Vol. 21, No. 1

JOURNAL OF VIROLOGY, Jan. 1977, p. 347-357 Copyright © 1977 American Society for Microbiology

Printed in U.S.A.

Proteins of Hepatitis B Surface Antigen J. WAI-KUO SHIH AND JOHN L. GERIN* Molecular Anatomy Program, Oak Ridge National Laboratory, Rockville, Maryland 20852

Received for publication 5 August 1976

Purified 22-nm forms of hepatitis B surface antigen (HBsAg) representing the three major antigenic subtypes (adw, ayw, and adr) were analyzed for their constituent polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No consistent difference in either the number or relative distributions of the polypeptides was observed for the various subtypes. Seven polypeptides were designated as P-1 through P-7 in order of their decreasing mobilities. By comparison with protein standards, their molecular weights were estimated as 23, 29.5, 36, 41.5, 53.5, 72, and 97 thousand. The P-1 and P-2 components represented the major polypeptides; P-2 and P-5 might be glycoproteins, based on their reaction with periodic acid-Shiff reagent. Each polypeptide contains cysteine residues. HBsAg was radiolabeled with 3H or 14C by reductive methylation or iodinated with 125I by the chloramine-T or lactoperoxidase procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled HBsAg yielded patterns identical to those obtained with protein stain. Comparison of HBsAg labeled by the chloramine-T and lactoperoxide procedures indicated that there was no distinction between internal or external components within the 22nm structure.

Hepatitis B surface antigen (HB8Ag) is synthesized in the cytoplasm of the infected hepatocyte and circulates in the plasma of chronic HBrAg carriers in a number of particulate forms. One form, the Dane particle (8), probably represents the virion of hepatitis B virus (HBV). The 22-nm spherical and filamentous forms share the antigenic HBrAg determinants with the lipoprotein coat of the virion and probably represent coat protein produced in excess by the infected hepatocytes. The 22-nm form can be readily purified from carrier plasma by large-scale techniques (13, 15) and has been successfully used as a subviral vaccine in animal (17, 25) and limited human (22) studies. The recognized antigens of HB1Ag appear to be specified by the HBV genome and consist of group-specific (a) and type-specific (d ory, w or r) determinants (1, 19). Chronic carriers of each of the four possible phenotypes (adw, adr, ayw and ayr) have been found in human populations, although the ayr phenotype is exceptionally rare and therefore not epidemiologically important. Subspecificities may also exist within the group-specific a complex (33). The 22-nm form of HB.Ag contains structural protein antigens that are gene products of HBV. In this paper we report data concerning the polypeptide compositions of purified 22-nm forms of three major HB8Ag phenotypes. (Some of these data were presented in a pre-

liminary manner at a symposium on hepatitis and blood transfusion in 1972 [14]). MATERIALS AND METHODS Source of purified HB,Ag. Plasmas containing either the adw, ayw, or adr subtypes of HB,Ag were obtained by plasmapheresis of asymptomatic chronic carriers and stored frozen at -20°C until used for antigen purification. The approximately 22nm form of HBsAg was isolated from each plasma by a four-step zonal centrifuge procedure as previously described (13-15). Briefly, the 22-nm form was purified from serum components and the other HB,Ag forms by two cycles of isopycnic banding in cesium chloride, followed by rate-zonal centrifugation in sucrose and a final banding in CsCl.

Polyacrylamide gel electrophoresis (PAGE) was performed by a modification of the method of Maizel (21). The polymerized gels contained either 5% or 7.5% (wt/vol) acrylamide, 0.125 or 0.180% (wt/vol) methylene bisacrylamide, 4 M urea, 0.1% (wt/vol) ammonium persulfate, 0.05% (vol/vol) N,N,N',N'tetramethylethylene diamine, and 0.1% (wt/vol) sodium dodecyl sulfate (SDS) in 0.1 M sodium phosphate buffer, pH 7.2. The electrophoretic buffer consisted of 0.1 M sodium phosphate buffer, pH 7.2, and 0.1% (wt/vol) SDS; 0.1% (vol/vol) 2-mercaptoethanol was freshly added to the upper chamber, and the gels were subjected to electrophoresis for 30 min before application of the sample. Samples of 50 or 100 1.l volume containing approximately 25 ,ug of protein were evaporated to dryness and solubilized by the addition of 50 ,ul of a solution of 8 M urea, 0.1% (wt/vol) SDS, and 1% (vol/vol) 2347

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mercaptoethanol. After 2 h at room temperature, the samples were heated to 90°C for 2 min. One drop of glycerol containing 0.05% (wt/vol) brom phenol blue was added to each sample, which was then applied to the top of the gel by underlayering. The gels were subjected to electrophoresis for 2.5 to 3 h at 8 mA/gel. Proteins were stained with Coomassie blue by the method of Weber and Osborn (35) and polysaccharides were stained by a modification of the periodic acid-Schiff (PAS) reaction as described by Bolognesi and Bauer (3). For the detection of polysaccharides, gels were fixed in 12.5% trichloroacetic acid for 1 h and incubated for 10 h at room temperature in 1% periodic acid. After the gels were washed for 2 h in running tap water and 2 h in 10% acetic acid, they were stained with a fresh preparation of Schiff reagent (Feulgen solution) for 2 h and destained with 1% aceitc acid. Densitometric tracings of Coomassie blue or PAS-stained gels were made using the linear transport accessory of the Gilford 2400 spectrophotometer at 550 or 520 nm respectively. For radioactivity determinations, gels were sectioned into 1-mm slices and prepared for counting as described below. Molecular-weight estimations. Estimates of the molecular weights of the polypeptides were made in 5% SDS-polyacrylamide gels by the method of Shapiro et al. (30) using the following protein standards; bovine serum albumin (BSA, molecular weight, 68,000), BSA dimer (molecular weight, 140,000), catalase (subunit molecular weight, 57,500), ovalbumin (molecular weight, 43,000), aldolase (subunit molecular weight, 40,000), yeast alcohol dehydrogenase (subunit molecular weight, 37,000), lysozyme Ch (molecular weight, 23,400), and myoglobin (molecular weight, 17,200). BSA, ovalbumin, and myoglobin were purchased from Schwarz/Mann (Orangeburg, N.Y.), beef liver catalase, rabbit muscle aldolase and yeast alcohol dehydrogenase were from Worthington Biochemical Corp. (Freehold, N.J.), and lysozyme Ch was a gift from John H. Hash of Vanderbilt University. The molecular weights of the standard proteins were taken from Weber and Osborn (35), and that of lysozyme Ch was from Shih and Hash (31). The gels were stained with Coomassie blue to locate the proteins, and the center of the tracking dye was the reference point for calculations of relative migration. Radioactive labeling of proteins and measurements of radioactivity. The procedure of Rice and Means (27) was used to label HBrAg proteins in vitro by the introduction of methyl- 14C or methyl-3H groups. A preparation of purified HB.Ag (100 to 180 lAg) cooled in ice was alkylated with 20 ,ul of 50 mM of ["4C]formaldehyde (10 mCi/mM) or [3H]formaldehyde (87.5 mCi/mM) in 0.25 ml of 0.2 M sodium borate buffer (pH 9.0) for 1 min and reduced with five sequential additions of 5 ,ul of sodium borohydride (5 mg/ml); excess reagents were subsequently removed by dialysis. The use of tritiated sodium borohydride (250 mCi/mM) together with [3Hlformaldehyde resulted in labeled HB1Ag with a specific activity of 7.4 x 106 cpm/mg of protein. HBSAg was iodinated with 125I by either chloramine-T (CT) (18) or lactoperoxidase (LP) (9) proce-

J. VIROL.

dures. In the first procedure, 10 ,g of CT was added to a 50-,l volume of HB.Ag (18 to 40 ,ug of protein) containing 0.25 M sodium phosphate buffer, pH 6.55, and 0.2 to 0.8 mCi of Na'25I (>14 mCi/,g, Amersham/Searle Corp.). After 5 min at room temperature, the reaction was terminated by the addition of 20 ,ug of sodium metabisulfite followed by 100 Al of KI (10 mg/ml). The reaction mixture was desalted by passage through a Sephadex G-50 column (0.9 by 1.5 cm); the void volume fractions containing the radioactivity were pooled. For the LP procedure, the HB.Ag was incubated with 7.5 ,ug of LP-Sepharose 2B in 50 ,ul of 0.25 M sodium phosphate buffer, pH 6.55, containing 0.2 to 0.5 mCi of Na'25I. Three aliquots of 9 x 10-5 M H202 were added at 10-min intervals. The reaction was stopped after 30 min at room temperature by the addition of 200 ,l of 0.025 M!NaN3 and 0.05 M KI in PBS. The labeled HB.Ag was recovered after desalting as described above. The LP-Sepharose 2B was prepared as described by David (9) and contained 1.5 ,ug of LP protein (Calbiochem, San Diego, Calif.) per ,l of suspended gel. The radioactivity of 14C and 3H was measured in a Beckman LS-250 liquid scintillation spectrometer using a toluene-based scintillation fluid with 10% (vol/vol) BBS-3 solubilizer (Beckman, Fullerton, Calif.). Polyacrylamide gel slices (1 mm) were solubilized with 0.2 ml of H202 (30%, wt/wt) in 20-ml scintillation vials overnight at 37°C. After cooling to room temperature, 10 ml of the cocktail described above was added to each vial. Samples containing 125I were counted directly in a Nuclear Chicago model 1100 gamma scintillation counter. Chemical treatment and analysis. HB.Ag was reduced with 2-mercaptoethanol and alkylated with iodoacetamide or [14C]iodoacetamide (New England Nuclear Corp., Boston, Mass.) diluted to 0.17 mCi/ mM as described by Crestfield et al. (7); additional 1% (wt/vol) SDS was added to the solubilization medium. Lipids were extracted from purified HB1Ag with chloroform-methanol (2:1) as described by Bligh and Dyer (2). Protein determinations were made by the procedure of Lowry et al. (20) using crystalline BSA as a standard.

RESULTS Analytical electrophoresis of HB1Ag proteins on polyacrylamide gels. Preparations of 22-nm forms of the adw, ayw, and adr antigenic subtypes of HB,Ag, as purified from human plasma by four-step zonal centrifuge procedures, were solubilized under reducing conditions, and subjected to electrophoresis in polyacrylamide gels containing SDS and urea. Analysis of an adw (JM) and an ayw (Ytr) preparation (Fig. 1) revealed the presence of up to seven distinct protein bands, which were designated P-1 through P-7 in order of decreasing electrophoretic mobility. Comparison with known protein standards solubilized and subjected to electrophoresis under identical conditions (Fig. 2) resulted in molecular-weight estimates of 23, 29.5, 36, 41.5, 53.5, 72, and 97

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FIG. 7. Glycoproteins of HB,Ag. Densitometric tracings of gels described in Fig. 6. The Coomassie blueand PAS-stained gels were scanned at 550 and 520 nm, respectively. The scans of the 630198 gels are in the left panel; those of Draa are in the right panel.

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FIG. 8. Isopycnic banding of tritiated HB,Ag in CsCl. The Ytr (ayw) antigen was labeled in vitro with 3H, mixed with unlabeled HB,Ag, and banded in CsCl for 21 h at 30,000 rpm and 5°C in the SW41 rotor. Gradient fractions were assayed for absorbance at 280 nm, 3H activity, complement-fixing (CF) antigen (25), and CsCl density by refractometry. region of the CsCl gradient

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the same density on rebanding for both the CTand LP-labeled HB,Ag. HB,Ag iodinated by the LP procedure had a buoyant density in CsCl identical to that of unlabeled antigen, whereas that of the CT-labeled HBSAg was slightly higher. The LP procedure resulted in HBsAg

with specific activities ranging from 0.3 to 1.0 ,uCi/,4g; the range of specific activities for the CT procedure was 2 to 6 ,uCi/,ug. Preparations labeled by either procedure were precipitable (>90%) with anti-HB0 by double-antibody radioimmunoprecipitation (32). SDS-PAGE of the iodinated material re-

VOL. 21, 1977

HB,Ag PROTEINS

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FIG. 9. SDS-PAGE of [3H]HB,Ag. [3H]HB,Agl ayw (Ytr) was mixed with the unlabeled Ytr antigen, solubilized, and subjected to elecrophoresis on 5% SDS-acrylamide gels. Each of two gels was loaded with a mixture containing 5,400 cpm of [3H]HB,Ag and 28 ug of unlabeled antigen. One gel was stained with Coomassie blue and scanning at 550 nm ( ). The other gel was sliced into 1-mm sections and assayed for 3H (-) as described in Methods and Materials.

covered from the tops of the gradients described above revealed two peaks (molecular weights, 16,000 and 7,000) in the CT-labeled material and one peak (molecular weight, 7,000) in the LP-labeled sample. These components were presumed to be the same as those shown in Fig. 10A and C and were resistant to Pronase or nuclease digestion and acid or alkaline hydrolysis. These radioactive components were not precipitable with anti-HB, by double-antibody radioimmunoprecipitation. DISCUSSION Purified 22-nm forms of the three major HBAg subtypes (adw, ayw, and adr) consisted of five to seven polypeptides when analyzed by SDS-polyacrylamide gel electrophoresis. The polypeptides P-1 through P-7 ranged in apparent molecular weight from 23,000 to 97,000, and these values were not dependent on the degree of cross-linking in the gels. No consistent difference was observed between the major subtypes in regard to the number or

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FIG. 10. SDS-PAGE of [1'251HB,Ag. A preparation of HB,Ag/adw was iodinated in vitro by the CT or LP procedures, solubilized, and subjected to electrophoresis on 7.5% SDS-acrylamide gels. Gel slices (1 mm) were assayed for 125I. (A) and (C) Patterns of 125I for samples iodinated by the CT and LP procedures, respectively. The positions of the P-1 through P-7 polypeptides are indicated at the top. (B) Same as A, except that the gel was fixed in 12.5% trichloroacetic acid and washed in 10% trichloroacetic acid before it was sliced into 1-mm sections.

relative distribution of the polypeptides. The polypeptides P-1 (molecular weight, 23,000) and P-2 (molecular weight, 29,500) represented the major protein components, although P-6 (molecular weight, 72,000) was also a major com-

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ponent of some preparations. Cysteine residues were found to be proportionally distributed among the polypeptides. Several laboratories have reported the presence of carbohyrates in HB,Ag (4, 5, 23), and some polypeptides are thought to be glycoproteins due to reactions with PAS reagent (23). In our hands, two of the polypeptides (P-2 and P-5) reacted with PAS reagent and might be glycoproteins. However, we hesitate to make a more definite statement for several reasons. First, there are nonspecific reactions in the use of PAS reagent with SDS gels (16). Second, molecular-weight estimates of glycoproteins vary with the extent of gel cross-linkage (28, 29), and our estimates for P-2 and P-5 were identical in 5, 7.5, and 10% acrylamide gels. Third, amino acid analyses of purified HB,Ag and its isolated P-2 component (unpublished data) failed to reveal the presence of amino sugars. More data must be obtained in this regard before a definite conclusion about glycoproteins can be made. In any case, the carbohydrate component does not appear to contribute to the antigenicity of the recognized HB,Ag determinants (16a, 32) as suggested by Chairez et al. (5). HB,Ag labeled with 3H, 14C, or 125I retained its biophysical, biochemical, and antigenic properties. SDS-PAGE of labeled HB,Ag demonstrated that the radioactivity comigrated with the polypeptides and in proportion to their protein content. Pronase digestion of iodinated material extracted from the P-1 through P-7 region of the gel established that the 1251 was indeed associated with the polypeptides. SDSPAGE electrophoresis of HB,Ag iodinated by either the CT or LP procedures revealed that a significant proportion of the 125I label was incorporated into low-molecular-weight material. The inability to fix the material in trichloroacetic acid and its resistance to Pronase digestion indicated that it was not protein in nature. This material corresponds to two (P-5 and P-6) of the six iodinated polypeptides described in an earlier report by Dreesman et al. (10). As recovered from the top of CsCl gradients, it possessed no antigenic activity recognized by antibodies to the established HBSAg determinants and probably represents unsaturated fatty acids iodinated during the CT and LP procedures. The molecular-weight estimates of the polypeptides described in this report are in reasonably good agreement with the major ones described earlier (13) and by others (6, 34). We have been unable to detect antigenic activity in HB,Ag components other than the P-1 through P-7 polypeptides, in contrast to the reports of others (11, 26).

No significant difference in the patterns of the radiolabeled polypeptides was observed after SDS-PAGE of HBSAg iodinated by either the CT or LP procedure. These data indicated that all of the polypeptides were accessible by both procedures and suggests that none of the polypeptides can be designated as internal or external to the structural organization of the 22-nm form of HBsAg. In other words, the probabilities that the tyrosine residues of the various polypeptides are on the particle surface are equal. In this context, the 22-nm form may be considered as a structure, the organization of which depends upon the relative affinities of the polypeptides to one another and is governed by the most favorable energy conditions for equilibrium. This is supported by the observation that these forms exist as a range of sizes in a normal distribution about a mean of 22 nm and may explain why the relative proportions of the polypeptides vary among the preparations. ACKNOWLEDGMENTS The Rockville Laboratory of the Molecular Anatomy Program is supported by the National Institute of Allergy and Infectious Diseases under Union Carbide's contract with the U. S. Energy Research and Development Administration. LITERATURE CITED 1. Bancroft, W. H., F. K. Mundon, and P. K. Russell. 1972. Detection of additional antigenic determinants of hepatitis B antigen by immunoprecipitation. J. Immunol. 109:420-425. 2. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917. 3. Bolognesi, D. P., and H. Bauer. 1970. Polypeptides of Avian RNA tumor viruses. 1. Isolation and physical

and chemical analysis. Virology 42:1097-1112. 4. Burrell, C. J., E. Proudfoot, G. A. Keen, and B. P. Marmion. 1973. Carbohydrates in hepatitis B antigen. Nature (London) New Biol. 243:260-262. 5. Chairez, R., S. Steiner, J. L. Melnick, and G. R. Dressman. 1973. Glycoproteins associated with hepatitis B antigen. Intervirology 1:224-228. 6. Chairez, R., F. B. Hollinger, J. P. Brunschwig, and G. R. Dressman. 1975. Comparative biophysical studies of hepatitis B antigen, subtypes adw and ayw. J. Virol. 15:182-190. 7. Crestfield, A. M., S. Moore, and W. H. Stein. 1963. The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J. Biol. Chem. 238:622-627. 8. Dane, D. S., C. H. Cameron, and M. Briggs. 1970. Virus-like particles in serum of patients withAustralia-antigen-associated hepatitis. Lancet 1:695698. 9. David, G. S. 1972. Solid state lactoperoxidase: a highly stable enzyme for simple, gentle iodination of proteins. Biochem. Biophys. Res. Commun. 48:464-471. 10. Dreesman, G. R., F. B. Hollinger, J. R. Suriano, R. S. Fujioka, J. P. Brunschwig, and J. L. Melnick. 1972. Biophysical and biochemical heterogeneity of purified hepatitis B antigen. J. Virol. 10:469-476. 11. Dreesman, G. R., F. B. Hollinger, R. M. McCombs, and J. L. Melnick. 1973. Alterations of hepatitis B

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24. Purcell, R. H., P. V. Holland, J. H. Walsh, D. C. Wong, and R. M. Chanock. 1969. A complementfixation test for measuring Australia antigen and antibody. J. Infect. Dis. 120:383-386. 25. Purcell, R. H., and J. L. Gern. 1975. Hepatitis B subunit vaccine: a preliminary report of safety and efficacy tests in chimpanzees. Am. J. Med. Sci. 270:395399. 26. Rao, K. R., and G. N. Vyas. 1973. Hepatitis B antigen activity in protein subunits produced by sonication. Nature (London) New Biol. 241:240-241. 27. Rice, R. H., and G. E. Means. 1971. Radioactive labeling of proteins in vitro. J. Biol. Chem. 246:831-832. 28. Russ, G., and K. Polakova. 1973. The molecular weight determination of proteins and glycoproteins of RNA enveloped viruses by polyacrylamide gel electrophoresis in SDS. Biochem. Biophys. Res. Commun. 55:666-672. 29. Segrest, J. P., R. J. Jackson, E. P. Andrews, and V. T. Marchesi. 1971. Human erythrocyte membrane glycoprotein: a re-evaluation of the molecular weight as detemined by SDS-polyacrylamide gel electrophoresis. Biochem. Biophys. Res. Commun. 44:390-395. 30. Shapiro, A. L., E. Vinuela, and J. V. Maizel, Jr. 1967. Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun. 28:815-820. 31. Shih, J. W.-K., and J. H. Hash. 1971. The N, 0-diacetylmuramidase of Chalaropsis species. III. Amino acid composition and partial structural formula. J. Biol. Chem. 246:994-1006. 32. Shih, J. W.-K., and J. L. Gerin. 1975. Immunochemistry of hepatitis B surface antigen (HB,Ag): preparation and characterization of antibodies to the constituent polypeptides. J. Immunol. 115:634-639. 33. Soulier, J. P., and A. M. Courouce-Pauty. 1973. New determinants of hepatitis B antigen (Au or HB antigen). Vox Sang. 25:212-234. 34. Vyas, G. N., E. W. Williams, G. G. B. Klaus, and H. E. Bond. 1972. Hepatitis-associated Australia antigen, proteins, peptides, and amino acid composition of purified antigen with its use in determining sensitivity of hemagglutination test. J. Immunol. 108:11141118. 35. Weber, K., and M. Osborn. 1969. The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4412.