17
Virus Research, 16 (1990) l-26 Elsevier
VIRUS 00568
Bluetongue virus: surface exposure of VP7 Samuel A. Lewis and Marvin J. Grubman United States Department of Agriculture, Agricultural Research Service, Plum Island Animal Disease Center, P. 0. Box 848, Greenport, NY 11944, U.S.A. (Accepted 4 December 1989)
The exposed proteins of bluetongue virus serotype 17 were determined using surface labeling and reactivity with monoclonal antibodies. Iodination of amino groups predominately labeled VP2; however, iodination of tyrosine residues labeled both VP2 and VP5, with VP7 labeled to a significantly lesser degree. To investigate the exposure of VP7 on the intact virion further, monoclonal antibodies that reacted with this protein were used. At least two antibodies, reacting with different epitopes on VP7, bound to intact Cons, as determined by adsorption of infectious particles, electron microscopic observation of antibody-bound virus, and co-sedimentation of antibody and virus. Surface iodination of viral cores was used to show that VP7 and VP3 are major exposed proteins on these particles. We conclude that a major core protein, VP7, has at least two epitopes exposed on the virus surface. Bluetongue virus; Surface protein; Monoclonal tion; Electron microscopy
antibody;
VP7; Immunoprecipita-
Introduction
Bluetongue virus (BTV), like all orbiviruses, has a protein coat that surrounds a viral core (Verwoerd et al., 1972; Spence et al., 1984). VP2 and VP5 have been identified as the major coat proteins and VP2 is primarily responsible for serotype specificity (Huismans and Erasmus, 1981; Appleton and Letchworth, 1983). These coat proteins are of interest since they are the targets for virus neutralization. Correspondence IO: M.J. Grubman, USDA-ARS-NAA, 848, Greenport, NY 11944-0848, U.S.A. 0168-1702/90/%03.50
Plum Island Animal Disease Center, P.O. Box
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
18
Huismans et al. (1987a) have demonstrated that a subunit vaccine consisting of VP2 isolated from purified virus can protect sheep from homologous virus challenge albeit only in amounts significantly greater than the quantity of this protein present in an inactivated whole virus vaccine. In addition, Letchworth and Appleton (1983) have shown that a neutralizing monoclonal antibody that reacts with VP2 can passively protect sheep from a challenge infection. Thus, it is of interest to examine the virus surface proteins carefully and to identify their exposed epitopes. VP7 is one of the major viral structural polypeptides and is the group reactive antigen (Huismans and Erasmus, 1981; Gumm and Neuman, 1982; Appleton and Letchworth, 1983). Previously, in vitro surface labeling experiments suggested that VP7 is an inner core protein (Martin et al., 1973). More recently, both immunological (Hyatt and Eaton, 1988) and indirect biochemical studies (Huismans et al., 1987b) have demonstrated that VP7 is instead exposed on the surface of core particles. Furthermore, Hyatt and Eaton (1988) have suggested that an epitope of VP7 may also be exposed on the surface of some viral particles. In the experiments reported herein, we demonstrate, by use of surface radiolabeling and reactivity with monoclonal antibodies, that at least two epitopes of VP7 are exposed on the virus surface. In addition, surface iodination of cores showed that VP7 and VP3 are major polypeptides exposed on these structures.
Materials and Methods Virus and cells
Bluetongue virus serotype 17 was originally obtained from Dr T.L. Barber, USDA, Denver, Colorado (Letchworth and Appleton, 1983). The virus was grown in baby hamster kidney (BHK-21) cells. Preliminary experiments determined that this serotype was relatively stable in 5 mM triethanolamine (TEA) or 2 mM Tris, pH 8.0 to 8.8 with or without sucrose, but was less stable in greater than 10 mM NaCl or at a lower pH. Virus purification and viral core generation
Virus was purified essentially as described by Mecham et al. (1986). Briefly, 8% polyethylene glycol (8,000) was used to precipitate virus from supernatant fluids of infected cells. Resuspended virus was treated with Triton X-100, pelleted through a 40% (wt/wt) sucrose cushion, resuspended, extracted with freon, pelleted as above, and banded on a 4-4056 (wt/wt) sucrose gradient. Material from the band was assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and shown to contain all structural proteins and genomic RNA. Viral cores and subviral particles @VP) were produced from purified virus as described by Van Dijk and Huismans (1980).
