Protein components of two strains of granulosis virus of the armyworm, Pseudaletia unipuncta (Lepidoptera, Noctuidae)

JOURNAL

OF

INVERTEBRATE

PATHOLOGY

32, 158-170 (1978)

Protein Components of Two Strains of Granulosis Virus of the Armyworm, Pseudaletia unipuncta (Lepidoptera, Noctuidae) T. YAMAMOTO'AND Division

of Entomology

and Parasitology,

Y.TANADA

University

of California,

Berkeley,

California

94720

Received January 26, 1978 A synergistic Hawaiian (GVH) and a nonsynergistic Oregonian (GVO) strain of a granulosis virus infect the armyworm, Pseudaletia unipuncta. The protein components ofthe enveloped virions and of the capsule (inclusion body) were compared between the two strains. When the enveloped virions of both strains were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the protein patterns were similar except for minor peaks in the higher molecular weight region. On the other hand, the capsule proteins differed between the two strains when examined with immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis. The capsule proteins of GVH were composed of two major proteins, one a structural protein and the other the protein of the synergistic factor. The capsule protein of GVO, however, had only the structural protein. The rocket immunoelectrophoresis and affinity chromatography indicated that the structural proteins of the two strains were partially dissimilar. The molecular weight of the structural protein of GVO was 29,100 f 500 and that of GVH was 28,700 ? 500. The amount of synergistic factor in a GVH capsule was about 5% of the dissolved capsule components. KEY WORDS: Pseudaletia unipuncta; granulosis virus; protein capsule; Baculovirus; virions enveloped; SDS-polyacrylamide gel electrophoresis; synergistic factor; virus structural protein; molecular weight baculovirus; rocket immunoelectrophoresis. (GV)

INTRODUCTION

The granulosis and nuclear polyhedrosis viruses infecting insects and other anthropods are classified as members of Baculovirus. Their enveloped virions (nucleocapsids) are occluded in a protein matrix which forms the inclusion (occlusion) body. The inclusion body of the granulosis virus is the capsule and that of the nuclear polyhedrosis virus is the polyhedron. Since these viruses are promising candidates for the control of insect pests, an increased number of studies have appeared on their characterization and identity, especially on the protein component of the inclusion body. The inclusion bodies dissociate under alkaline conditions into molecules of approximately 1l- 12 S (Bergold, 1947; Scott et al., 1971; Longworth et al., 1972; Harrap, 1972; Rohrmann, 1977) with molecular weights estimated at 276,000-378,000 ’ On leave from the Asahi Chemical Industry Co., Tokyo, Japan. 0022/201 l/78/0322-0158$01.00/0 Copyright All rights

0 1978 by Academic Ress, Inc. of reproduction in any form reserved.

(Bergold, 1947, 1963) and at 180,000 (Summers and Egawa, 1973). The dissociated molecules are capable of forming higher order polymers (Longworth et al., 1972; Yamamoto and Tanada, 1978). The presence of an alkaline proteinase in the inclusion body facilitates the degradation of the inclusion body protein (Yamafuji et al., 19411 1942; Kozlov et al., 1975b; Eppstein et al., 1975; Eppstein and Thoma, 1975; Summers and Smith, 1975; McCarthy and Liu, 1976; Norton and DiCapua, 1976; Rohrmann, 1977; Crawford and Kalmakoff, 1977; Yamamoto and Tanada, 1978). The basic unit of the proteins is approximately 25,000-29,000 daltons (Kozlov et al., 1975a; Eppstein et al., 1975; Summers and Smith, 1975; Rohrmann, 1977; Guelpa et al., 1977; Yamamoto and Tanada, 1978). The inclusion body proteins contain at least two antigens by immunodiffusion (Krywienczyk and Bergold, 1961; Tanada and Watanabe, 1971; Longworth et al., 1972; Scott and Young, 1973; Chang and Tanada, 158

PROTEIN

COMPONENTS

OF TWO

1978). Those of a number of nuclear polyhedrosis viruses share two antigens and possess one unique antigen by immunodiffusion (Krywienczyk and Bergold, l%l). The am~ywor~~~, Pseudaletia unipuncta, is susceptible to two strains of a granulosis virus (GV), the synergistic Hawaiian (GVH) and the nonsynergistic Oregonian strain (GVO) (Tanada and Hukuhara, 1968). In addition to the differences in their synergistic properties, the strains differ in the size of their virions . The GVH strain has an enzyme like factor that enhances the infection of several baculoviruses (Hara et al., 1976). This factor is found in the proteinaceous matrix of the capsule (Tanada et al., 1973; Hara et al., 1976) and has a molecular weight of about 126,000. We have further studied the two strains by using biochemical and serological techniques to differentiate the protein components of the enveloped virions and of the capsules. MATERIALS

