JOURNAL OF VIROLOGY, Jan. 1975, P. 208-216 Copyright ( 1975 American Society for Microbiology

Vol. 15, No. 1 Printed in U.S.A.

Characterization of Type 5 Adenovirus Fiber Protein PRESTON H. DORSETT' AND HAROLD S. GINSBERG2* Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19174 Received for publication 23 August 1974

Type 5 adenovirus fiber protein was purified and subjected to chemical characterization. Equilibrium sedimentation ultracentrifugation analysis indicated that the intact fiber has a molecular weight of approximately 183,000. Denaturation and chemical analyses implied that the fiber consists of three polypeptide chains, each of about 61,000 mol wt. Mapping of tryptic peptides and electrophoretic separation of the constituent chains suggested that the intact fiber consists of two identical and one unique polypeptide chains. The type 5 adenovirus fiber, a major capsid protein, exists as a shaft (2 by 16 nm) terminated on one end by a sphere 4 nm in diameter (22, 25). The shaft inserts into the penton base unit to form a penton, 12 of which then form the vertexes of the icosahedron. Morphologically, fiber purified from adenovirus-infected cell lysates, which contain a great excess of unassembled capsid proteins, appears identical to that seen in the intact virion (5, 24, 25). Hemagglutination studies indicate that the fiber contains both a type-specific and a subgroup-specific antigen (16, 17, 22). The typespecific antigen has been tentatively assigned to the terminal knob, whereas the subgroupspecific antigen is thought to reside in the shaft. Although several laboratories have purified and characterized the fiber, its precise molecular weight and subunit composition remains uncertain. The sedimentation coefficient of the native fiber protein has been determined to be 5.8 to 6.1S, both in sucrose gradients and in the analytical ultracentrifuge (10, 18). However, the molecular weight of the native fiber has been variously estimated from 60,000 to 500,000. Kohler (7), Valentine and Pereira (22), and Pettersson et al. (18) determined the molecular weight of the native fiber molecule to be 60,000 to 80,000; Sundquist et al. (20) determined by analytical ultracentrifugation that the molecular weight of the native fiber was approximately 200,000 and that the intact structure consisted of three polypeptide chains, each of 60,000 to

65,000 mol wt; and Maizel and his co-workers (13) estimated that the native fiber molecule consisted of eight polypeptide chains, each of which were 61,000. Such ambiguities in the molecular weight and subunit composition of the fiber protein presented additional difficulties in understanding the regulation of capsid protein synthesis and morphogenesis of the virion. Therefore, studies were initiated to investigate the physical and chemical structure of the type 5 adenovirus fiber protein. This communication reports the results of those studies.

MATERIALS AND METHODS Tissue culture cells and virus. Type 5 adenovirus was grown in suspension cultures of KB cells. The methods for cell growth, viral propagation, and extraction of viral antigens have been previously described (1, 9, 10, 24). Radioactive labeling of viral protein. KB suspension cultures (3.0 x 105 cells/ml) were infected and maintained in Eagle minimum essential medium containing one-half the normal concentration of amino acids and 5% (vol/vol) dialyzed calf serum. Twelve to 14 h after infection, when host cell protein synthesis was minimal (1), "4C-labeled amino acid mixture (New England Nuclear Corp., Boston, Mass.) was added to a final concentration of 0.5 gCi/ml. The infected cells were harvested 24 to 48 h later, and the viral proteins were extracted. Purification of type 5 adenovirus fiber protein. The fiber protein was purified by the procedure previously described (10) for adenovirus antigens with the following modification. After sequential chromatography on columns of hydroxylapatite and DEAEcellulose, the fiber protein was concentrated by pres1 Present address: Department of Microbiology, sure dialysis to approximately 2 ml and dialyzed The University of Tennessee Center for the Health against 0.1% (vol/vol) ampholine, pH 7 to 10. The protein solution was then subjected to isoelectric Sciences, Memphis, Tenn. 38163. 2Present address: College of Physicians and focusing in ampholine-sucrose, pH 5 to 8, by the Surgeons, Columbia University, New York, N.Y. method of Svensson (21). The potential was increased 10032. gradually to 600 V and maintained for 48 h. At the 208