19
Gel electrophoresis and metabolic radiolabeling SDS-PAGE was performed essentially as described by Laemmli (1970). Metabolic radiolabeling of virus infected cells using [35S]methionine has been described (Devaney et al., 1988). Virus was radiolabeled with [3H]uridine as follows: BHK cells, in roller bottles, were infected with BTV at a multiplicity of 0.3 PFU/cell. Infected cells were incubated for 24 h and [ 3H]uridine (1 mCi/lO’ cells) was added for an additional 28 h. Virus was purified as above. Monoclonal antibodies Production of anti-VP7 monoclonal antibodies lB3A.6 (1B) and 8A3B.6 (8A) as well as an anti-VP2 antibody, 6C2A.4.2 (6C) has been described (Appleton and Letchworth, 1983). Control antibodies 2PD11.12.8.1 (2P) and 2FF11.11.4 (2F) recognize foot-and-mouth-disease virus antigens and have been described (Baxt et al., 1984). All monoclonal antibodies used were either IgG,, or IgG,,. Monoclonal antibodies were purified by either HPLC (HPHT column, Bio-Rad Laboratories, Richmond, CA) or ammonium sulfate precipitation (O-35% cut). Purity was verified by SDS-PAGE. Immunoprecipitation of virus [ 3H]Uridine-labeled, purified virus and cores were immunoprecipitated with monoclonal antibodies using Staphylococcus aureus (S. aureus) as described (Kessler, 1975; Grubman et al., 1984). Competition assay To determine if antibodies 1B and 8A recognized different epitopes, a solid-phase competition assay was used. Partially purified virus was diluted in phosphate buffered saline (PBS) and dried onto 96-well plates. Epitopes were blocked by addition of ascites fluid containing monoclonal antibody for 30 min and then probed with purified, iodinated monoclonal antibody for 30 min. All incubations were at 37 o C. Co-sedimentation of virus and bound antibody Iodinated monoclonal antibodies were mixed with purified virus and incubated for 3 h on ice in 5 mM TEA, pH 8.0. Unbound antibody was separated by banding virus on a 4-40% (wt/wt) sucrose gradient as described above. Electron microscopic observation of virus bound by monoclonal antibodies Antibody, Schenectady,
at 20 pg/ml in PBS, was adsorbed to formvar (Ernest F. Fullam, NY) coated 400 mesh grids for 15 min. The grids were washed,
20
incubated on drops of virus at 30 pg/ml in 5 mM TEA, pH 8.0 with 0.01% Triton X-100 for 15 min, washed three times with TEA and stained with 2% phosphotungstic acid. Preliminary titrations of antibody and virus concentrations were used to establish conditions where there was virtually no binding of virus to grids treated with control antibody. The number of bound intact vu-ions was determined by counting the particles over at least four separated grid holes on each of two grids. Micrographs were taken of particles selected at low magnification. Adsorption
of infectious virus by monoclonal
antibodies
Ten micrograms of each antibody was bound to 100 ~1 of S. aweus previously incubated with 10 pg of rabbit anti-mouse Ig (Cappel, West Chester, PA). Approximately, 0.2 ng (10,000 PFU) of virus in 100 ~1 was adsorbed 3 times with monoclonal antibodies/S. aureus. All incubations were on ice for 45 min in 5 mM TEA, pH 8.0 with 0.01% Triton X-100. Following dilution of the supernatant fluid, the number of unadsorbed infectious virus was determined by plaque assay using Mengeling-Vaughn porcine kidney cells. Iodination
of virus, cores and antibody
Purified virus (2 pg), in 5 mM TEA, pH 8.5, was iodinated with 0.1 mCi of 1251 using two procedures. To label tyrosine groups, I+ was generated using a 5-fold molar excess of N-bromosuccinimide as described by Reay (1982). After reaction with virus for 1 min, a lo-fold molar excess of I- and 4-hydroxyphenylacetic acid was added to stop the reaction. Lysine groups were labeled using S-SHPP [sulfosuccinimidyl-3-(4-hydroxyphenyl) propionate] (Thompson et al., 1987). S-SHPP was as deiodinated with a 1.5-fold molar excess of 1251 using N-bromosuccinimide scribed above. After reaction with virus for 10 min at room temperature, the reaction was quenched for 10 min with a 500-fold molar excess of lysine. Cores were iodinated as described above. Virus and cores were also iodinated using IODO-GEN which also labels tyrosine residues (Fraker and Speck, 1978). Monoclonal antibodies were iodinated using N-bromosuccinimide as described above. Following iodination, virus, cores or antibody were passed over a Sephadex G-25 column, (equilibrated with 2 mM Tris, pH 8.8) and virus or cores were banded on a 4-40% (wt/wt) sucrose gradient.
Results Anti-VP7
antibodies
that immunoprecipitate
virus
Thirteen monoclonal antibodies that recognized VP7 were screened for to immunoprecipitate [3H]uridine labeled virus and viral cores. The precipitated virus to varying degrees although all precipitated cores well shown). Two of these antibodies that precipitated virus well, 1B and
the ability antibodies (data not 8A, were
21
TABLE
1
Immunoprecipitation
of [3H]uridine-labeled
Antibody
virus and viral cores
CPM Virus
1B 8A 2PD (control) 6C (anti-VP2)
Cores
1,100 1,400 0 5,400
Input counts
6,200 3,600 0 0
12,000
WfJO
Radiolabeled virus and cores were immunoprecipitated with anti-VP7 and control antibodies (as&tic fluid) using .S. aureus, as described in Materials and Methods. Background counts were subtracted from all values.
