or represents altered forms of the polypeptide backbone remains to be determined. In addition to the three major structural
polypeptides, we consistently noted two minor high-molecular-weight components of 105,000 and 95,000 daltons. When analyzed by two-dimensional (nonreducing and reducing) SDS-PAGE, the larger of the two was determined to be a dimer of El, and the second was shown to be a heterodimer of El and E2. Ho-Terry and Cohen (1980) recently reported a single glycoprotein which they called A that migrated closely to the combined molecular weights of their two major glycoproteins and seemed susceptible to reduction. They proposed that A might be a disulfide-bonded dimer of El and E2, but they made no attempt to characterize it further. Delineation of whether the homo- and heterodimers are present in the virion or are artifacts induced in the preparation of the proteins for SDS-PAGE will require further investigation. However, the presence of three distinct forms of El (El, El-El, El-E2) could well prove to be important in modulating the function of this protein. We were able to select two monoclonal antibodies which inhibit hemagglutination of rubella virus to very high titer. Immunoprecipitation patterns of radiolabeled viral proteins and analysis by SDSPAGE showed that both minor high-molecular-weight proteins and the 62,000
202
WAXHAM
a
AND WOLINSKY
b
C
d
e
w62 9P54 9P47 ~38
FIG. 6. Immunological identification of reduced and carboxymethylated rubella virus proteins. Lysates of [“Slmethionine-labeled rubella virus were immunoprecipitated with cerebrospinal fluid from a patient with progressive rubella panencephalitis (lane a), hyperimmune rabbit anti-rubella antibody (lane b), monoclonal antibody (lanes c and d), or protein A-Sepharose alone (lane e) and analyzed on 10% SDS-polyacrylamide gels.
dalton protein were recognized by these antibodies and by definition share the same antigenic site. This is because each of the proteins contains the El glycoprotein in either the dimeric (El-El), heterodimeric (El-E2), or monomeric form (El). This interpretation was supported by the demonstration that following conditions which disrupted disulfide linked proteins only the El protein was specifically immunoprecipitated. These results show that the El protein of rubella virus must include determinants involved in hemagglutinin function, which is consistent with both HoTerry and Cohen’s preliminary digestion studies with rubella virus (1980) and the finding that monoclonal antibodies generated against the El glycoprotein of Sindbis virus, a togavirus of the alphavirus subgroup, also inhibit hemagglutination (Chanas et aL, 1982). In summary, our studies show that rubella virus contains three major structural proteins, El, E2, and C, and that the hemagglutinin function is associated with the El glycoprotein. ACKNOWLEDGMENTS The authors thank Drs. David C. Merz and Alfred C. Server for critical comments during the course of
these studies and Ms. Linda Kelly for preparation of the manuscript. This work was aided by the United Cerebral Palsy Research and Educational Foundation and a Research Career Development Award, NS00443 (JSW) from the National Institutes of Neurological and Communicative Disorders and Stroke. REFERENCES BELLINI, W. J., TRUDGETT,A., and MCFARLIN, D. E. (1979). Purification of measles virus with preservation of infectivity and antigenicity. J. Gen Viral. 43, 633-639.
BONNER,W. M., and LASKEY, R. A. (1974). A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83-88.
BORDIER,C., and CRETTOL-JARVINEN, A. (1979). Peptide mapping of heterogeneous protein samples. J. Biol. Chm.
254, 2565-2567.
BARDELETTI,G., KESSLER,N., and AYMARD-HENRY, M. (1975). Morphology, biochemical analysis and neuraminidase activity of rubella virus. Arch Virol 49, 175-186. BROWN,D. T. (1980). The assembly of alphaviruses. In “The Togaviruses” (R. W. Schlesinger, ed.), pp. 473-501. Academic Press, New York. CHANAS, A. C., GOULD, E. A., CLEGG,J. C. S., and VARMA, M. G. R. (1982). Monoclonal antibodies to Sindbis virus glycoprotein El can neutralize, enhance infectivity, and independently inhibit haemagglutination or haemolysis. J. Gen. Viral. 58,3746.
RUBELLA
HEMAGGLUTININ
CLEVELAND,D. W., FISCHER,S. G., KIRSCHNER,M. W., and LAEMMLI, U. K. (1977). Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem 252, 1102-1106. FENNER, F. (1976). Classification and nomenclature 7. of viruses. Intervirology HOBBINS,T. E., STEWART,G. L., PARKMAN, P. D., HOPPS,H. E., and MEYER, H. M., JR. (1967). Biophysical and biochemical properties of rubella virus hemagglutinin. Fed Proc 26,421. HO-TERRY,L., and COHEN,A. (1980). Degradation of rubella virus envelope components. Arch ViroL 65, 1-13. KORNFELD,R., and KORNFELD,S. (1980). Structure of glycoproteins and their oligosaccharide units. In “The Biochemistry of Glycoproteins and Proteoglycans” (W. J. Lennarz, ed.), pp. l-34. Plenum, New York. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680-685. LIEBHABER, H. (1970). Measurement of rubella antibody by hemagglutination inhibition. Variables affecting rubella hemagglutination. J. Immunol 194, 818825. LIEBHABER,H., and GROSS,P. A. (1972). The structural proteins or rubella virus. Virology 47, 684693.
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tial fragmentation with CNBr. Anal Biochem. 97, 382-386.
SERVER,A. C., MERZ,D. C., WAXHAM,M. N., and WOLINSKY,J. S. (1982). Differentiation of mumps virus strains with monoclonal antibody to the HN glycoprotein. InLfect.Immun 35,179-186. SIMONS,K., GAROFF,H., and HELENIUS,A. (1980). Alphavirus proteins. In “The Togaviruses” (R. W. Schlesinger, ed.), pp. 317-333. Academic Press, New York. SMITH, G. W., and HIGHTOWER,L. E. (1981). Identification of the P proteins and other disulfide-linked and phosphorylated proteins of Newcastle Disease virus. J. ViroL 37, 256-267. STEWART,G. L., PARKMAN,P. D., HOPPS,H. E., DoucLAS, R. D., HAMILTON,J. P., and MEYER, H. M., JR. (1967). Rubella virus hemagglutination-inhibition test. New EngL J. Med 276,554-557. TRUDEL, M., RAVAORINORO,M., and PAYMENT, P. (1980). Reconstitution of rubella hemagglutinin on liposomes. Can J. MicrobioL 26,899-904. VAHERI, A., and HOVI, T. (1972). Structural proteins and subunits of rubella virus. J. ViroL 9, 10-16. VAN ALSTYNE,D., KRYSTAL,G., KETTYLS,G. D., and BOHN,E. M. (1981). The purification of rubella virus (RV) and determination of its polypeptide composition. Virologl/ 108, 491-498. WOLINSKY, J. S., WAXHAM, M. N., HESS,J. L., TOWNSEND,J. J., and BARINGER,J. R. (1982). Immunochemical features of a case of progressive rubella panencephalitis. Clin Exp. ImmunoL 48, 359-366.