AND METHODS

Virus propagation. The GV and nuclear polyhedrosis virus (NPV) were propagated in armyworm larvae obtained from a stock colony which had been reared for several generations in the laboratory. The colony had been started from field-collected adults. The larvae were usually infected in the fourth or fifth instar, and the virus was collected from larvae in the sixth instar when the signs and symptoms were most pronounced (Hara et al., 1976). Preparation of virus components. The inclusion bodies (capsules and polyhedra) were purified according to the method described by Hara et al. (1976). In order to isolate the inclusion body components, the inclusion bodies were first dissolved in 0.02 N NaOH, which took less than 1 min, and then dialyzed against 10 mM Tris-HCl buffer (pH 8). The dialysates were centrifuged at 30,OOOg for 15 min, and the supematants and sediments were separated. The supematants of GV and NPV, which contained the inclusion body components, were analyzed.

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The sediment containing the enveloped virions of NPV was discarded and that of GV was resuspended in 0.02 N NaOH and centrifuged at 30,OOOg for 15 min. This centrifugation was repeated two times by resuspending the sediment with fresh NaOH at each time. The final precipitate was suspended in water and washed three times by differential centrifugations at 6000 and 30,OOOg for 15 min. The enveloped virions of the two GVs were filtered through membrane filters in a 25-mm Swinnex holder containing a glass-fiber prefilter (Millipore Filter Corp., Bedford, Mass.). The membrane filter used in the holder for GVH filtration had a pore size of 0.45 pm and that for the GVO had a pore size of 0.22 pm. The Swinnex holder was attached to a plastic syringe which was operated by hand pressure. Each virus was filtered three times. The purified viruses were collected by filtering GVH on a 0.22~pm pore size filter and GVO on a 0. l-pm filter. The viruses on the filter membrane were rinsed with distilled water from a pipet and collected. The aqueous virus suspensions were centrifuged at 30,OOOg for 15 min and the virus sediments were resuspended in a minimum amount of water for analysis. The synergistic factor was obtained from the capsule components by the method described by Hara et al. (1976). The protein concentrations of the viruscomponent preparation were determined by the method of Lowry et al. (1951) with bovine serum albumin as a standard. Antibody preparation. Virgin females of the New Zealand White rabbits were used for antibody production. The capsule components (4 mg as protein) and the synergistic factor (2 mg as protein) were each mixed with equal volumes of complete Freund’s adjuvant and inoculated into separate rabbits. The initial inoculation of antigen was at two different sites, subcutaneous and intramuscular. A week later, 2 mg of the antigen was inoculated intravenously and this inoculation was repeated at weekly intervals for 3 weeks. A week after the final inocula-

160

YAMAMOTO

tion, the blood was collected by heart puncture and centrifuged at 1,500g for 15 min, and the serum was collected. Agarose gel electrophoresis. A 10 x 10 cm glass plate supported the agarose gel. There were two sections in the gel: One contained 50 mM Tris-acetate buffer, pH 8.2, and occupied 8 x 10 cm of the glass plate; the other contained 10 mM Tris-acetate buffer and occupied 2 x 10 cm of the plate. Six holes were made at the base of the 10 mM buffer section. The holes were filled with 3 ~1 of either dissolved capsule components or a solution of the synergistic factor. Electrophoresis was run at constant 100 V. After the tracking dye (bromphenol blue) had reached the end of the plate facing the positive electrode, the electrophoresis was stopped. The gel was cut into six strips and transferred to a different glass plate for immunoelectrophoresis or to a polyacrylamide gel for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Zmmunoelectrophoresis. Two methods of immunoelectrophoresis, two-dimensional (Clarke and Freeman, 1966) and rocket (Laurell, 1972), were carried out in 1% agarose gel containing 50 mM Tris-acetate buffer, pH 8.2, and antiserum. The electrophoresis was conducted at 50 V for 12 hr at 4°C. SDS-Polyacrylamide gel electrophoresis (SDS-PAGE). An apparatus similar to that

described by Reid and Bieleski (1968) was used except that one plate was frosted glass. For the molecular weight determinations, 10% gel, containing 50 mM T&-acetate buffer, pH 8.2, was solidified between two glass plates without a stacking gel. The same buffer was used as the electrode buffer. For the examinations of the components of the enveloped virions, the gel slab consisted of two sections: the stacking 5% gel and the resolving 10% gel. The resolving gel contained 0.1% SDS and 100 mM Tris-HCl buffer, pH 8.8. In the stacking gel, the concentration of the buffer was reduced to 30 mM. The electrode buffer wells were filled with 100 mM Tris-acetate buffer, pH 8.2,

AND

TANADA

containing 0.1% SDS. After electrophoresis, the gel was stained with Coomassie brilliant blue R-250 (0.25% dye in 10% TCA and 30% methanol), rinsed in a solution of 7% acetic acid and 30% methanol, and traced with a densitometer at 550 nm. Throughout these procedures, the gel remained on the frosted glass plate to prevent shrinking or swelling. The second-dimensional electrophoresis was performed as follows. The agarose gel strip was dipped for 5 min in 10 mM Tris-HCl buffer, pH 8, containing 1% SDS and then laid on a 10% polyacrylamide gel containing 10 mM Tris-HCl buffer, pH 8.8, and 0.1% SDS. The electrophoresis was run at 30 mA for 2 hr. Ajjinity chromatography. The method described by Cuatrecasas (1970) was used to prepare the capsule component-conjugated agarose gel. The gel (Bio-Gel A-50m from Bio-Rad Laboratories, Richmond, Calif.) was activated with BrCN. At least 90% of the protein added as a ligand was bound to the gel. The ligand-conjugated agarose gel was packed in a column (2.3 x 5 cm) and washed with 0.5 M NaCl and 0.01 N NaOH before use. The serum was fractionated with 50% ammonium sulfate. It was applied on the column that had been equilibrated with 10 mM Tris-HCl buffer,pH 8. The column was washed with 0.5 M NaCl buffered with 10 mM Tris-HCl, pH 8. The antisera were eluted with 0.01 N NaOH and the elute was immediately neutralized with 0.1 vol of 1 M Tris-HCl buffer. pH 7. RESULTS Enveloped

Virions

The electron micrographs (Fig. 1) of the enveloped vu-ions isolated from the capsule proteins showed no apparent adverse effect caused by the 0.02 N NaOH, pH about 12. The enveloped vii-ions of GVO passed through a 0.22~pm membrane filter, whereas those of GVH were retained by this filter. The filtration method not only reduced the process of purification, but also the envel-

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OF TWO GRANULOSIS

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161

FIG. 1. Electron micrographs of the enveloped virions of two strains of granulosis virus. The enveloped virions were stained with 2% uracyl acetate for 1 min. (A) Hawaiian strain; (B) Oregonian strain. Each bar indicates 100 nm.

oped virions obtained by filtration displayed fewer numbers of bands with SDS-PAGE (Fig. 2) as compared to those prepared by differential centrifugation (unpubl.). When the patterns of the enveloped virions of GVH and GVO were compared, they were similar (Fig. 2). Both had a major band in the smaller molecular weight region and several bands in the larger molecular weight region. The only apparent difference occurred in a small peak at 60,000 daltons for GVH and at 70,000 daltons for GVO. Furthermore, when the capsules were washed with 1 M NaCl prior to dissolution with NaOH, the patterns were still similar. The patterns, however, were slightly different when the enveloped virions were purified by sucrose density centrifugation (lo-80%, w/v) rather than by filtration. The volumes of the minor bands were reduced, possibly

by the breakage of the envelopes when the virions were transferred for dialysis from the dense sucrose solution to water. This breakage was confirmed by the observation of many naked virions under an electron microscope. Agarose Gel Electrophoresis The electrophoretic patterns of the capsule components are shown in Figure 3. There were two preparations of the capsules of each virus strain: one treated with 1 M NaCl to remove the proteinase associated with the virus and the other untreated and acted upon by the proteinase. The GVO capsule components produced one major band in the presence and absence of the proteinase, whereas the GVH produced two bands in the presence and one band in the absence of the enzyme.

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60

60

50

AND TANADA

40 MW

30 x 10-3

20

FIG. 2. Patterns of SDS-PAGE of the enveloped virions of two strains of granulosis virus. The resolving gel (10% polyacrylamide) was 10 x 12 cm and 1 mm thick. The stacking gel (5% polyacrylamide, 2 cm in height) had 14 sample slots and was solidified on the resolving gel. Twenty microliters ofenveloped virions suspended in 50 mM Tris-HCl buffer, pH 8, was mixed with 5 ~1 of 10% SDS containing 5% /3-mercaptoethanol and heated at 100°C for 1 min. The sample was then mixed with 5 ~1 of 50% glycerol, and 3 ~1 was charged on the stacking gel. Electrophoresis was performed at 30 mA for 2 hr. The gel was stained with Coomassie brilliant blue R-250 (0.25% in a mixture of 10% TCA and 30% methanol) and then rinsed and traced with a densitometer (Helena Quick Scan Fhn-Vis) equipped with a 550-nm filter (Kodak gelatin filter type 99). GVO, Oregonian strain; GVH, synergistic Hawaiian strain.

Two-Dimensional

Electrophoresis

The gel was cut into strips after the agarose gel electrophoresis, and the gel strips were transferred to another plate on which had been cast an agarose gel containing serum. The capsular components of GVH, from which the proteinase had not been removed with NaCl, produced two independent precipitin lines with the antiserum of the GVH capsule components (Fig. 4A). When free of proteinase, the GVH components, even though they formed only one major peak with agarose gel electrophoresis, also produced two independent precipitin lines with the antiserum of the GVH capsule components (Fig. 4C). The two lines, however, were much broader and shorter than those produced by the capsule components containing active proteinase. Only the smaller of the two precipitin lines reacted with the antiserum of the synergistic factor (Fig. 4E). Thus the band migrating the shorter distance on the agarose gel electrophoresis was produced by the synergistic factor. This was further confirmed by another twodimensional electrophoresis in which SDSPAGE was the second-dimensional electro-

GVO

0

2

4 Migratlon

GVH

0 2 Distance

4 (cm)

FIG. 3. Patterns of agarose gel electrophoresis of the capsule components of two strains of granulosis virus in the presence and absence of a proteinase. A glass plate (10 x 10 cm) was covered with two sections of 1% agarose gel: one with 50 mM Tris-acetate buffer, pH 8.2, in 8 x 10 cm ofthe plate, and the other with 10 mM Tris-acetate buffer,pH 8.2, in 2 x 10 cm of the plate. Both gels were 1 mm thick. Holes were punched in the smaller section and each hole was filled with 3 ~1 of 2 mg/ml antigen. After electrophoresis at 100 V for 1 hr, the gel was soaked in 0.8% NaCI for 24 hr, stained with Coomassie brilliant blue R-250, and traced as in Fig. 2.

PROTEIN

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OF TWO GRANULOSIS

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163

FIG. 4. Two-dimensional immunoelectrophoresis of capsule components of two strains of granulosis virus. The agarose gel strip was transferred to another glass plate which was covered with 1% agarose gel (8 x 10 cm of the plate) containing serum and 50 mM Tris-acetate buffer, pH 8.2. Electrophoresis was performed at 50 V for 2 hr. (A) GVH strain with proteinase; (B) GVO strain with proteinase; (C) GVH strain without proteinase; (D) GVO strain without proteinase. In A-D, the serum was anti-GVH at l~Ycm2. (E) GVH strain with proteinase, and the serum was anti-synergistic factor at 1.5 pYcm2.

phoresis. The short migrating band was a large molecule and was apparently the synergistic factor (Fig. 5). The capsule components of GVO reacted weakly with the antiserum of the GVH capsule components. Both the proteinasepresent and -absent components formed only one precipitin line (Figs. 4B, D).

Rocket Immunoelectrophoresis

The two-dimensional immunoelectrophoresis showed that the GVO capsule components produced weaker precipitin lines than those of the GVH. Since this profile of the second dimension depends on the sharpness of the bands in the first dimension,

164

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AND TANADA

FIG. 5. Two-dimensional SDS-PAGE of capsule components of the GVH strain. AtIer agarose gel electrophoresis, the gel was cut into a strip which was dipped for 5 min in 10 mM Tris-HCl buffer, pH 8, containing 1% SDS. The gel strip was then laid on 10% polyacrylamide gel (10 x 12 cm), and electrophoresis was carried out at 30 mA for 2 hr. The polyacrylamide gel was stained with Coomassie brilliant blue R-250 and rinsed as in Fig. 2.

comparison of the two GV strains was further investigated with rocket immunoelectrophoresis. The capsule components of the two strains were compared antigenically with the antiserum of the capsule proteins of GVH. The test also included the synergistic factor and the polyhedron components of a nuclear polyhedrosis virus (NPV) of the armyworm. The precipitin lines produced by the four antigens r?re given in Figure 6. The NPV produced little, if any, precipitin lines. GVO produced precipitin lines almost twice as high and weaker than those of GVH. The GVH produced two precipitin lines; the shorter line was formed by the synergistic factor which reacted with its antibody in the GVH antiserum. On the basis of a comparison between the precipitin lines produced by a given volume of the synergistic factor against a given volume of capsule compon-

ents, the amount of the synergistic factor in a GVH capsule was calculated as 5% of the total dissolved capsule components. The effect of proteinase on the capsule components, expressed as precipitin lines, is given in Figure 7A. The lines were shorter and more distinct in the absence of than in the presence of this enzyme. The presence or absence of proteinase in the GVH capsule components did not alter the height and sharpness of the precipitin lines produced by the synergistic factor (Fig. 7B). Aflinity

Chromatography

The antiserum of the GVH capsule components was fractionated by affinity chromatography for immunological cross-reactions between GVH and GVO. The antibodies of the GVH capsule components were isolated from the GVH capsule-conjugated agarose

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FIG. 6. Rocket immunoelectrophoresis of the inclusion body components of a nuclear polyhedrosis and two strains of a granulosis virus. The capsule and the polyhedra were dissolved in 0.02 N NaOH and dialyzed against 10 mM Tris-HCl buffer,pH 8. After centritiigation at 30,OOOgfor 15 min, the supematant (3 ~1) was charged on an agarose gel containing serum at 1.5 p&cm*. The synergistic factor (3 ~1) isolated from capsules was also charged on the gel. Electrophoresis was conducted at 50 V for 12 hr. The concentrations of antigens are: (Sl) 0.107 mg/ml; (S2) 0.175 mg/ml; (S3) 0.313 mg/ml; (Hl, 01, Nl) 1 mg/ml; (H2, 02, N2) 2 mg/ml; (H3, 03, N3) 3 mg/ml. S, Synergistic factor; H, granulosis virus, synergistic Hawaiian strain; 0, granulosis virus, Oregonian strain; N, nuclear polyhedrosis virus.

gel. Three peaks were eluted from the column (Fig. 8A). The third peak was eluted with 0.01 N NaOH and neutralized with 0.1 buffer, pH 7. It was exvol of 1 M Tris-HCl amined by rocket immunoelectrophoresis, and the pattern showed that this peak had two types of antibodies, one against the structural proteins of the capsule and the other against the synergistic factor (Fig. 9A). The afiinity chromatography of the antibodies of the GVH capsule proteins with the GVO capsule-conjugated agarose gel produced the elution protile shown in Fiie 8B. The first peak which had not reacted with the column was examined by rocket immunoelectrophoresis. The volume of the antibody against the structural protein was reduced but had not been completely removed from the capsule components of GVO (Fig. 9B). Even after additional repeated chromatography with GVO capsule-

conjugated agarose gel, the antibody against the structural protein could not be further removed. Molecular

Weight of Structural

Protein

The molecular weights of the structural proteins in the capsules of GVH and GVO were determined by SDS-PAGE. The proteinase associated with the capsule was removed by washing the intact capsules with 1 M NaCl. After dissolution with 0.02 N NaOH, the capsule components were heated in 2% SDS containing 1% &mercaptoethanol at 100°C for 3 min. They were then analyzed by SDS-PAGE. There was one band in the electrophoretic pattern of GVO and two bands in that of GVH (Fig. 10). The molecular weight of the structural proteins was calculated as 29,100 + 500 for GVO and as 28,700 + 500 for GVH (Fig.

166

YAMAMOTO

P+l

p+2

P-1

P-2

AND TANADA

P+l

P+52

P-l

P-2

FIG. 7. Comparison of the antigenicity of capsule components of the granulosis virus, Hawaiian strain, in the absence and presence of a proteinase. The capsules were dissolved in 0.02 N NaOH and dialyzed against 10 mM Tris-HCl buffer,pH 8. For the preparation of the nonproteinase treatment, the capsules were first washed with 1 M NaCl to remove the proteinase before dissolution. The solution was centrifuged at 30,OOOg for 15 mitt, and the supematant (3 ~1) was charged on an agarose gel containing the antiserum or antibodies. The conditions for electrophoresis were the same as in Fig. 6. (A) Anti-GVH serum at 1 pYcm*; (B) antibodies (6 pg/cm2) against GVH. P+, Proteinase present; P-, proteinase absent: P + 1 and P - 1, antigen at 1 mg/ml; P + 2 and P - 2, antigen at 2 mg/ml.

10). These calculations were based on the results of four separate tests. DISCUSSION

The liberation of the enveloped virions by treatment with 0.02 N NaOH caused no apparent disruption to the envelopes, which appeared undistorted and free of inclusion body proteins when observed under an electron microscope. Moreover, SDS-PAGE analysis indicated the absence of inclusion body proteins in the preparation of the enveloped virions and the absence of envelope proteins in the inclusion body preparations. The purification of the liberated enveloped virions by filtration results in less distortion or disruption of the envelopes than purification by sucrose gradient centrifugation. The purified enveloped virions of GVH and GVO produce similar patterns when analyzed by SDS-PAGE. They differ in two minor peaks, one of 60,000 daltons for GVH and the other of 70,000 daltons for GVO. The

significance of these minor peaks awaits further studies. The molecular weights of the basic subunit protein of the inclusion bodies have been reported as 28,000 (Eppstein et al., 1975; Kozlov et al., 1975a, b; Summers and Smith, 1975, 1975/1976; Yamamoto and Tanada, 1978) and 26,000 (Rohrmann, 1977). The molecular weight of GVH is 28,700 ? 500 and that of GVO is 29,100 f 500. This difference is slight but we believe it to be significant. The difference, however, should be confirmed by amino acid analysis and peptide mapping. The GVH and GVO capsule components have one group-specific antigen. Norton and DiCapua (1976) also have reported one group-specific antigen in the polyhedra of several nuclear polyhedrosis viruses. The GVH, however, has a second antigen produced by the synergistic factor (Tanada and Watanabe, 1971; Chang and Tanada, 1978). An endogenous proteinase facilitates the dissolution of inclusion bodies of baculovi-

‘In

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OF TWO GRANULOSIS

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167

the results of a previous study (Yamamoto and Tanada, 1978). The synergistic factor forms about 5% of the dissolved capsule components. According to the results of rocket and two‘.5 dimensional immunoelectrophoresis, the capsular components (structural proteins) of the two strains, in the presence and absence of proteinase, differ from each other regardless of the enzyme. This difference, 0 120 240 360 0 however, may be caused by the differently Elution Volume (ml) aggregated antigens. Highly aggregated proFIG. 8. Affinity chromatography for the fractionation teins are more easily precipitated by the of antibodies of two strains of granulosis virus. Serum antibody than dissociated ones. The aggrega(75 ml) was mixed with 75 ml of saturated ammonium tion of inclusion body components has been sulfate. The precipitate was collected by centrifugation and dissolved in 10 mM Tris-HCl buffer, pH 8. The observed also by others (Summers and solution was dialyzed against 10 mM Tris-HCI buffer, Egawa, 1973; Summers and Smith, 1975; pH 8, and 12 ml of solution containing 53 mg/ml protein Bppstein et al., 1975; Yamamoto and was collected and applied to aGVH capsule-conjugated Tanada, 1978). In order to avoid such agagarose gel column. Capsule components of GVH (100 gregation problems, we resorted to affinity mg of protein) were conjugated on BrCi+activated to study the immunological agarose gel (25 ml). The column was washed with 10 chromatography mM Tris-HCl buffer, pH 8, followed by 0.5 M NaCl relationship of the antigens in GVO and buffered with 10 mM Tris-HCl,pH 8. Antibodies were GVH. The advantages of affinity chromatogeluted from the column with 0.01 N NaOH and neutralraphy are that it is affected neither by the ized with 0.1 vol of 1 M Tris-HCl buffer, pH 7. The aggregation of the antigens nor by the ratio elution profile is shown in A. Elute was concentrated in cellophane tubing and tested by rocket immunoelectroof antigen to antibody. In affinity chromaphoresis. Four milliliters of concentrated solution, tography, the antigen is immobilized by containing 5.18 mg/ml protein, was applied on a GVO covalent bonds onto the agarose gel matrix capsule-conjugated agarose gel column. Chromatogand may trap the antibody with only a single raphy was performed as described for the GVH site of attachment rather than forming the capsule-conjugated agarose gel column. The first peak shown in B was concentrated and tested by rocket antigen-antibody complex involving multiimmunoelectrophoresis. ple binding sites. Thus, the ratio of antigen to antibody does not play an important role. rns. The proteinase, however, does not Inasmuch as the afImity chromatographic destroy the group-specific antigen of the and rocket immunoelectrophoretic methods polyhedron proteins (Norton and DiCapua, used in our study detected differences in 1976; Crawford and Kalmakoff, 1977) and of the structural proteins of the inclusion body the capsule proteins (Chang and Tanada, even in two strains of a virus in a common 1978). We also observed that the grouphost, these methods may serve to identify specific antigen of GV is not completely insect viruses. However, differences in the destroyed by the proteinase. When the cap- enveloped virions may not be detectable by sule component, however, is broken down these methods. Bergold (1958) has discussed the possible by this enzyme, there is an alteration in the origin and formation of the inclusion body molecular structure of the antigenic comproteins of baculoviruses, i.e., whether they ponent because the precipitin lines in rocket immunoelectrophoresis become longer and are host- or virus-directed proteins. The less distinct than those of the antigens unaf- presence of the synergistic factor in the capfected by the enzyme. The synergistic factor sule proteins and the dissimilarity between is unatfected by the enzyme. This confirms the capsular proteins of the two GV strains

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FIG. 9. Rocket immunoelectrophoresis conducted with GVH capsule components and the antibodies fractionated by affinity chromatography. Agarose gel (1%) contained 50 mM Tris-acetate buffer,pH 8.2, and 6 &cm* antibodies. Holes were filled with 3 ~1 of capsule components of GVH. Electrophoresis was conducted at 50 V for 12 hr. Concentrations of antigens are given in milligrams per milliliters. In A, the third peak shown in Fig. 8A, and in B, the first peak shown in Fig. 8B, were used as antibodies. The antigen concentrations in A and B were 0.1,0.5, 1, and 5 m&l, Born the left to the right of the figure.

100

60

40

30

20

IO

MW x 1O-3 FIG. 10. Patterns of SDS-PAGE of structural proteins of capsules of two granulosis virus strains. Fifty microliters of 3 mg/ml dissolved capsules, which had been treated with 1 M NaCl to remove the proteinase, was mixed with 12.5 ~1 of 10% SDS containing 5% &mercaptoethanol. The mixture was heated at 100°C for 3 min and then 12.5 ~1 of 50% glycerol was added. The final mixture of 3 ~1 was applied on a 10% polyacrylamide gel which contained 0.1% SDS and 50 mM Tris-acetate buffer, pH 8.2. After electrophoresis at 100 V for 2 hr, the gel was stained with Coomassie brilliant blue R-250, rinsed, and traced with a densitometer as described in Fig. 2. During the entire process, the gel was supported on a frosted glass plate. The following proteins were used as molecular weight references: BSA, bovine serum albumin, 68,000; EA, egg albumin, 43,000; CA, carbonic anhydrase from bovine erythrocytes, 29,000; RNase, ribonuclease from bovine pancreas, 13,700; Cyt C, cytochrome c from horse heart, 12,400; GVO, Oregonian strain; GVH, synergistic Hawaiian strain.

PROTEIN

COMPONENTS

OF TWO GRANULOSIS

within the same host support the hypothesis that the formation of the inclusion body proteins is controlled by the virus rather than the host insect. ACKNOWLEDGMENTS We wish to thank Roberta M. Hess, Esther M. Omi, and Ralph J. Michels for their technical assistance. A portion of this study was supported by a grant from the National Science Foundation.

REFERENCES BERGOLD, G. H. 1947. Die Isolierung des PolyederVirus und die Natur der Polyeder. Z. Naturfirsch., 2b, 122-143. BERGOLD, G. H. 1958. Viruses of insects. In “Handbuch der Virusforschung” (C. Hallauer and K. F. Meyer, eds.), Vol. 4, pp. 60-142. Springer, Vienna. BERGOLD, G. H. 1963. The nature of nuclearpolyhedrosis viruses. In “Insect Pathology, An Advanced Treatise” (E. A. Steinhaus, ed.), Vol. 1 pp. 413-456. Academic Press, Inc. New York. CHANG, P. -M., AND TANADA, Y. 1978. Serological study on the transmission of a granulosis virus of the armyworm, Pseudaletia unipuncta (Lepidoptera: Noctuidae), to other lepidopterous species. J. Znvertebr. Pathol., 31, 106- 117. CLARKE, H. G. M., AND FREEMAN, T. 1966. A quantitative immuno-electrophoresis method (Laurell electrophoresis). Protides Biol. Fluids, Proc. Colloq. 14, 503-509. CRAWFORD, A. M., AND KALMAKOFF, J. 1977. Effect of alkaline protease on the antigenic nature of Wiseana nuclear polyhedrosis virus polyhedron protein. J. Viral., 24, 412-415. CIJATRECASAS, P. 1970. Protein purilication by affmity chromatography. Derivatizations of agarose and polyacrylamide beads. J. Biol. Chem., 245, 3059.3865. EP~~TEIN, D. A., AND THOMA, J. A. 1975. Alkaline protease associated with the matrix protein of a virus infecting the cabbage looper. Biochem. Biophys. Res.

Commun.,

62, 478-484.

EP~~TEIN, D. A., THOMA, J. A., SCOTT, H. A., AND YOUNG III, S. Y. 1975. Degradation of matrix protein from a nuclear-polyhedrosis virus of Trichoplusia ni by an endogenous protease. Virology, 67, 591-594. GUELPA,

B.,

BERGOIN,

M.,

AND CROIZIER,

G.

1977.

La proteine d’inclusion et les prottines du virion du Baculovirus du dip&e Tipula paludosa (Meigen). C. R. Acad.

Sci. (Paris),

284, 779-782.

HARA, S., TANADA, Y., AND OMI, E. M. 1976. Isolation and characterization of a synergistic enzyme from the capsule of a granulosis virus of the armyWOIYII,

Pseudaletia

27, 115- 124.

unipuncta.

J. Znvertebr.

Pathol.,

VIRUSES

169

HARRAP, K. A. 1972. The structure of nuclear polyhedrosis viruses. I. The inclusion body. Virology, 50, 114-123. KOZLOV, E. A., LEVITINA, T. L., SIDOROVA, N. M., RADAVSKI, Yu. L., AND SEREBRYANI, S. B. 1975a. Comparative chemical studies of the polyhedral proteins of the nuclear polyhedrosis viruses of Bombyx mori and Galleria mellonella. J. Znvertebr. Pathol., 25, 103- 107. KOZLOV, E. A., SIDOROVA, N. M., AND SEREBRYANI, S. B. 1975b. Proteolytic cleavage of polyhedral protein during dissolution of inclusion bodies of nuclear polyhedrosis ‘viruses of Bombyx mori and Galleria mellonella under alkaline conditions. J. Znvertebr. Pathol., 25, 97- 101. KRYWIENCZYK, J., AND BERGOLD, G. H. l%l. Serological studies of inclusion body proteins by agar diffusion technique. J. Znvertebr. Pathol., 3, 15-28. LAURELL, C. -B. 1972. Electroimmuno assay. Stand. J. Clin. Lab. Invest., 29(Suppl. 124), 21-37. LONGWORTH, J. F., ROBERTSON, J. S., AND PAYNE, C. C. 1972. The purification and properties of inclusion body protein of the granulosis virus of Pieris brassicae. J. Znvertebr. Pathol., 19, 42-50. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275.

W. J., AND LIU, S. -Y. 1976. Electrophoretie and serological characterization of Porthetria dispar polyhedron protein. J. Znvertebr. Pathol., 28, 57-65. NORTON, P. W., AND DICAPUA, R. A. 1976. Nuclear polyhedrosis virus group antigen: Resistance to endogenous protease degradation. In “Proceedings, 1st International Colloquium on Invertebrate Pathology: 9th Annual Meeting, Society for Invertebrate Pathology,” pp. 325-326. Queen’s Univ. Kingston, Canada. REID, M. S., AND BIELESKI, R. L. 1968. A simple apparatus for vertical flat-sheet polyacrylamide gel electrophoresis. Anal. Biochem., 22, 374-381. ROHRMANN, G. F. 1977. Characterization of Npolybedrin of two baculovirus strains pathogenic for Orgyia pseudotsugata. Biochemistry, 16, 16311634. SCOTT, H. A., AND YOUNG, S. Y. 1973. Antigens associated with a nuclear polyhedrosis virus from cabbage looper larvae. J. Znvertebr. Pathol., 21, 315317. SCOTT, H. A., YOUNG III, S. Y., AND MCMASTERS, J. A. 1971. Isolation and some properties of components of nuclear polyhedra from the cabbage looper, Trichoplusia ni. J. Znvertebr. Pathol., 18, 177-182. SUMMERS, M. D., AND EGAWA, K. 1973. Physical and chemical properties of Trichoplusia ni granulosis virus granulin. J. Virol., 12, 1092-1103. SUMMERS, M. D., AND SMITH, G. 1975. Trichoplusia

MCCARTHY,

170

YAMAMOTO

ni granulosis virus granulin: A phenol soluble, phosphorylated protein. J. Viral., 16, 1108- 1116. SUMMERS, M. D., AND SMITH, G. E. 1975/1976. Comparative studies of baculovirus granulins and polyhedrhrs. Intervirology, 6, 168- 180. TANADA, Y., HIMENO, M., AND OMI, E. M. 1973. Isolation of a factor, from the capsule of a granulosis virus, synergistic for a nuclear-polyhedrosis virus of the armyworm. J. Invert&. P&ho/. , 21, 31-40. TANADA, Y., AND HUKUHARA, T. 1968. A nonsynergistic strain of a granulosis virus of the armyworm, Pseudaletia unipuncta. J. Znvertebr. Pathol., 12, 262-268.

AND TANADA TANADA, Y., AND WATANABE, H. 1971. Disc electrophoresis and serological studies of the capsule proteins obtained from two strains of a granulosis virus of the armyworm, Pseudaletia unipuncta. J. Znvertebr. Pathol., 18, 307-312. YAMAFUJI, K., So, K., AND Soo, K. 1941/1942. Die Wirkung des Seidenraupenpolyedervirus auf die Atmung und Katalase der Hefe. Biochem. Z., 311, 203-208. YAMAMOTO, T., AND TANADA, Y. 1978. Phospholipid, an enhancing component in the synergistic factor of a granulosis virus of the armyworm, Pseudaletia unipuncta. J. Znvertebr. Pathol., 31, 48-56.