studied in more detail (Table 1). The positive control, antibody 6C (anti-VP2), neutralized virus (Letchworth and Appleton, 1983), but did not precipitate cores (or SVP, data not shown). None of the anti-VP7 antibodies tested neutralized virus. Antibodies 1B and 8A recognized different epitopes based on competition assays. These antibodies were able to block homologous binding to antigen completely, but heterologous binding only partially (Fig. 1). Co-sedimentation
of virus and anti-VP7
antibodies
Iodinated anti-VP7 antibodies were incubated with purified virus and then banded on a sucrose gradient (Fig. 2). Anti-VP7 antibodies sedimented with virus whereas control antibody did not.
do00
b
$*__ ~;:. : EA-‘*s
4000 2000 60000
I
6A-‘2’
::\
A-A
I
\,lB
BA
0
1:20.000
1:lOOO
1:50
DILUTION
Fig. 1. Antibody
0
OF BLOCKING
1:zOOo
1:lOO
1:5
ANTIBODY
competition for epitopes. Antibodies 1B (open symbols) and 8A (solid symbols) used to block binding of ‘*‘I labeled 1B and 8A to BTV.
were
22
I
h
8A
FRACTIONS Fig. 2. Binding of monoclonal antibodies to virus. Fifty microgram of virus was mixed with iodinated antibody (200,ooO cpm, approx. 2 ng). Binding of antibody was assessed after separation of unbound antibody on a 4-40% sucrose gradient. Virus sedimented to the same position in all four gradients as determined by A,,,,. Arrows indicate the peak virus fractions. The four top fractions contain most of the antibody and are not shown on the graphs.
Electron microscopic observation of virus bound by anti-VP7 antibodies
To determine if the virions bound by antibodies appeared intact, they were specifically adsorbed to electron microscope grids using monoclonal antibodies. Antibody-specific binding of virus was demonstrated by the number of virus bound by each antibody (Table 2). The virions bound by anti-VP7 antibodies were indistinguishable from those bound by an anti-VP2 antibody (Fig. 3). Adsorption of infectious virus by anti-VP7 antibodies
Monoclonal antibodies, bound to S. aureus, were used to adsorb virus. Over 98% of infectious virus was adsorbed by anti-VP7 antibodies as well as by an anti-VP2
TABLE
2
Binding
of virus to antibody
Antibody 1B 8A 2F (control) 6C (anti-VP2)
coated
grids Number 11 94 0.8 12
f
of virus per grid hole f SD 5.6 35.0 1.3 9.1
Antibodies adsorbed to electron microscope grids were used to capture virus as described in Materials and Methods. Viis were counted in a minimum of 4 grid holes on each of 2 grids (400 mesh).
23
6C
18
0A
Fig. 3. Electron micrographs of virus bound by monoclonal antibodies shown in Table 2. Virus adsorbed by anti-VP7 antibodies (1B and 8A) was indistinguishable from that bound by anti-VP2 (6C). A minimum of four particles were photographed for each antibody none of which appeared defective.
antibody (Table 3). Trace amounts of iodinated antibodies were included to determine that all antibody remained bound to the S. aureuS during the incubations and washes.
Surface iodination Iodination of purified virus using S-SHPP labeled primarily VP2 and to a lesser extent VP5 and VP7 (Fig. 4, lane A). In contrast, iodination using I+ labeled not only VP2 but also VP5 as well as a small amount of VP7 (Fig. 4, lane B). Identical results were obtained using IODO-GEN (data not shown).
TABLE
3
Adsorption
of infectious
virus by monoclonrd
antibodies
Antibody
Plaques
1B 8A 2P (control)
0, O,l
6C (anti-VP2)
0, 030
Virus was removed from suspension number of infectious virus remaining assay performed in triplicate.
1, L2 109,117,107
by adsorption in suspension,
to antibodies that were bound after dilution, was determined
to S. aureur. The by using a plaque
24
C
D
Fig. 4. Fluorogram of iodinated BTV surface proteins. Purified virus was iodinated using A) S-SHPP or B) I+. Purified cores were iodinated using C) I+ or D) S-SHPP. [“S]Methionine labeled BTV was used as a marker for the viral proteins. The positions of VP2 and VP3 for serotype 17 have been reversed to conform to the convention that the outer capsid protein is VP2 (Grubman et al., 1983). Proteins were resolved by 10% SDS-PAGE.
Iodination of viral cores resulted in labeling of VP7 as well as VP3 and VP6 (Fig. 4, lanes C and D). An additional band of unknown origin, with a lower molecular weight than VP7, was also observed when cores were labeled with S-SHPP (Fig. 4, lane D).
Discussion Our results demonstrate that at least two epitopes of VP7 are exposed on the virion surface, and suggest that VP2 is the major exposed protein. Apparently, VP5 and VP7 have limited areas exposed to the surface. VP7 has recently been suggested to have an epitope exposed on the virions’ surface based on colloidal-gold labeling of purified virus; however, labeling of virus freshly extruded from cells and fixed onto grids was inconclusive (Hyatt and Eaton,
25
1988). More recently, it was found that fresh unfixed virus was convincingly labeled (B. Eaton, personal communication). We used anti-VP7 monoclonal antibodies to demonstrate that VP7 was exposed on all virus particles and that these particles are intact in the following three ways. (1) Co-sedimentation of virus and anti-VP7 antibodies showed that the antibodies were binding a uniform population of virus and not a subpopulation of damaged particles (such as the more stable core particles). (2) Electron microscopic observation of purified virus bound by anti-VP7 monoclonal antibodies showed that the virions were morphologically uniform, intact, and indistinguishable from virions bound by a neutralizing monoclonal antibody reactive with VP2. (3) Adsorption of essentially all infectious virus demonstrated that VP7 was present on the surface of all these particles. Furthermore, all infectious particles were adsorbed by antibody 6C which does not bind to SVP or viral cores. Iodination of virions slightly labeled VP7. The degree of labeling was insufficient to establish the surface exposure of VP7 by this method alone. More obviously, iodination of tyrosine residues on the surface of BTV resulted in labeling of VP2 as well as VP5, in agreement with Martin et al. (1973); in contrast, when lysine residues were iodinated, VP5 was labeled to a much lower extent (not appreciably more than VP7). Exposed lysine residues more accurately reflect the amount of exposed polypeptide since the e-amino groups of these residues are almost always exposed to solvent (on soluble proteins) whereas the iodinated carbons of tyrosine are rarely exposed (Lee and Richards, 1971). Additional evidence suggesting that VP5 is only minimally exposed on the virion surface is the failure of chymotrypsin, trypsin or S. aureuS V8 protease to cleave VP5 (Van Dijk and Huismans, 1980; unpublished observation) and the selective removal of VP2 at appropriate pH and salt concentrations (Huismans et al., 1987a). Apparently, VP2 is the major BTV surface protein and VP5 is a minor surface protein that has a fortuitously positioned tyrosine(s). Surface iodination of cores resulted in labeling of VP7 as well as VP3 and VP6 suggesting that these proteins are exposed on the surface of BTV cores. This is in agreement with the results of Huismans et al. (1987b) and Hyatt and Eaton (1988), but contradicts the results of Martin et al. (1973) who iodinated VP1 and VP3 on the surface of cores. A problem that we cannot address directly is the potential alteration of virus during purification (Hyatt and Eaton, 1988). Circumstantial evidence indicates that the purification used does not damage the virus since: (1) Damage to virions should allow labeling by S-SHPP of internal proteins especially VP5 (a major coat protein in which lysine residues account for more than 7% of the amino acids in the two sequenced serotypes [Purdy et al. 1986, Gould and Pritchard 1988, and Wade-Evans et al. 19881). (2) The neutralizing monoclonal antibody, 6C, recognizes the purified virus but does not recognize cores or SVP. Acknowledgements We thank Robert P. Goldsmith and Marla K. Zellner for their technical support. We also thank Mary A. Wigmore for preparation of the manuscript.
26
References Appleton, J.A. and Letchworth, G.J. (1983) Monoclonal antibody analysis of serotype-restricted and unrestricted bluetongue viral antigenic determinants. Virology 124, 286-299. Baxt, B., Morgan, D.O., Robertson, B.H. and Timpone, C.A. (1984) Epitopes on foot-and-mouth disease virus outer capsid protein VP1 involved in neutralization and cell attachment. J. Virol. 51, 298-305. Devaney, M.A., Kendall, J. and Grubman, M.J. (1988) Characterization of a non-structural phosphoprotein of two orbiviruses. Viis Res. 11, 151-164. Fraker, P.J. and Speck, J.C. (1978) Protein and cell membrane iodinations with a sparingly soluble chloroamide 1, 3, 4, 6-tetrachloro-3a, 6a-diphenylglycoluril. B&hem. Biophys. Res. Commun. 80, 849-857. Gould, A.R. and Pritchard, L.I. (1988) The complete nucleotide sequence of the outer coat protein VP5, of the Australian bluetongue virus (BTV) serotype 1, reveals conserved and non-conserved sequences. Virus Res. 9, 285-292. Grubman et al. (1983) Identification of bluetongue virus type 17 genome segments coding for polypeptides associated with virus neutralization and intergroup reactivity. Virology 131, 355-366. Grubman, M.J. et al. (1984) Biochemical map of polypeptides specified by foot-and-mouth disease virus. J. Virol. 50, 579-586. Gumm, I.D. and Newman, J.F.E. (1982) The preparation of purified bluetongue virus group antigen for use as a diagnostic reagent. Arch. Virol. 72, 83-93. Huismans, H. and Erasmus, B.J. (1981) Identification of the serotype-specific and group-specific antigens of bluetongue virus. Onderstepoort J. Vet. Res. 48, 51-58. Huismans, H., Van der Walt, N.T., Cloete, M. and Erasmus, B.J. (1987a) Isolation of a capsid protein of bluetongue virus that induces a protective immune response in sheep. Virology 157, 172-179. Huismans, H., Van Dijk, A.A. and Els, H.J. (1987b) Uncoating of parental bluetongue virus to core and subcore particles in infected L cells. Virology 157, 180-188. Hyatt, A.D. and Eaton, B.T. (1988) Ultrastructural distribution of the major capsid proteins within bluetongue virus and infected cells. J. Gen. Virol. 69, 805-815. Kessler, SW. (1975) Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J. Immunol. 115, 1617-1624. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature (London) 227, 680-685. Lee, B. and Richards, F.M. (1971) The interpretation of protein structures: estimation of static accessibility. J. Mol. Biol. 55, 379400. Letchworth, G.J. and Appleton, J.A. (1983) Passive protection of mice and sheep against bluetongue virus by a neutralizing monoclonal antibody. Infect. Immun. 39, 208-212. Martin, S.A., Pett, D.M. and Zweerink, H.J. (1973) Studies on the topography of reovirus and bluetongue virus capsid proteins. J. Virol. 12, 194-198. Mecham, J.O., Dean, V.C. and Jochim, M.M. (1986) Correlation of serotype specificity and protein structure of the five U.S. serotypes of bluetongue virus. J. Gen. Virol. 67, 2617-2624. Purdy, M.A., Ritter, G.D. and Roy, P. (1986) Nucleotide sequence of cDNA clones encoding the outer capsid protein, VP5, of bluetongue virus serotype 10. J. Gen. Virol. 67, 957-962. Reay, P. (1982) Use of N-bromosuccinimide for the iodination of proteins for radioimmunoassay. Ann. Clin. B&hem. 19, 129-133. Spence, R.P. et al. (1984) The biochemistry of orbiviruses (Brief review). Arch. Virol. 82, l-18. Thompson, J.A., Lau, A.L. and Cunningham, D.D. (1987) Selective radiolabeling of cell surface proteins to a high specific activity. Biochemistry 26, 743-750. Van Dijk, A.A. and Huismans, H. (1980) The in vitro activation and further characterization of the bluetongue virus-associated transcriptase. Virology 104, 347-356. Verwoerd, D.W. et al. (1972) Structure of the bluetongue virus capsid. J. Virol. 10, 783-794. Wade-Evans, A.M. et al. (1988) Sequence analysis and in vitro expression of a cDNA clone of genome segment 5 from bluetongue virus, serotype 1 from South Africa. Virus Res. 11, 227-240. (Received
12 September
1989; revision
received
4 December
1989)
Virus Research, 16 (1990) l-26 Elsevier
VIRUS 00568
Bluetongue virus: surface exposure of VP7 Samuel A. Lewis and Marvin J. Grubman United States Department of Agriculture, Agricultural Research Service, Plum Island Animal Disease Center, P. 0. Box 848, Greenport, NY 11944, U.S.A. (Accepted 4 December 1989)
The exposed proteins of bluetongue virus serotype 17 were determined using surface labeling and reactivity with monoclonal antibodies. Iodination of amino groups predominately labeled VP2; however, iodination of tyrosine residues labeled both VP2 and VP5, with VP7 labeled to a significantly lesser degree. To investigate the exposure of VP7 on the intact virion further, monoclonal antibodies that reacted with this protein were used. At least two antibodies, reacting with different epitopes on VP7, bound to intact Cons, as determined by adsorption of infectious particles, electron microscopic observation of antibody-bound virus, and co-sedimentation of antibody and virus. Surface iodination of viral cores was used to show that VP7 and VP3 are major exposed proteins on these particles. We conclude that a major core protein, VP7, has at least two epitopes exposed on the virus surface. Bluetongue virus; Surface protein; Monoclonal tion; Electron microscopy
antibody;
VP7; Immunoprecipita-
Introduction
Bluetongue virus (BTV), like all orbiviruses, has a protein coat that surrounds a viral core (Verwoerd et al., 1972; Spence et al., 1984). VP2 and VP5 have been identified as the major coat proteins and VP2 is primarily responsible for serotype specificity (Huismans and Erasmus, 1981; Appleton and Letchworth, 1983). These coat proteins are of interest since they are the targets for virus neutralization. Correspondence IO: M.J. Grubman, USDA-ARS-NAA, 848, Greenport, NY 11944-0848, U.S.A. 0168-1702/90/%03.50
Plum Island Animal Disease Center, P.O. Box
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
18
Huismans et al. (1987a) have demonstrated that a subunit vaccine consisting of VP2 isolated from purified virus can protect sheep from homologous virus challenge albeit only in amounts significantly greater than the quantity of this protein present in an inactivated whole virus vaccine. In addition, Letchworth and Appleton (1983) have shown that a neutralizing monoclonal antibody that reacts with VP2 can passively protect sheep from a challenge infection. Thus, it is of interest to examine the virus surface proteins carefully and to identify their exposed epitopes. VP7 is one of the major viral structural polypeptides and is the group reactive antigen (Huismans and Erasmus, 1981; Gumm and Neuman, 1982; Appleton and Letchworth, 1983). Previously, in vitro surface labeling experiments suggested that VP7 is an inner core protein (Martin et al., 1973). More recently, both immunological (Hyatt and Eaton, 1988) and indirect biochemical studies (Huismans et al., 1987b) have demonstrated that VP7 is instead exposed on the surface of core particles. Furthermore, Hyatt and Eaton (1988) have suggested that an epitope of VP7 may also be exposed on the surface of some viral particles. In the experiments reported herein, we demonstrate, by use of surface radiolabeling and reactivity with monoclonal antibodies, that at least two epitopes of VP7 are exposed on the virus surface. In addition, surface iodination of cores showed that VP7 and VP3 are major polypeptides exposed on these structures.
Materials and Methods Virus and cells
Bluetongue virus serotype 17 was originally obtained from Dr T.L. Barber, USDA, Denver, Colorado (Letchworth and Appleton, 1983). The virus was grown in baby hamster kidney (BHK-21) cells. Preliminary experiments determined that this serotype was relatively stable in 5 mM triethanolamine (TEA) or 2 mM Tris, pH 8.0 to 8.8 with or without sucrose, but was less stable in greater than 10 mM NaCl or at a lower pH. Virus purification and viral core generation
Virus was purified essentially as described by Mecham et al. (1986). Briefly, 8% polyethylene glycol (8,000) was used to precipitate virus from supernatant fluids of infected cells. Resuspended virus was treated with Triton X-100, pelleted through a 40% (wt/wt) sucrose cushion, resuspended, extracted with freon, pelleted as above, and banded on a 4-4056 (wt/wt) sucrose gradient. Material from the band was assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and shown to contain all structural proteins and genomic RNA. Viral cores and subviral particles @VP) were produced from purified virus as described by Van Dijk and Huismans (1980).
19
Gel electrophoresis and metabolic radiolabeling SDS-PAGE was performed essentially as described by Laemmli (1970). Metabolic radiolabeling of virus infected cells using [35S]methionine has been described (Devaney et al., 1988). Virus was radiolabeled with [3H]uridine as follows: BHK cells, in roller bottles, were infected with BTV at a multiplicity of 0.3 PFU/cell. Infected cells were incubated for 24 h and [ 3H]uridine (1 mCi/lO’ cells) was added for an additional 28 h. Virus was purified as above. Monoclonal antibodies Production of anti-VP7 monoclonal antibodies lB3A.6 (1B) and 8A3B.6 (8A) as well as an anti-VP2 antibody, 6C2A.4.2 (6C) has been described (Appleton and Letchworth, 1983). Control antibodies 2PD11.12.8.1 (2P) and 2FF11.11.4 (2F) recognize foot-and-mouth-disease virus antigens and have been described (Baxt et al., 1984). All monoclonal antibodies used were either IgG,, or IgG,,. Monoclonal antibodies were purified by either HPLC (HPHT column, Bio-Rad Laboratories, Richmond, CA) or ammonium sulfate precipitation (O-35% cut). Purity was verified by SDS-PAGE. Immunoprecipitation of virus [ 3H]Uridine-labeled, purified virus and cores were immunoprecipitated with monoclonal antibodies using Staphylococcus aureus (S. aureus) as described (Kessler, 1975; Grubman et al., 1984). Competition assay To determine if antibodies 1B and 8A recognized different epitopes, a solid-phase competition assay was used. Partially purified virus was diluted in phosphate buffered saline (PBS) and dried onto 96-well plates. Epitopes were blocked by addition of ascites fluid containing monoclonal antibody for 30 min and then probed with purified, iodinated monoclonal antibody for 30 min. All incubations were at 37 o C. Co-sedimentation of virus and bound antibody Iodinated monoclonal antibodies were mixed with purified virus and incubated for 3 h on ice in 5 mM TEA, pH 8.0. Unbound antibody was separated by banding virus on a 4-40% (wt/wt) sucrose gradient as described above. Electron microscopic observation of virus bound by monoclonal antibodies Antibody, Schenectady,
at 20 pg/ml in PBS, was adsorbed to formvar (Ernest F. Fullam, NY) coated 400 mesh grids for 15 min. The grids were washed,
20
incubated on drops of virus at 30 pg/ml in 5 mM TEA, pH 8.0 with 0.01% Triton X-100 for 15 min, washed three times with TEA and stained with 2% phosphotungstic acid. Preliminary titrations of antibody and virus concentrations were used to establish conditions where there was virtually no binding of virus to grids treated with control antibody. The number of bound intact vu-ions was determined by counting the particles over at least four separated grid holes on each of two grids. Micrographs were taken of particles selected at low magnification. Adsorption
of infectious virus by monoclonal
antibodies
Ten micrograms of each antibody was bound to 100 ~1 of S. aweus previously incubated with 10 pg of rabbit anti-mouse Ig (Cappel, West Chester, PA). Approximately, 0.2 ng (10,000 PFU) of virus in 100 ~1 was adsorbed 3 times with monoclonal antibodies/S. aureus. All incubations were on ice for 45 min in 5 mM TEA, pH 8.0 with 0.01% Triton X-100. Following dilution of the supernatant fluid, the number of unadsorbed infectious virus was determined by plaque assay using Mengeling-Vaughn porcine kidney cells. Iodination
of virus, cores and antibody
Purified virus (2 pg), in 5 mM TEA, pH 8.5, was iodinated with 0.1 mCi of 1251 using two procedures. To label tyrosine groups, I+ was generated using a 5-fold molar excess of N-bromosuccinimide as described by Reay (1982). After reaction with virus for 1 min, a lo-fold molar excess of I- and 4-hydroxyphenylacetic acid was added to stop the reaction. Lysine groups were labeled using S-SHPP [sulfosuccinimidyl-3-(4-hydroxyphenyl) propionate] (Thompson et al., 1987). S-SHPP was as deiodinated with a 1.5-fold molar excess of 1251 using N-bromosuccinimide scribed above. After reaction with virus for 10 min at room temperature, the reaction was quenched for 10 min with a 500-fold molar excess of lysine. Cores were iodinated as described above. Virus and cores were also iodinated using IODO-GEN which also labels tyrosine residues (Fraker and Speck, 1978). Monoclonal antibodies were iodinated using N-bromosuccinimide as described above. Following iodination, virus, cores or antibody were passed over a Sephadex G-25 column, (equilibrated with 2 mM Tris, pH 8.8) and virus or cores were banded on a 4-40% (wt/wt) sucrose gradient.
Results Anti-VP7
antibodies
that immunoprecipitate
virus
Thirteen monoclonal antibodies that recognized VP7 were screened for to immunoprecipitate [3H]uridine labeled virus and viral cores. The precipitated virus to varying degrees although all precipitated cores well shown). Two of these antibodies that precipitated virus well, 1B and
the ability antibodies (data not 8A, were
21
TABLE
1
Immunoprecipitation
of [3H]uridine-labeled
Antibody
virus and viral cores
CPM Virus
1B 8A 2PD (control) 6C (anti-VP2)
Cores
1,100 1,400 0 5,400
Input counts
6,200 3,600 0 0
12,000
WfJO
Radiolabeled virus and cores were immunoprecipitated with anti-VP7 and control antibodies (as&tic fluid) using .S. aureus, as described in Materials and Methods. Background counts were subtracted from all values.
studied in more detail (Table 1). The positive control, antibody 6C (anti-VP2), neutralized virus (Letchworth and Appleton, 1983), but did not precipitate cores (or SVP, data not shown). None of the anti-VP7 antibodies tested neutralized virus. Antibodies 1B and 8A recognized different epitopes based on competition assays. These antibodies were able to block homologous binding to antigen completely, but heterologous binding only partially (Fig. 1). Co-sedimentation
of virus and anti-VP7
antibodies
Iodinated anti-VP7 antibodies were incubated with purified virus and then banded on a sucrose gradient (Fig. 2). Anti-VP7 antibodies sedimented with virus whereas control antibody did not.
do00
b
$*__ ~;:. : EA-‘*s
4000 2000 60000
I
6A-‘2’
::\
A-A
I
\,lB
BA
0
1:20.000
1:lOOO
1:50
DILUTION
Fig. 1. Antibody
0
OF BLOCKING
1:zOOo
1:lOO
1:5
ANTIBODY
competition for epitopes. Antibodies 1B (open symbols) and 8A (solid symbols) used to block binding of ‘*‘I labeled 1B and 8A to BTV.
were
22
I
h
8A
FRACTIONS Fig. 2. Binding of monoclonal antibodies to virus. Fifty microgram of virus was mixed with iodinated antibody (200,ooO cpm, approx. 2 ng). Binding of antibody was assessed after separation of unbound antibody on a 4-40% sucrose gradient. Virus sedimented to the same position in all four gradients as determined by A,,,,. Arrows indicate the peak virus fractions. The four top fractions contain most of the antibody and are not shown on the graphs.
Electron microscopic observation of virus bound by anti-VP7 antibodies
To determine if the virions bound by antibodies appeared intact, they were specifically adsorbed to electron microscope grids using monoclonal antibodies. Antibody-specific binding of virus was demonstrated by the number of virus bound by each antibody (Table 2). The virions bound by anti-VP7 antibodies were indistinguishable from those bound by an anti-VP2 antibody (Fig. 3). Adsorption of infectious virus by anti-VP7 antibodies
Monoclonal antibodies, bound to S. aureus, were used to adsorb virus. Over 98% of infectious virus was adsorbed by anti-VP7 antibodies as well as by an anti-VP2
TABLE
2
Binding
of virus to antibody
Antibody 1B 8A 2F (control) 6C (anti-VP2)
coated
grids Number 11 94 0.8 12
f
of virus per grid hole f SD 5.6 35.0 1.3 9.1
Antibodies adsorbed to electron microscope grids were used to capture virus as described in Materials and Methods. Viis were counted in a minimum of 4 grid holes on each of 2 grids (400 mesh).
23
6C
18
0A
Fig. 3. Electron micrographs of virus bound by monoclonal antibodies shown in Table 2. Virus adsorbed by anti-VP7 antibodies (1B and 8A) was indistinguishable from that bound by anti-VP2 (6C). A minimum of four particles were photographed for each antibody none of which appeared defective.
antibody (Table 3). Trace amounts of iodinated antibodies were included to determine that all antibody remained bound to the S. aureuS during the incubations and washes.
Surface iodination Iodination of purified virus using S-SHPP labeled primarily VP2 and to a lesser extent VP5 and VP7 (Fig. 4, lane A). In contrast, iodination using I+ labeled not only VP2 but also VP5 as well as a small amount of VP7 (Fig. 4, lane B). Identical results were obtained using IODO-GEN (data not shown).
TABLE
3
Adsorption
of infectious
virus by monoclonrd
antibodies
Antibody
Plaques
1B 8A 2P (control)
0, O,l
6C (anti-VP2)
0, 030
Virus was removed from suspension number of infectious virus remaining assay performed in triplicate.
1, L2 109,117,107
by adsorption in suspension,
to antibodies that were bound after dilution, was determined
to S. aureur. The by using a plaque
24
C
D
Fig. 4. Fluorogram of iodinated BTV surface proteins. Purified virus was iodinated using A) S-SHPP or B) I+. Purified cores were iodinated using C) I+ or D) S-SHPP. [“S]Methionine labeled BTV was used as a marker for the viral proteins. The positions of VP2 and VP3 for serotype 17 have been reversed to conform to the convention that the outer capsid protein is VP2 (Grubman et al., 1983). Proteins were resolved by 10% SDS-PAGE.
Iodination of viral cores resulted in labeling of VP7 as well as VP3 and VP6 (Fig. 4, lanes C and D). An additional band of unknown origin, with a lower molecular weight than VP7, was also observed when cores were labeled with S-SHPP (Fig. 4, lane D).
Discussion Our results demonstrate that at least two epitopes of VP7 are exposed on the virion surface, and suggest that VP2 is the major exposed protein. Apparently, VP5 and VP7 have limited areas exposed to the surface. VP7 has recently been suggested to have an epitope exposed on the virions’ surface based on colloidal-gold labeling of purified virus; however, labeling of virus freshly extruded from cells and fixed onto grids was inconclusive (Hyatt and Eaton,
25
1988). More recently, it was found that fresh unfixed virus was convincingly labeled (B. Eaton, personal communication). We used anti-VP7 monoclonal antibodies to demonstrate that VP7 was exposed on all virus particles and that these particles are intact in the following three ways. (1) Co-sedimentation of virus and anti-VP7 antibodies showed that the antibodies were binding a uniform population of virus and not a subpopulation of damaged particles (such as the more stable core particles). (2) Electron microscopic observation of purified virus bound by anti-VP7 monoclonal antibodies showed that the virions were morphologically uniform, intact, and indistinguishable from virions bound by a neutralizing monoclonal antibody reactive with VP2. (3) Adsorption of essentially all infectious virus demonstrated that VP7 was present on the surface of all these particles. Furthermore, all infectious particles were adsorbed by antibody 6C which does not bind to SVP or viral cores. Iodination of virions slightly labeled VP7. The degree of labeling was insufficient to establish the surface exposure of VP7 by this method alone. More obviously, iodination of tyrosine residues on the surface of BTV resulted in labeling of VP2 as well as VP5, in agreement with Martin et al. (1973); in contrast, when lysine residues were iodinated, VP5 was labeled to a much lower extent (not appreciably more than VP7). Exposed lysine residues more accurately reflect the amount of exposed polypeptide since the e-amino groups of these residues are almost always exposed to solvent (on soluble proteins) whereas the iodinated carbons of tyrosine are rarely exposed (Lee and Richards, 1971). Additional evidence suggesting that VP5 is only minimally exposed on the virion surface is the failure of chymotrypsin, trypsin or S. aureuS V8 protease to cleave VP5 (Van Dijk and Huismans, 1980; unpublished observation) and the selective removal of VP2 at appropriate pH and salt concentrations (Huismans et al., 1987a). Apparently, VP2 is the major BTV surface protein and VP5 is a minor surface protein that has a fortuitously positioned tyrosine(s). Surface iodination of cores resulted in labeling of VP7 as well as VP3 and VP6 suggesting that these proteins are exposed on the surface of BTV cores. This is in agreement with the results of Huismans et al. (1987b) and Hyatt and Eaton (1988), but contradicts the results of Martin et al. (1973) who iodinated VP1 and VP3 on the surface of cores. A problem that we cannot address directly is the potential alteration of virus during purification (Hyatt and Eaton, 1988). Circumstantial evidence indicates that the purification used does not damage the virus since: (1) Damage to virions should allow labeling by S-SHPP of internal proteins especially VP5 (a major coat protein in which lysine residues account for more than 7% of the amino acids in the two sequenced serotypes [Purdy et al. 1986, Gould and Pritchard 1988, and Wade-Evans et al. 19881). (2) The neutralizing monoclonal antibody, 6C, recognizes the purified virus but does not recognize cores or SVP. Acknowledgements We thank Robert P. Goldsmith and Marla K. Zellner for their technical support. We also thank Mary A. Wigmore for preparation of the manuscript.
26
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received
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