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TYPE 5 ADENOVIRUS FIBER

end of electrofocusing, 1.5-ml fractions were collected from the bottom of the column, and each was assayed for pH, protein, and the viral antigens. A single homogeneous fiber peak was obtained with an isoelectric point of 6.15. Analytical ultracentrifugation. Sedimentation coefficients and molecular weight determinations were performed in a Spinco model E ultracentrifuge equipped with an RTIC temperature control and an electronic speed control. All experiments were conducted in 0.01 M Tris-hydrochloride (pH 7.6) containing 0.1 M NaCl and 0.005 M EDTA. Sedimentation coefficients were determined in a single-sectored cell with a 12-mm solution column. Rotor speed was 52,640 rpm, and the sedimentation boundary was photographed at 8-min intervals for the first 40 min and 16-min intervals thereafter. Temperature corrections and computerized sedimentation coefficient determinations were done according to Incardona et al. (4). Sedimentation equilibrium determinations of molecular weight were done in a double-sectored synthetic-boundary cell by the miniscus depletion technique of Yphantis (26). Interference optics were utilized, and photographs were made every 4 h. Equilibrium was determined to be attained when there was less than 0.01 fringe shift per h. The rotor speed of 12,000 or 14,000 rpm was determined as described by Yphantis (26). The partial specific volume of the fiber protein was calculated from the amino acid composition (19). Tryptic digestion and peptide mapping. Radioactive fiber protein (5 x 10' counts/min) in distilled water was denatured by boiling for 3 to 4 min. One-tenth volume of 1 M NH4HCO,-NH4OH buffer, pH 8.6, was added, and the protein was digested for 6 h with 1% (wt/wt) trypsin (TPCK-treated, Calbiochem). After digestion, the solution was dried in a stream of nitrogen, and the peptides were suspended in 0.01 M NH4OH. The tryptic fragments were separated by peptide mapping on a cellulose thin-layer plate (Brinkman Instruments, Inc., Westbury, N.Y.). The plate was sprayed evenly with electrophoresis buffer (pyridineacetic acid-water; 50:2:948, pH 6.5) and subjected to electrophoresis at 800 V for 10 min at 5 C. The plate was then blotted to remove excess buffer and the sample (2 to 5 gliters) was applied with a Drummon microcapillary pipette. Electrophoresis was performed at 800 V for 45 min at 5 C, after which the plate was dried at 40 C. Chromatography was performed at right angles to the line of electrophoresis in pyridine-n-butanol-acetic acid-water (12:15:3:12) until the solvent front reached the top of the plate. After drying, the plate was processed for autoradiography. Autoradiography of peptide maps. Kodak NoScreen N54T X-ray film was placed in contact with the peptide map and sealed in a Kodak exposure holder. Uniform contact of the plate and film was maintained by placing the holder in a press and applying pressure. Autoradiograms were exposed for 14 to 30 days, after which the film was developed in Kodak X-ray developer.

209

Amino acid analysis. Amino acid analysis was performed on a Beckman 120 C amino acid analyzer by the two-column method (15). Tryptophan was measured spectrophotometrically (2). Cysteine was determined as S-carboxymethyl cysteine after reduction and alkylation. Sepharose column chromatography. Gel filtration on a column (0.9 cm by 92 cm) of Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, N.J.) in the present of 5M guanidine-hydrochloride (GuHCl; Schwartz-Mann Chemicals, Orangeburg, N.Y.) was performed by the method of Fish et al. (3) except that the column buffer consisted of 0.05 M Tris-acetic acid, pH 8.6, containing 0.01 M EDTA and 5M GuHCl. V. and Vt were marked with blue dextran and phenol red, respectively. Reduced and alkylated bovine serum albumin, ovalbumin, and cytochrome c were employed as protein markers. Molecular weights were calculated by plotting the log of the molecular weight against the distribution constant of the standards (3). Sucrose gradient analysis. Fiber protein was sedimented through 4.6-ml linear gradients of 5 to 20% sucrose in 0.1 M Tris-acetic acid, pH 8.6, containing 0.01 M EDTA and 0.1 M NaCl. In some experiments, 9 M urea or 6 M GuHCl was included in the gradient. Centrifugation was carried out in an SW65 rotor at 15 C. Approximate sedimentation coefficients were calculated by the method of Martin and Ames (14) with bovine serum albumin as a reference (11). Polyacrylamide gel electrophoresis. Nondenaturing gels consisted of 6.0% (wt/vol) acrylamide, 0.2% (wt/vol) N,N'methylene-bisacrylamide and 0.1 M Tris-glycine, pH 9.0. Polymerization was catalyzed by ammonium persulfate (0.07% wt/vol) and N,N,N', N'-tetramethylethylenediamine (0.1% vol/vol). Electrophoresis buffer consisted of 0.1 M Tris-glycine, pH 9.0, containing 0.01 M EDTA and 0.1% (vol/vol) 2-mercaptoethanol. The gels were subjected to electrophoresis at 1 mA/gel for 2 h to remove catalyst and nonpolymerized material, after which the protein sample, containing 6% sucrose and bromophenol blue, was layered on top of the gels. Electrophoresis was performed at 1 V/cm for 1 h, after which the voltage was increased to 3 V/cm and maintained at that voltage until the dye marker approached the bottom of the gel. For some experiments, this gel system was modified to contain denaturants. (i) Sodium dodecyl sulfate (SDS) gels were prepared according to the method of Kiehn and Holland (6) except that the gels and buffers contained 8 M urea. The SDS gels were subjected to electrophoresis at 3 mA/gel for 1 h, after which the sample containing 6% (wt/vol) sucrose and bromophenol blue was applied to the top of the gel. Electrophoresis was performed at 3 V/cm until the marker dye neared the end of the gel, usually about 15 h. (ii) Urea gels (9 M) were prepared as follows. After polymerization, the gels, containing 9 M deionized urea, were removed from the tubes and immersed in 0.1 M Tris-glycine buffer, pH 9.0, containing 9 M urea, 0.001 M dithiothreitol (DTT) and 0.2% (wt/vol) Brij 35. After soaking for 48 h in two buffer changes,

210

DORSETT AND GINSBERG

the gels were replaced into slightly larger gel tubes by suction. The protein sample in 0.01 M Tris-glycine buffer, pH 9.0, containing 10 M urea, 0.001 M DTT, 0.2% (wt/vol) Brij 35, 6% (wt/vol) sucrose, and bromophenol blue was heated at 100 C for 3 min, cooled, and layered on top of the gels. Electrophoresis was performed as described above. Analysis of acrylamide gels. Acrylamide gels containing radioactive protein were processed as previously described (6). Gels containing 125I were counted in a Packard auto-gamma counter and gels containing 14C or 3H were measured in a Packard liquid scintillation spectrometer. Reduction and alkylation of fiber protein. Fiber protein was dissolved in 0.1 M Tris-acetic acid buffer, pH 8.6, containing 0.01 M EDTA and 6 M GuHCl. The reaction vial was flushed with N2, after which DTT (15.4 mg/ml) was added. Reduction was allowed to proceed for 2 h at 25 C under a N2 atmosphere, after which iodoacetic acid (22.2 mg/ml) was added, and then the sample was incubated at 37 C for 2 h. lodination of fiber protein. Velicer and Ginsberg modification (23) of the method of McConahy and Dixon (12) was employed to conjugate 125I with the purified fiber protein.

RESULTS

Characteristics of the purified fiber protein. Before characterization, the purified fiber was analyzed for contaminating material. When 106 counts/min of "C-labeled amino acidlabeled uninfected cell lysate was added to 3H-labeled amino acid-labeled infected cell lysate, and the fiber was purified, 14C could not be detected above background. This purified fiber protein then was analyzed by acrylamide gel electrophoresis under nondenaturing conditions. A single peak of 3H-labeled fiber was obtained which was not contaminated with "4C-labeled host protein (Fig. 1A). Since the predominant antigenic determinant of the fiber is type specific, immunoelectrophoresis of fiber was performed and developed with antisera prepared against lysates of KB cells infected with type 2 or type 5 adenovirus. A single precipitin line was obtained with antiserum against type 5 adenovirus-infected cells (Fig. 1B). By these criteria, the purified fiber protein was judged to be free of contaminating material of either host or viral origin. Molecular weight of the intact fiber. The sedimentation coefficient of fiber was determined in the analytical ultracentrifuge. Two protein concentrations (2.58 and 1.22 mg/ml) were centrifuged in separate runs. Schlieren photographs showed that the fiber sedimented as a single symmetrical peak, indicating a lack of aggregation and demonstrating further the purity of the protein. The s21W values were calcu-

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lysate.

lated to be 5.8 and 6.0, respectively. These data were essentially in agreement with a 52o,w of 6.2 calculated for the fiber from sucrose gradients. Equilibrium centrifugation according to Yphantis (26) was employed to determine the molecular weight of the native molecule. Three protein concentrations were employed (2.58, 1.22, and 0.66 mg/ml) in separate experiments. A plot of the log of the fringe displacement (concentration) versus the square of the radius yielded a straight line for all concentrations (Fig. 2), which also indicates that the fiber was pure and did not tend to aggregate under these conditions. The calculated molecular weights were 162,000, 180,000, and 184,000 for the respective protein dilutions. Chemical disruption of the fiber protein. Chemical denaturants other than detergents were utilized to disrupt the fiber protein into its constituent polypeptide chains. Purified fiber was subjected to denaturation with both 10 M urea and 6 M GuHCl, each of which disrupts most proteins into random coils. 1251-labeled

VOL. 15, 1975

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reduction and alkylation of the protein in 6 M GuHCl yields the approximate molecular weights of the polypeptide chains, if the column previously has been calibrated with protein standards (3). "C-labeled fiber was reduced and alkylated in 6 M GuHCl and subjected to chromatography on a Sepharose 4B column containing 5M GuHCl. Reduced and alkylated markers were chromatographed concomitantly with the fiber to serve as internal standards. The fiber subunits eluted as a single homogeneous peak with an elution volume slightly greater than bovine serum albumin (Fig. 4). The apparent molecular weight of this polypeptide chain was 60,500. Polyacrylamide gel electrophoresis in the presence of SDS, which separates proteins on the basis of molecular size, was also employed to determine the size of the fiber subunit. "C-labeled fiber was reduced and alkylated, in the presence of 10 M urea, and dialyzed against 10 M urea for 18 h. SDS was added to yield a 1% (wt/vol) concentration, and the solution was dialyzed for 4 to 5 days against 0.01 M Tris-glycine buffer, pH 9.0, containing 0.1% (wt/vol) SDS, 10 M urea, 0.1% (vol/vol) 2-mercaptoethanol and 0.01 M EDTA. After dialysis, the

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fiber in 0.01 M Tris-hydrochloride, pH 8.5, was made to contain either 6 M GuHCl or 10 M urea and 0.2% (wt/vol) Brij 35. DTT (0.1 mg/ml) was added to each solution to prevent disulfide interchange, after which the fiber solution in 10 M urea was placed in boiling water for 3 min. The denatured proteins were analyzed by centrifugation on sucrose gradients containing either 9 M urea, 0.2% (wt/vol) Brij 35 and 0.1 mg DDT per ml, or 6 M GuHCl and 0.1 mg DTT per ml. The 6S fiber molecule was disrupted into approximately 3S subunits by either treatment (Fig. 3). Since some subsequent analyses required the absence of guanidine, heating in 10 M urea, 0.2% (wt/vol) Brij 35 and 0.1 mg of DTT per ml was routinely utilized to disrupt the fiber molecule. Estimation of the size of the fiber subunit. The previous data indicated that the fiber consists of two or three polypeptide chains, each having approximately the same molecular weight. Therefore, the molecular weight of the constituent polypeptide chains of the fiber protein was determined by two independent techniques. Gel filtration of agarose in 5M GuHCl after

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FIG. 4. Chromatography of guanidine-denatured fiber on Sepharose 4B in the presence of 5M GuHCI, pH 8.6. Denatured 14C-labeled fiber was chromatographed concomitantly with similarly denatured bovine serum albumin and ovalbumin. V0 was marked with blue dextran and Vt was marked with phenol red. The column was equilibrated and developed with 0.1 M Tris-acetic acid, pH 8.6, containing 0.01 EDTA and 5M GuHCI. Symbols: 0, "4C-labeled fiber protein; and --, markers.

protein-SDS solution was subjected to SDSacrylamide gel electrophoresis. Reference proteins in the same buffer were also included in the same electrophoretic run. The electrophoretic migration of the fiber corresponded to a protein with a molecular weight of about 61,000 (Fig. 5). Thus, these data, in conjunction with the analytical ultracentrifugation, indicate that the intact fiber molecule is composed of three polypeptide chains of approximately 61,000 daltons. These findings are consistent with those reported for the characteristics of the type 2 adenovirus fiber (20). Amino acid analysis. Table 1 shows the average of five separate amino acid analyses of the fiber protein. Amino acid analysis of fiber alkylated in the presence of 6 M GuHCl yielded the same results as material analyzed after reduction and alkylation in 6 M GuHCl, indicating that the half-cystine residues, which are present on the average of one residue per polypeptide chain, exist in the free-sulfhydryl form. It is noteworthy that these data are similar to, but not identical with, the amino acid content of the type 2 adenovirus fiber (20), which shares subgroup antigens with the type 5 fiber (22). For example, the cysteine content of the type 5 fiber was about one-half of that reported for type 2 (20). Peptide mapping of fiber. Enzymatic and

J. VIROL.

chemical cleavage of the fiber protein was employed in conjunction with amino acid analysis in order to determine the chemical uniqueness of the three fiber polypeptide chains. Since amino acid analysis indicated that there was a, mean of 34 residues of arginine and lysine calculated per polypeptide chain of 61,000 daltons (Table 1), one would expect, after tryptic digestion, a minimum of approximately 35 peptides if the 3 chains were identical, or a greater number, depending on the number of distinct peptides and chemically unique polypeptide chains. "C-labeled fiber protein was subjected to tryptic digestion, and the resulting peptides were separated by peptide mapping. At least 72 different peptides were apparent (Fig. 6). This would indicate that the intact molecule must be composed of more than one chemically distinct polypeptide chain, and would be consistent with two species of polypeptide chains. Separation of the fiber polypeptide chains. The previous data indicate that the fiber molecule is composed of more than one chemically 6 0-

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TYPE 5 ADENOVIRUS FIBER

VOL. 15, 1975

TABL.E 1. Amino acid compositiona of the fiber protein from type 5 adenovirus Residues Amino acid

Mol%

(61o,l000 protein)

Lysine .................... Histidine .................. Arginine ................... Aspartic acid ............... Threonineb ................. Serineb .................... Glutamic acid .............. Proline .................... Glycine .................... Alanine .................... Valine .................... One-half cystinec ........... Methionine ................ Isoleucine .................. Leucine .................... Tyrosine ................... Phenylalanine ............. Tryptophand ...............

6.1 1.2 1.4 12.2 11.4 8.1 6.3 4.4 8.9 8.1 5.9 0.4 1.3 4.5 13.3 2.3 3.8 1.1

27.4 6.1 6.2 57.0 54.1 36.8 30.6 19.0 42.2 37.8 27.0 1.1 7.2 20.4 63.7 7.3 18.1 5.1

Mean of determination of five samples. b Extrapolated to zero hydrolysis. c Determined as S-carboxymethyl cysteine after reduction and alkylation. d Determined spectrophotometrically by the method of Bencze and Schmid (2). a

unique polypeptide chain. Since peptide mapping data may yield erroneous estimates of the total peptides due to incomplete cleavage or selective loss of peptides, experiments were performed to separate the fiber subunits on the basis of electrical charge. "4C-labeled fiber was denatured in 10 M urea, Brij 35, and DTT and applied to denaturing acrylamide gels containing 9 M urea, Brij 35 and DTT (Fig. 7). Two distinct polypeptide chains were resolved by this method. The slower migrating polypeptide chain contained approximately twice the radioactivity as the faster migrating chain. Thus, these data are in accord with the peptide mapping data and indicate that the fiber molecule consists of three polypeptide chains, of which two are identical. It cannot be stated with certainty that the slower component was not merely an aggregate of the more highly charged protein, but this possibility seems unlikely since (i) the electrophoresis was performed in the presence of denaturant and nonionic detergent, and (ii) the separation was not dependent on size of the component, but upon ionic charge. DISCUSSION There is an apparent lack of correlation between the molecular weight and the sedimen-

213

tation coefficient for the fiber. If the fiber is assumed to be a prolate ellipsoid of 183,000 mol wt with an axial ratio of 8, the calculated sedimentation coefficient would be 8.3S. The 6S sedimentation coefficient observed for the fiber protein could result partially from concentration dependence, but this could not account for the entire discrepancy in S value. Since the fiber is not a true prolate ellipsoid, it may not behave according to these theoretical treatments. It seems clear, however, that the fiber cannot be a single polypeptide chain of 60,000 to 80,000 mol wt nor an aggregate with a molecular weight of 500,000 and exhibit a sedimentation coefficient of 6S. A molecular weight of 183,000 for the type 5 adenovirus fiber is in accord with that presented by Sundquist et al. (20) for type 2 adenovirus fiber, although these investigators did not determine whether the subunits were identical. The data presented in this communication are in conflict, however, with previous estimates of the fiber molecular weight. Valentine and Pereira (22) calculated a molecular weight of 70,000 for the type 5 adenovirus fiber by estimating the volume of the molecule from electron micrographs. Such an estimate could be affected by shrinkage or distortion of the molecule during fixation and staining. Two other earlier studies with analytical ultracentrifugation determined the molecular weight of the native molecule to be 70,000 to 80,000 (7, 18). These studies employed the determination of a diffusion constant and subsequent calculation of the molecular weight. Such determinations have a greater dependence on protein concentration and are more prone to error than the equilibrium ultracentrifugation techniques utilized in this investigation and by Sundquist et al. (20). Maizel and his co-workers (13) calculated that the native molecule consisted of 8 subunits of 62,000 mol wt each, thereby producing a molecule of approximately 500,000 mol wt. These calculations were made from SDS-acrylamide gel electrophoerograms by determining the proportion of radioactivity migrating as the fiber subunit when virions labeled with 3H-labeled amino acids were disrupted and analyzed. Such determinations are dependent on the fiber polypeptide chains being clearly separated from any other virion polypeptide chain. The molecular weight of the penton has been estimated to be 400,000 to 500,000 with a sedimentation coefficient of about 10.5S (U. Pettersson and S. Hoglund, personal communication, cited by Velicer and Ginsberg [23]). The molecular weight of the penton base has been estimated to be 210,000 (8) and 220,000 (22),

214

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FIG. 6. Peptide mapping of fiber. Heat-denatured fiber was subjected to digestion by trypsin (1% wt/wt); the resulting peptides were separated by peptide mapping on cellulose thin-layer plates. A 5 uliter sample of the tryptic digest, containing 37,500 'IC counts/min, was subjected to electrophoresis at 800 V for 45 min in pyridine-acetic acid-water buffer, pH 6.5, followed by ascending chromatography in n-butanol--acetic acidpyridine-water. The origin is in the lower central portion of the plate. The direction of electrophoresis was horizontal and that of chromatography was vertical. The peptides were detected by autoradiography, and each peptide was marked on the film with a dot. Some of the peptides that were visible on the film were not reproduced clearly on the photograph.

but it seems likely that this is underestimated. Thus, a molecular weight of 180,000 for the fiber would be reasonable since the penton consists of one base unit and one fiber. In addition, Maizel et al. (13) indicated that the protein ratio of fiber

to penton is approximately 1 in SDS gels of disrupted virons. The data presented indicate that the type 5 adenovirus fiber is composed of three polypeptide chains of 61,000 mol wt, which are noncova-

TYPE 5 ADENOVIRUS FIBER

VOL. 15, 1975

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tyrosine and tryptophan in proteins. Anal. Chem. 29:1193-1196. Fish, W. W., K. G. Mann, and C. Tanford. 1969. The estimation of polypeptide chain molecular weights by gel filtration in 6 M guanidine hydrochloride. J. Biol. Chem. 241:4989-4994. Incardona. N. L., N. Notarius, and J. B. Flanagan. 1971. Measurement of temperature within the sample cell during sedimentation velocity experiments. Anal. Biochem. 40:267-280. Ishibashi, M., and J. V. Maizel, Jr. 1974. The polypeptides of adenovirus. VI. Early and late glycoproteins. Virology 58:345-361. Kiehn, E. D., and J. J. Holland. 1970. Synthesis and cleavage of enterovirus polypeptides in mammalian

cells. J. Virol. 5:358-367.

7. Kohler, K. 1965. Reinigung and characterisierung zweier proteine des adenovirus Type 2. Z.. Naturforsch.

(-) (+) FRACTION 20b:747-752. FIG. 7. Acrylamide gel electrophoresis of the fiber subunits. rC-labeled fiber was denatured in 10 M 8. Laver, W. G.. and N. G. Wrigley. 1969. Removal of pentons from particles of adenovirus type 2. Virology urea cotaining0.2% Brj 35 and an 0.01 MDTT urea 0.2% Brij M DTT by containing 39:599-605. boiling for 3 min. After denaturation, the polypeptide 9. Lawrence, W. C., and H. S. Ginsberg. 1967. Intracellular uncoating of type 5 adenovirus deoxyribonucleic acid. chains were separated by electrophoresis on 6% acrylJ. Virol. 1:851-867. amide gels containing 9 M urea, 0.21% Brij 35 and 10. Levine, A. J., and H. S Ginsberg. 1967. Mechanism by 0.001 M DTT, pH 9.0. which fiber antigen inhibits multiplication of type 5

bound into a multimeric molecule of

lently lentlyimately approximately

mol wt. Both peptide 183,000 mol Both peptide

adenovirus. J. Virol. 1:747-757.

11. Loeb, G. I., and H. A. Sheraga. 1956. Hydrodynamic and thermodynamic properties of bovine serum albumin at

low pH. J. Phys. Chem. 60:1633-1644. mapping and electrophoretic separation of the polypeptide chains suggest that two of the 12. McConahy, P. J., and F. J. Dixon. 1966. A method of trace iodination of proteins for immunologic studies. Int. chains in each molecule are identical, but the Arch. Allergy Appl. Immunol. 29:185-189. 1 Maizel, other chain has a different primary sequence. 13. J. V., Jr., D. 0. White, and M. D. Scharff. 1968. The polypeptides of adenovirus. II. