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The purification and properties of inclusion body protein of the granulosis virus of Pieris brassicae
JOURNAL OF INVEBTEBRATEl PATHOLOGY 19, 42-50 (1972)
The Purifbation
and Properties Granulosis
Virus
of Inclusion of Pieris
Body
Protein
of the
brassicae
J. F. LONGWORTH, J. S. ROBBXTSON, AND C. C. PAYNE N.E.R.C.
Insect Pathology Unit, Commonwealth Forestry Institute,
University of Oxford, England
Received iUay 28,1&71 Inclusion bodies of the granulosis virus of Pieris brassicae were dissolved in alkali, and three components were obtained-inclusion body protein, enveloped virus particles, and the inclusion body envelope. These were separated by gel chromatography and density gradient centrifugation. The inclusion body envelope had a repeating structure different from the protein lattice of the inclusion body. Serological tests showed that two proteins were present in solutions of inclusion body protein. Both of these were present at the surface of the inclusion body but only one of these, protein B, was present at the surface of the enveloped virus particle. The two proteins were separated by gel chromatography and were shown to be serologically unrelated. Amino acid analyses confirmed that the two proteins were different. The isoelectric point of protein A was pH 5.8 and of B, 3.5. Protein A wss “typical” inclusion body protein; protein B present both at the surface of the inclusion body and the enveloped virus particle has different properties. The implications of these results in the serology of insect viruses are discussed.
preted as the presence of two different: proteins. No data have been presented on the antigenic properties of the inclusion body envelope, indeed its existence has been doubted (Smith, 1967). The present work is an examination of the components of inclusion body protein of Pieris brastiae granulosis virus with reference to their physical properties, antigenic structure and interrelationships.
When inclusion bodies of nuclear polyhedrosis and granulosis viruses are dissolved in alkali, three components are released; an envelope, virus particles, and inclusion body protein in solution. Solutions of inclusion body protein are monodisperse, and under certain salt and pH conditions the protein has a sedimentation constant of about 12.5 S (Bergold, 1947). The inclusion body envelopes and inclusion body protein have similar amino acid compositions (Wyatt, 1950). Inclusion body proteins possess several antigenic groups; two were common to all the proteins investigated (Krywienczyk and Bergold, 1961), and at least one was char-
MATERIALS
AND
METHODS
Purifiation of inclusion bodies. The capsules were sedimented from aqueous extracts of infected larvae at 10,000 g for 20 min and were purified either by three cycles of centrifugation on l@-70% (v/v) glycerol gradients for 20 min at 3,500 g or on lo-60 % (w/v) sucrose gradients in an “A” type zonal rotor at 4,500 rpm for 90 min. Inclusion bodies were dissolved for 2 hr in a buffer containing sodium carbonate (0.03 M) and sodium chloride (0.05 M) at 20-30 me/ml of capsules. After 2 hr the pH of the preparation was adjusted from 11.0 to 8.0.
acteristic of each protein. They concluded that unfractionated inclusion body protein of Bombyx mori nuclear polyhedrosis virus contained two components, one of which
could be removed by ultracentrifugation. This was thought to be an aggregate of the 12.5 S protein, but the evidence from immunodiffusion plates could equally be inter42
INCLUSION
BODY
PROTEIN
Puti&zation of virions. Enveloped virus particles were removed from the dissolution mixture by centrifugation at 30,000 g for 1 hr. The pellets were resuspended in distilled water and either centrifuged on lO-60% (w/v) sucrose gradients at 45,000 g for 1 hr or alternatively virus particles were purified on 5-50% (w/v) sucrose gradients in a B XIV zonal rotor at 17,000 rpm for 1 hr. The gradients were monitored at 254 nm. Virus preparations were passed through 50 X 2.5 cm Sepharose 2B columns to ensure complete removal of dissolved inclusion body protein. Preparation of inclusion body protein. Solutions of inclusion body proteins were filtered through Millipore filters of decreasing pore size down to 100 nm, given several cycles of precipitation at pH 5.7, and resuspended in 0.003 M ammonium hydrogen carbonate. Inclusion body protein was fractionated on a 100 X 2.5 cm Sepharose 6B column wit.h a borate buffer (pH 7.5, 0.005 M borax, 0.1s M boric acid) as the eluent. Samples of up to 25 ml at pH 7.4, were passed through the column by upward flow at 30 ml/hr. The eflluent was monitored at 254 nm and 5ml fractions were collected. Polyacrytamide gel electrophoresis. The Shandon disc electrophoresis equipment was used following the technique of Davis (1964). Both 3 % and 4 % (w/v) acrylamide gels were used with the proportions of acrylamide and bis-acrylamide according to the formula of Richards et al. (1965). The gel solution also contained 0.37 Y TrismHCl buffer, pH 8.9, 0.14% ammonium persulfate, and 0.06 % N,N,N’,N’-tetramethylethylenediamine (TEMED); 0.025 M Tris. glycine buffer, pH 8.4, was used in the reservoirs. The sample was applied in 0.5 ml reservoir buffer plus 0.02 ml 10 % (w/v) sucrose. The gels were prerun at 5 mA per tube (5 X 65 mm) for 2 hr, and electrophoresis was also at 5 mA per tube for 30 min. Gels were fixed and stained in 1% Naphthalene Black 10B in 7% (v/v) acetic acid
OF
GRANVLOSIS
VIRl?S
43
for 1 hr and destained electrophoretically or by shaking in 7 % acetic acid. Gel electrojocusing. The method used was similar to the one described by Wrigley (1969). Column electrojocusing. LKB 8100-10 Ampholine electrofocusing equipment was used; the sucrose-pH gradient, pH 3-10, was prepared by layering mixtures of decreasing density of the “light” and “heavy” solut.ions. The sample was introduced into the column in 1% glycine in place of the 3 ml of ihe “light” solution. The cathode was at the base of the column. Current was passed for 1 hr at 200 V and a further 71 1~ at 300 V. The sucrose-pH gradient was monitored at 254 nm, to locate the protein band. For pH determinations 2 ml fractions were then collected. Serology. Antisera were prepared in guinea pigs which were immunized intraperitoneally twice at an interval of 7 days, with antigens in 0.003 M ammonium hydrogen carbonate and with adjuvant. The animals were bled 3 weeks later and sera were stored at - 20°C without preservatives. Antisera were pre pared to inclusion bodies (50 mg), inclusion body protein (15 mg), virions (23 absorbance units), and to 15 mg inclusion body protein purified by Bergold’s method (Bergold, 1947). The rea&,ions between antisera and protein preparations were examined by grecipitation in gel. Determination of sedimentalion coe@cie~~ts. Sedimentation coefficients were determined at 20°C in a Spinco Model E analytical ultracentrifuge fitted with bot.h schiirrcn and UV absorption optics. Amino acid analyses. Anti0 acids were analyzed in an E.E.L. amino acid analyzer using essentially the procedure of Hirs et al. (1954). Electron microscopy. Samples were placed on Formvar grids, negatively stained with 2% (w/v) uranyl acetate, and examined on an AEI EMGB electron microscope at an accelerating voltage of 60 kV.
44
LONGWORTH,
ROBERTSON,
RESULTS Purified inclusion bodies were dissolved in alkali and aliquots were examined at intervals in the electron microscope. Untreated capsules were electron dense but were swollen and more electron transparent after 15 set in alkali. After 1 min some protein was released and virions were observed outside the inclusion body envelope which had swollen, to double the size of the untreated capsule. After 5 min, many virions were present and much of the protein had been released. The protein was in the form of large masses of finely dispersed amorphous material with no repeating structure. The inclusion body and virus particle envelope released their contents only when physically broken and were relatively insoluble in alkali. The inclusion body envelope showed a hexagonal arrangement of holes 6 nm in diameter (Fig. l), and this is typical of a membranous surface structure. Previous workers discarded inclusion body envelopes and protein aggregates from their preparations and so the antigenic properties of these fractions has not been determined. In view of their relative insolubility in alkali, the possibility that a different protein was present was examined. Andigens Present in Inclusion Body Protein Figure 2 shows the reaction of antisera to whole inclusion bodies and purified virions with partially purified inclusion body protein as antigen. The inclusion body antiserum produced two precipitin lines in gel, only one of which was produced by the antiserum to virions. This suggests that two antigens (A and B) are present on the surface of the inclusion body and that only one of these (B) is present on the surface of the virion; both antigens are present in an alkaline solution of inclusion body protein. None of the antisera reacted with extracts of healthy P. brassicae larvae, and antisera to
AND
PAYNE
extracts of healthy P. brassicae larvae and pupae did not react with inclusion body protein. Fraction&m of Inclusion column Chromatography
Body Protein by
When inclusion body protein was prepared by Bergold’s method only one main peak was produced in a Sepharose 6B column. In the analytical ultracentrifuge this was shown to consist of a major 12.5 S component; rarely a second component of 16 S was detected and frequently 24 S components were present. Immunodiffusion tests showed that both antigens A and B were present. When the protein aggregates which were removed in Bergold’s procedure were examined in the 6B column, a further peak was produced near the void volume. This contained only antigen B. This component did not sediment homogeneously in the analytical ultracentrifuge and in the 2B column was shown to consist of a range of molecular weights covering the fractionation range of the gel. Figure 3 shows the fractionation of a dissolution of inclusion bodies on the 6B column. The void volume peak contained virions and antigen B. The subsequent peak contained both antigens. Antigen A was also present in the third small peak, but the proteins present in the last two peaks did not react with any of the antisera. Analysis of Inclusion Body Protein by Polyacrylamide Gel Ekxtrophoreti All inclusion body protein preparations produced several bands in the gels. Figure 4 shows a densitometer tracing of stained protein patterns of purified inclusion body protein. The relative proportions of the bands were variable, but the fastest moving band (band 1) was always the most concentrated and corresponded to the 12.5 S molecule. Assuming bands II, III, IV, and V to be dimer, trimer, tetramer, and pentamer of the 12.5 S molecule a straight line relationship was derived between the log RI of the
INCLUSION
FIG.
BODY
PROTEIN
OF
GRAh’uLOSIS
VIRUS
1. Pier-is brasticae granulosis virus inclusion body envelope negatively
stained with many1
acetate.
bands and the assumed polymerization number. This indicates t.hat the bands are likely to be polymers of the same molecule. These aggregates were not caused by the conditions of the gel, for when band II was isolated and rerun, bands I and II were produced, and isolating and rerunning band III gave bands I, II, and III. Thus the dimer and trimer were disaggregating under these conditions. When the four bands in unstained gels were tested with antiserum to whole inclusion bodies, antigens A and B were present in each, indicating that antigen B is not merely an aggregate of antigen A and if the two are separate proteins, in the monomer form, both may have similar charge/density relationships.
FIG. 2. Immunodiffusion plate showing the reaction of antiserum to whole inclusion bodies (IB) and virions (VP) with partially purified inclusion body protein (P) as antigen.
Antigen B, produced from 6B column fractionation of inclusion body protein, proved too large to move through poly-
LONGWORTH,
ROBERTSON,
100
AND
PAYNE
300
Vol.
Eluent
(ml.)
3. Fractionation of a dissolution of Pieris brmsicae granulosis virus inclusion Sepharose 6B column. V = virions; A = antigen A; B = antigen B. FIQ.
acrylamide gel at both pH 5.7 and 8.9; it is unlikely that in this form B was disaggregating to any significant extent. Only rarely were antigens A and B separated in acrylamide gel, for example, after one run at pH 8.9, antigen A moved slightly faster than B. When the t,wo bands were tested against antiserum to whole inclusion bodies, two precipitin lines were formed which crossed over without interference, indicating that the two antigens were serologically unrelated. In inclusion body protein purified by Bergold’s method, antigen B was only detected in polyacrylamide gel in concentrated samples of 8 mg/ml. This supports the conclusion that antigen B is less soluble in alkali than A and is present in the dissolution predominantly in an aggregated form. Serological Relationship of Antigens A and B Figure 2 shows the reaction of antisera to virions and to whole inclusion bodies with
bodies on a
partially purified inclusion body protein. Clearly two serologically unrelated antigens are involved. The “B” precipitin line is nearer the inclusion body antiserum well in this case, and this is probably a concentration effect. In Figure 5 a 4-fold dilution series of a whole dissolution mixture of 10 mg/ml is in the upper row of wells (from right to left), and in the lower row of wells is an increasing concentration of inclusion body antiserum with 1: 16 dilution at the right and neat antiserum at the left. Extreme concentrations of antiserum elicit only one precipitin line with opposite extremes of antigen concentration, and each line is formed by a separate set of antigens; B is detected only in high antigen concentration and A is inhibited at high antigen concentration with dilute antiserum; both are detected at intermediate antiserum and antigen concentrations. At optimum proportions, the A and B precipitin lines are midway between the antigen and antiserum wells.
INCLUSIOK
BODY
PROTEIN
OF
GIL4NcLOSIS
Polyymerisation
VIRUS
47
number.
FIG. 4. Densitometer trace of stained protein patterns in a 4% polyacrylamide gel after electrophoresis of inclusion body protein. Inset: Plot of log R, of each band and the polymerization number, assuming band I to be a monomer.
Isoelectric Points of Antigens A and B The isoelectric point of the A protein was determined by gel electrofocusing of band I from a polyacrylamide gel electrophoresis of inclusion body protein. The absence of aggregates was found to improve resolution. The p1 of the A protein was in the region of pH 5.7 to 6.0. This was confirmed by analytical polyacrylamide gel electrophoresis of a mixture of proteins A and B and pH 5.7, using 0.1 M disodium hydrogen phosphate and 0.05 M citric acid in place of t,he Tris .
HCl and Trismglycine buffers. Figure 6 shows that at this pH, antigen A did not migrate in the gel whereas B st.ill bears a slight negative charge as it has moved slightly toward the anode. Further, the A and B precipitin lines intersect in a pattern of nonidentity, confirming that A and B are serologically unrelated. This suggested that the isoelectric point of B was less than pH 5.7. The sample contained small molecular weight proteins, presumably breakdown products of inclusion body protein. These
48
LONGWORTH, ROBERTSON, AND PAYNE
FIG. 5. Immunodiffusion plate showing the reaction of a 4-fold dilution series of 10 mg/ml of a whole dissolution mixture of inclusion body protein (upper wells, right to left) and a a-fold dilution series of neat antiserum to whole inclusion bodies (lower wells, left to right).
4%
p+ylamide
gel.
i Fro. 6. Immunodiffusion of antigens from inclusion body protein electrophoresed on a 4% polyacrylamide gel in 0.1 M disodium hydrogen phosphate 0.05 M citric acid, pH 5.7, and antiserum to whole inclusion bodies.
do not all have the same isoelectric point as A and form a separate precipitin line, suggesting that additional antigen groups may be exposed as the 12.5 S molecule is degraded. Purified B protein had too great a molecular weight for isoelectric focusing in gel even at the lowest gel concentrations and the p1 was therefore determined by column electrofocusing in a sucrose-pH gradient. The protein precipitated in the column
suite early in the test so that there was a broad peak between pH 3.0 and 4.6 with a maximum at pH 3.5. No precipitation occurred at pH 5.7, indicating that protein B had been completely freed from protein A. When a mixture of antigens A and B was focused on a pH 3.0 to 10.0 gradient there was precipitation over the range of pH 3.0 to 7.0. That occurring at the lower pH’s was shown serologically to be protein B only. I. Amino Acid Analysis Samples were selected for amino acid analysis from each peak in a Sepharose 6B fractionation of inclusion body protein. These were protein B from the leading peak, protein A, and the smaller molecular weight components. The analyses are presented in Table 1. Clearly proteins A and B were very different. “A” had a typical protein absorption spectrum, whereas “B” had a relatively low absorption at 280 nm, presumably because of its low tyrosine content. Neither of the two low molecular weight proteins reacted with the antisera available and though their amino acid analyses show some resemblances to that of protein A, there are sufficient differences to warrant a further examination of their nature and function. DISCUSSION
The protein composition of the inclusion bodies of the granulosis virus of P. brastiae is more complex than previous published work on this group of viruses would suggest. In addition to the protein described by Bergold, a second protein is present with a lower p1 and a different amino acid composition; it is serologically unrelated to the more alkali soluble “typical” inclusion body protein. Krywienceyk and Bergold (1961) showed that antiserum to the unpurified polyhedral protein of B. mmi nuclear polyhedra gave two precipitin lines in gel with the homolo-
TRCLUSION
BODY
PROTEIN
gous antigen and with highly purified polyhedron protein. Antiserum to the latter detected only one precipitin line with both antigens. It was suggested that the line common to both preparations was caused by the 12.5 S protein and that the aggregates of this protein were responsible for the second line, these being removed in purification. Our results are similar, but two serologitally unrelated proteins are involved, one of which is selectively removed during purification of the other. Harrap (1969) has shown with the nuclear polyhedrosis virus of Aglais urticae and Porthetria d&par that two proteins are present in the inclusion body, one of which is relatively insoluble in alkali; the two have a different amino acid composition and are not serologically identical. With P. brassicae granulosis virus, protein B was common to the surfaces of the inclusion body and of the intact virion. It is significant that this protein is relatively insoluble in alkali and that both structures are resistant to alkali. The relatively small quantities of prot.ein B are only associated with these surfaces. Only protein B was detected in P. brmsicae inclusion body protein with antiserum to purified virions. If such proteins are present on the outer envelope of virions of all nuclear polyhedrosis and granulosis viruses, this could explain why Krywienczyk and Bergold (1960) concluded that there was little or no serological relationship between virions and inclusion body proteins. Such cross reactions that could be detected may be explained on the basis of low levels of B protein in preparations which predominantly contained A protein. Indeed their cross reactions were often not fully reciprocal and were usually greatest when virions were reacted with polyhedron protein antisera. In these cases cross reactions of up to 50% of the homologous comparison were recorded. The sturcture of the nuclear polyhedrosis virus particles has recently been investigated by Harrap (1971). He has shown that
OF
GRANULOSIS
,49
VIRUS
TABLE
1
AMINO ACID ANALYSES OF “A” AND “B” PROTEINS, AND THE LAST Two PROTEINACEOUS PE.4KS ISOLATED BROM A SEPHAROSE 6B FRACTIONATION OF PURIFIED INCLUSION BODY PROTEINS
Amino acids
I
7
T
Proteins -1
Peak 4L 1?eak 5
rrgr,
._ Aspartic Threonine Serine Glutamic Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine
255.0 128.4 50.3 272.0 172.5 160.0 90.5 59.2 150.0 46.8 140.2 228.0 94.4 157.7 52.3 149.8 126.8
-94.4
48.7 78.6 75.9 50.2 100.0 50.8 40.8 2.1 37.0 43.7 14.6 24.5 34.3 32.5 33.3
316.8 185.0 119.3 305.0 197.0 100.0 105.8 133.9 161.3 145.8 69.0 100.0 30.4 51.6 49.2
236.5 74.6 50.8 33.9 174.3 100.0 50.0 19.7 73.1 8.6 69.4 58.1 23.0 39.8 53.2 72.5 25.3
a All figures are represented as percentages, with glycine taken as lOO’%. the double lipoprotein membrane surrounding the “inner” membrane, or nucleocapsid, is covered by a layer of flat, disc-shaped subunits; these in turn are enclosed in a thin structureless layer that obscures the structures beneath. It is possible that this structureless layer is the location of the B protein, but further work is necessary to determine whether this is the only protein present in this layer. On the basis of the present work, one must conclude that (1) previous comparative studies on inclusion body protein may have been concerned only with one component of at least a two-component system; and (2) comparative studies on the virions may have been, at least in part, studies on a protein which is also a constituent of the inclusion
body.
50
LONQWORTH, ROBINSON, AND PAYNE
There is a need therefore to examine the antigenic structure of the inclusion body proteins of selected nuclear polyhedrosis viruses, their distribution within the polyhedron and the relationship of these proteins to the intact virus particle. Further, it is essential to examine the antigenic structure of the components of the virion with a view to determining the most satisfactory basis for serological comparison of these complex viruses. ACKNOWLEDGMENT During the period of this investigation, Mr. C. C. Payne was in receipt of a Christopher Welch Scholarship. We are indebted to Dr. I. 0. Walker, Department of Biochemistry, University of Oxford, for determining the sedimentation coefficients and the amino acid analyses presented in this paper.
REFERENCES BERGOLD, G. H.
1947. Die Isolierung des Polyeder-Virus und die Natur der Polyeder. 2. Naturforsch., B 2, 122-143. DAYIS, B. J. 1964. Disc electrophoresis. II.
Methods and application to human serum proteins. Ann. N. Y. Acad. Sci., 121, 4o4-442. HARRAP, K. A. 1969. The structure and replication of some insect viruses. D. Phil. Thesis, University of Oxford, England. HARRAP, K. A. 1971. The structure of Porthetriu d&par nuclear polyhedrosis virus. PTOC. 4th Int. Colloq. Insect Pathol. 1970 (in Press). HIRS, G. H., STEIN, W. H., AND MOORE, S. 1954. The amino acid composition of ribonuolease. J. Biol. Chem., 211, 941-950. KRYWIENCZYK, J. AND BERGOLD, G. H. 1960. Serological relationships between insect viruses and their inclusion body proteins. J. Insect Pathol., 2, 113-132. KRYWIENC~~K, J., AND BERQOLD, G. H. 1961. Serological studies of inclusion body proteins by agar diffusion technique. J. Insect Pathol., 3,x-23. RICHARDS, E. G., COLL, J. A., AND GRATZER, W. B. 1965. Disc electrophoresis of ribonucleic acid in polyacrylamide gels. Anal. Biochem. 12,462. SMITH, K. M. 1967. Qrsect Virology,” p. 13. Academic Press, New York. WRIGLEY, C. 1969. Gel electrofocusing-a technique for analysing multiple protein samples by isoelectric focusing. Sci. Tools, 15, [Z], 17-22. WYATT, G. R. 1950. Studies on insect viruses and nucleic acids. Ph.D. Thesis, University of Cambridge, England.
The Purifbation
and Properties Granulosis
Virus
of Inclusion of Pieris
Body
Protein
of the
brassicae
J. F. LONGWORTH, J. S. ROBBXTSON, AND C. C. PAYNE N.E.R.C.
Insect Pathology Unit, Commonwealth Forestry Institute,
University of Oxford, England
Received iUay 28,1&71 Inclusion bodies of the granulosis virus of Pieris brassicae were dissolved in alkali, and three components were obtained-inclusion body protein, enveloped virus particles, and the inclusion body envelope. These were separated by gel chromatography and density gradient centrifugation. The inclusion body envelope had a repeating structure different from the protein lattice of the inclusion body. Serological tests showed that two proteins were present in solutions of inclusion body protein. Both of these were present at the surface of the inclusion body but only one of these, protein B, was present at the surface of the enveloped virus particle. The two proteins were separated by gel chromatography and were shown to be serologically unrelated. Amino acid analyses confirmed that the two proteins were different. The isoelectric point of protein A was pH 5.8 and of B, 3.5. Protein A wss “typical” inclusion body protein; protein B present both at the surface of the inclusion body and the enveloped virus particle has different properties. The implications of these results in the serology of insect viruses are discussed.
preted as the presence of two different: proteins. No data have been presented on the antigenic properties of the inclusion body envelope, indeed its existence has been doubted (Smith, 1967). The present work is an examination of the components of inclusion body protein of Pieris brastiae granulosis virus with reference to their physical properties, antigenic structure and interrelationships.
When inclusion bodies of nuclear polyhedrosis and granulosis viruses are dissolved in alkali, three components are released; an envelope, virus particles, and inclusion body protein in solution. Solutions of inclusion body protein are monodisperse, and under certain salt and pH conditions the protein has a sedimentation constant of about 12.5 S (Bergold, 1947). The inclusion body envelopes and inclusion body protein have similar amino acid compositions (Wyatt, 1950). Inclusion body proteins possess several antigenic groups; two were common to all the proteins investigated (Krywienczyk and Bergold, 1961), and at least one was char-
MATERIALS
AND
METHODS
Purifiation of inclusion bodies. The capsules were sedimented from aqueous extracts of infected larvae at 10,000 g for 20 min and were purified either by three cycles of centrifugation on l@-70% (v/v) glycerol gradients for 20 min at 3,500 g or on lo-60 % (w/v) sucrose gradients in an “A” type zonal rotor at 4,500 rpm for 90 min. Inclusion bodies were dissolved for 2 hr in a buffer containing sodium carbonate (0.03 M) and sodium chloride (0.05 M) at 20-30 me/ml of capsules. After 2 hr the pH of the preparation was adjusted from 11.0 to 8.0.
acteristic of each protein. They concluded that unfractionated inclusion body protein of Bombyx mori nuclear polyhedrosis virus contained two components, one of which
could be removed by ultracentrifugation. This was thought to be an aggregate of the 12.5 S protein, but the evidence from immunodiffusion plates could equally be inter42
INCLUSION
BODY
PROTEIN
Puti&zation of virions. Enveloped virus particles were removed from the dissolution mixture by centrifugation at 30,000 g for 1 hr. The pellets were resuspended in distilled water and either centrifuged on lO-60% (w/v) sucrose gradients at 45,000 g for 1 hr or alternatively virus particles were purified on 5-50% (w/v) sucrose gradients in a B XIV zonal rotor at 17,000 rpm for 1 hr. The gradients were monitored at 254 nm. Virus preparations were passed through 50 X 2.5 cm Sepharose 2B columns to ensure complete removal of dissolved inclusion body protein. Preparation of inclusion body protein. Solutions of inclusion body proteins were filtered through Millipore filters of decreasing pore size down to 100 nm, given several cycles of precipitation at pH 5.7, and resuspended in 0.003 M ammonium hydrogen carbonate. Inclusion body protein was fractionated on a 100 X 2.5 cm Sepharose 6B column wit.h a borate buffer (pH 7.5, 0.005 M borax, 0.1s M boric acid) as the eluent. Samples of up to 25 ml at pH 7.4, were passed through the column by upward flow at 30 ml/hr. The eflluent was monitored at 254 nm and 5ml fractions were collected. Polyacrytamide gel electrophoresis. The Shandon disc electrophoresis equipment was used following the technique of Davis (1964). Both 3 % and 4 % (w/v) acrylamide gels were used with the proportions of acrylamide and bis-acrylamide according to the formula of Richards et al. (1965). The gel solution also contained 0.37 Y TrismHCl buffer, pH 8.9, 0.14% ammonium persulfate, and 0.06 % N,N,N’,N’-tetramethylethylenediamine (TEMED); 0.025 M Tris. glycine buffer, pH 8.4, was used in the reservoirs. The sample was applied in 0.5 ml reservoir buffer plus 0.02 ml 10 % (w/v) sucrose. The gels were prerun at 5 mA per tube (5 X 65 mm) for 2 hr, and electrophoresis was also at 5 mA per tube for 30 min. Gels were fixed and stained in 1% Naphthalene Black 10B in 7% (v/v) acetic acid
OF
GRANVLOSIS
VIRl?S
43
for 1 hr and destained electrophoretically or by shaking in 7 % acetic acid. Gel electrojocusing. The method used was similar to the one described by Wrigley (1969). Column electrojocusing. LKB 8100-10 Ampholine electrofocusing equipment was used; the sucrose-pH gradient, pH 3-10, was prepared by layering mixtures of decreasing density of the “light” and “heavy” solut.ions. The sample was introduced into the column in 1% glycine in place of the 3 ml of ihe “light” solution. The cathode was at the base of the column. Current was passed for 1 hr at 200 V and a further 71 1~ at 300 V. The sucrose-pH gradient was monitored at 254 nm, to locate the protein band. For pH determinations 2 ml fractions were then collected. Serology. Antisera were prepared in guinea pigs which were immunized intraperitoneally twice at an interval of 7 days, with antigens in 0.003 M ammonium hydrogen carbonate and with adjuvant. The animals were bled 3 weeks later and sera were stored at - 20°C without preservatives. Antisera were pre pared to inclusion bodies (50 mg), inclusion body protein (15 mg), virions (23 absorbance units), and to 15 mg inclusion body protein purified by Bergold’s method (Bergold, 1947). The rea&,ions between antisera and protein preparations were examined by grecipitation in gel. Determination of sedimentalion coe@cie~~ts. Sedimentation coefficients were determined at 20°C in a Spinco Model E analytical ultracentrifuge fitted with bot.h schiirrcn and UV absorption optics. Amino acid analyses. Anti0 acids were analyzed in an E.E.L. amino acid analyzer using essentially the procedure of Hirs et al. (1954). Electron microscopy. Samples were placed on Formvar grids, negatively stained with 2% (w/v) uranyl acetate, and examined on an AEI EMGB electron microscope at an accelerating voltage of 60 kV.
44
LONGWORTH,
ROBERTSON,
RESULTS Purified inclusion bodies were dissolved in alkali and aliquots were examined at intervals in the electron microscope. Untreated capsules were electron dense but were swollen and more electron transparent after 15 set in alkali. After 1 min some protein was released and virions were observed outside the inclusion body envelope which had swollen, to double the size of the untreated capsule. After 5 min, many virions were present and much of the protein had been released. The protein was in the form of large masses of finely dispersed amorphous material with no repeating structure. The inclusion body and virus particle envelope released their contents only when physically broken and were relatively insoluble in alkali. The inclusion body envelope showed a hexagonal arrangement of holes 6 nm in diameter (Fig. l), and this is typical of a membranous surface structure. Previous workers discarded inclusion body envelopes and protein aggregates from their preparations and so the antigenic properties of these fractions has not been determined. In view of their relative insolubility in alkali, the possibility that a different protein was present was examined. Andigens Present in Inclusion Body Protein Figure 2 shows the reaction of antisera to whole inclusion bodies and purified virions with partially purified inclusion body protein as antigen. The inclusion body antiserum produced two precipitin lines in gel, only one of which was produced by the antiserum to virions. This suggests that two antigens (A and B) are present on the surface of the inclusion body and that only one of these (B) is present on the surface of the virion; both antigens are present in an alkaline solution of inclusion body protein. None of the antisera reacted with extracts of healthy P. brassicae larvae, and antisera to
AND
PAYNE
extracts of healthy P. brassicae larvae and pupae did not react with inclusion body protein. Fraction&m of Inclusion column Chromatography
Body Protein by
When inclusion body protein was prepared by Bergold’s method only one main peak was produced in a Sepharose 6B column. In the analytical ultracentrifuge this was shown to consist of a major 12.5 S component; rarely a second component of 16 S was detected and frequently 24 S components were present. Immunodiffusion tests showed that both antigens A and B were present. When the protein aggregates which were removed in Bergold’s procedure were examined in the 6B column, a further peak was produced near the void volume. This contained only antigen B. This component did not sediment homogeneously in the analytical ultracentrifuge and in the 2B column was shown to consist of a range of molecular weights covering the fractionation range of the gel. Figure 3 shows the fractionation of a dissolution of inclusion bodies on the 6B column. The void volume peak contained virions and antigen B. The subsequent peak contained both antigens. Antigen A was also present in the third small peak, but the proteins present in the last two peaks did not react with any of the antisera. Analysis of Inclusion Body Protein by Polyacrylamide Gel Ekxtrophoreti All inclusion body protein preparations produced several bands in the gels. Figure 4 shows a densitometer tracing of stained protein patterns of purified inclusion body protein. The relative proportions of the bands were variable, but the fastest moving band (band 1) was always the most concentrated and corresponded to the 12.5 S molecule. Assuming bands II, III, IV, and V to be dimer, trimer, tetramer, and pentamer of the 12.5 S molecule a straight line relationship was derived between the log RI of the
INCLUSION
FIG.
BODY
PROTEIN
OF
GRAh’uLOSIS
VIRUS
1. Pier-is brasticae granulosis virus inclusion body envelope negatively
stained with many1
acetate.
bands and the assumed polymerization number. This indicates t.hat the bands are likely to be polymers of the same molecule. These aggregates were not caused by the conditions of the gel, for when band II was isolated and rerun, bands I and II were produced, and isolating and rerunning band III gave bands I, II, and III. Thus the dimer and trimer were disaggregating under these conditions. When the four bands in unstained gels were tested with antiserum to whole inclusion bodies, antigens A and B were present in each, indicating that antigen B is not merely an aggregate of antigen A and if the two are separate proteins, in the monomer form, both may have similar charge/density relationships.
FIG. 2. Immunodiffusion plate showing the reaction of antiserum to whole inclusion bodies (IB) and virions (VP) with partially purified inclusion body protein (P) as antigen.
Antigen B, produced from 6B column fractionation of inclusion body protein, proved too large to move through poly-
LONGWORTH,
ROBERTSON,
100
AND
PAYNE
300
Vol.
Eluent
(ml.)
3. Fractionation of a dissolution of Pieris brmsicae granulosis virus inclusion Sepharose 6B column. V = virions; A = antigen A; B = antigen B. FIQ.
acrylamide gel at both pH 5.7 and 8.9; it is unlikely that in this form B was disaggregating to any significant extent. Only rarely were antigens A and B separated in acrylamide gel, for example, after one run at pH 8.9, antigen A moved slightly faster than B. When the t,wo bands were tested against antiserum to whole inclusion bodies, two precipitin lines were formed which crossed over without interference, indicating that the two antigens were serologically unrelated. In inclusion body protein purified by Bergold’s method, antigen B was only detected in polyacrylamide gel in concentrated samples of 8 mg/ml. This supports the conclusion that antigen B is less soluble in alkali than A and is present in the dissolution predominantly in an aggregated form. Serological Relationship of Antigens A and B Figure 2 shows the reaction of antisera to virions and to whole inclusion bodies with
bodies on a
partially purified inclusion body protein. Clearly two serologically unrelated antigens are involved. The “B” precipitin line is nearer the inclusion body antiserum well in this case, and this is probably a concentration effect. In Figure 5 a 4-fold dilution series of a whole dissolution mixture of 10 mg/ml is in the upper row of wells (from right to left), and in the lower row of wells is an increasing concentration of inclusion body antiserum with 1: 16 dilution at the right and neat antiserum at the left. Extreme concentrations of antiserum elicit only one precipitin line with opposite extremes of antigen concentration, and each line is formed by a separate set of antigens; B is detected only in high antigen concentration and A is inhibited at high antigen concentration with dilute antiserum; both are detected at intermediate antiserum and antigen concentrations. At optimum proportions, the A and B precipitin lines are midway between the antigen and antiserum wells.
INCLUSIOK
BODY
PROTEIN
OF
GIL4NcLOSIS
Polyymerisation
VIRUS
47
number.
FIG. 4. Densitometer trace of stained protein patterns in a 4% polyacrylamide gel after electrophoresis of inclusion body protein. Inset: Plot of log R, of each band and the polymerization number, assuming band I to be a monomer.
Isoelectric Points of Antigens A and B The isoelectric point of the A protein was determined by gel electrofocusing of band I from a polyacrylamide gel electrophoresis of inclusion body protein. The absence of aggregates was found to improve resolution. The p1 of the A protein was in the region of pH 5.7 to 6.0. This was confirmed by analytical polyacrylamide gel electrophoresis of a mixture of proteins A and B and pH 5.7, using 0.1 M disodium hydrogen phosphate and 0.05 M citric acid in place of t,he Tris .
HCl and Trismglycine buffers. Figure 6 shows that at this pH, antigen A did not migrate in the gel whereas B st.ill bears a slight negative charge as it has moved slightly toward the anode. Further, the A and B precipitin lines intersect in a pattern of nonidentity, confirming that A and B are serologically unrelated. This suggested that the isoelectric point of B was less than pH 5.7. The sample contained small molecular weight proteins, presumably breakdown products of inclusion body protein. These
48
LONGWORTH, ROBERTSON, AND PAYNE
FIG. 5. Immunodiffusion plate showing the reaction of a 4-fold dilution series of 10 mg/ml of a whole dissolution mixture of inclusion body protein (upper wells, right to left) and a a-fold dilution series of neat antiserum to whole inclusion bodies (lower wells, left to right).
4%
p+ylamide
gel.
i Fro. 6. Immunodiffusion of antigens from inclusion body protein electrophoresed on a 4% polyacrylamide gel in 0.1 M disodium hydrogen phosphate 0.05 M citric acid, pH 5.7, and antiserum to whole inclusion bodies.
do not all have the same isoelectric point as A and form a separate precipitin line, suggesting that additional antigen groups may be exposed as the 12.5 S molecule is degraded. Purified B protein had too great a molecular weight for isoelectric focusing in gel even at the lowest gel concentrations and the p1 was therefore determined by column electrofocusing in a sucrose-pH gradient. The protein precipitated in the column
suite early in the test so that there was a broad peak between pH 3.0 and 4.6 with a maximum at pH 3.5. No precipitation occurred at pH 5.7, indicating that protein B had been completely freed from protein A. When a mixture of antigens A and B was focused on a pH 3.0 to 10.0 gradient there was precipitation over the range of pH 3.0 to 7.0. That occurring at the lower pH’s was shown serologically to be protein B only. I. Amino Acid Analysis Samples were selected for amino acid analysis from each peak in a Sepharose 6B fractionation of inclusion body protein. These were protein B from the leading peak, protein A, and the smaller molecular weight components. The analyses are presented in Table 1. Clearly proteins A and B were very different. “A” had a typical protein absorption spectrum, whereas “B” had a relatively low absorption at 280 nm, presumably because of its low tyrosine content. Neither of the two low molecular weight proteins reacted with the antisera available and though their amino acid analyses show some resemblances to that of protein A, there are sufficient differences to warrant a further examination of their nature and function. DISCUSSION
The protein composition of the inclusion bodies of the granulosis virus of P. brastiae is more complex than previous published work on this group of viruses would suggest. In addition to the protein described by Bergold, a second protein is present with a lower p1 and a different amino acid composition; it is serologically unrelated to the more alkali soluble “typical” inclusion body protein. Krywienceyk and Bergold (1961) showed that antiserum to the unpurified polyhedral protein of B. mmi nuclear polyhedra gave two precipitin lines in gel with the homolo-
TRCLUSION
BODY
PROTEIN
gous antigen and with highly purified polyhedron protein. Antiserum to the latter detected only one precipitin line with both antigens. It was suggested that the line common to both preparations was caused by the 12.5 S protein and that the aggregates of this protein were responsible for the second line, these being removed in purification. Our results are similar, but two serologitally unrelated proteins are involved, one of which is selectively removed during purification of the other. Harrap (1969) has shown with the nuclear polyhedrosis virus of Aglais urticae and Porthetria d&par that two proteins are present in the inclusion body, one of which is relatively insoluble in alkali; the two have a different amino acid composition and are not serologically identical. With P. brassicae granulosis virus, protein B was common to the surfaces of the inclusion body and of the intact virion. It is significant that this protein is relatively insoluble in alkali and that both structures are resistant to alkali. The relatively small quantities of prot.ein B are only associated with these surfaces. Only protein B was detected in P. brmsicae inclusion body protein with antiserum to purified virions. If such proteins are present on the outer envelope of virions of all nuclear polyhedrosis and granulosis viruses, this could explain why Krywienczyk and Bergold (1960) concluded that there was little or no serological relationship between virions and inclusion body proteins. Such cross reactions that could be detected may be explained on the basis of low levels of B protein in preparations which predominantly contained A protein. Indeed their cross reactions were often not fully reciprocal and were usually greatest when virions were reacted with polyhedron protein antisera. In these cases cross reactions of up to 50% of the homologous comparison were recorded. The sturcture of the nuclear polyhedrosis virus particles has recently been investigated by Harrap (1971). He has shown that
OF
GRANULOSIS
,49
VIRUS
TABLE
1
AMINO ACID ANALYSES OF “A” AND “B” PROTEINS, AND THE LAST Two PROTEINACEOUS PE.4KS ISOLATED BROM A SEPHAROSE 6B FRACTIONATION OF PURIFIED INCLUSION BODY PROTEINS
Amino acids
I
7
T
Proteins -1
Peak 4L 1?eak 5
rrgr,
._ Aspartic Threonine Serine Glutamic Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine
255.0 128.4 50.3 272.0 172.5 160.0 90.5 59.2 150.0 46.8 140.2 228.0 94.4 157.7 52.3 149.8 126.8
-94.4
48.7 78.6 75.9 50.2 100.0 50.8 40.8 2.1 37.0 43.7 14.6 24.5 34.3 32.5 33.3
316.8 185.0 119.3 305.0 197.0 100.0 105.8 133.9 161.3 145.8 69.0 100.0 30.4 51.6 49.2
236.5 74.6 50.8 33.9 174.3 100.0 50.0 19.7 73.1 8.6 69.4 58.1 23.0 39.8 53.2 72.5 25.3
a All figures are represented as percentages, with glycine taken as lOO’%. the double lipoprotein membrane surrounding the “inner” membrane, or nucleocapsid, is covered by a layer of flat, disc-shaped subunits; these in turn are enclosed in a thin structureless layer that obscures the structures beneath. It is possible that this structureless layer is the location of the B protein, but further work is necessary to determine whether this is the only protein present in this layer. On the basis of the present work, one must conclude that (1) previous comparative studies on inclusion body protein may have been concerned only with one component of at least a two-component system; and (2) comparative studies on the virions may have been, at least in part, studies on a protein which is also a constituent of the inclusion
body.
50
LONQWORTH, ROBINSON, AND PAYNE
There is a need therefore to examine the antigenic structure of the inclusion body proteins of selected nuclear polyhedrosis viruses, their distribution within the polyhedron and the relationship of these proteins to the intact virus particle. Further, it is essential to examine the antigenic structure of the components of the virion with a view to determining the most satisfactory basis for serological comparison of these complex viruses. ACKNOWLEDGMENT During the period of this investigation, Mr. C. C. Payne was in receipt of a Christopher Welch Scholarship. We are indebted to Dr. I. 0. Walker, Department of Biochemistry, University of Oxford, for determining the sedimentation coefficients and the amino acid analyses presented in this paper.
REFERENCES BERGOLD, G. H.
1947. Die Isolierung des Polyeder-Virus und die Natur der Polyeder. 2. Naturforsch., B 2, 122-143. DAYIS, B. J. 1964. Disc electrophoresis. II.
Methods and application to human serum proteins. Ann. N. Y. Acad. Sci., 121, 4o4-442. HARRAP, K. A. 1969. The structure and replication of some insect viruses. D. Phil. Thesis, University of Oxford, England. HARRAP, K. A. 1971. The structure of Porthetriu d&par nuclear polyhedrosis virus. PTOC. 4th Int. Colloq. Insect Pathol. 1970 (in Press). HIRS, G. H., STEIN, W. H., AND MOORE, S. 1954. The amino acid composition of ribonuolease. J. Biol. Chem., 211, 941-950. KRYWIENCZYK, J. AND BERGOLD, G. H. 1960. Serological relationships between insect viruses and their inclusion body proteins. J. Insect Pathol., 2, 113-132. KRYWIENC~~K, J., AND BERQOLD, G. H. 1961. Serological studies of inclusion body proteins by agar diffusion technique. J. Insect Pathol., 3,x-23. RICHARDS, E. G., COLL, J. A., AND GRATZER, W. B. 1965. Disc electrophoresis of ribonucleic acid in polyacrylamide gels. Anal. Biochem. 12,462. SMITH, K. M. 1967. Qrsect Virology,” p. 13. Academic Press, New York. WRIGLEY, C. 1969. Gel electrofocusing-a technique for analysing multiple protein samples by isoelectric focusing. Sci. Tools, 15, [Z], 17-22. WYATT, G. R. 1950. Studies on insect viruses and nucleic acids. Ph.D. Thesis, University of Cambridge, England.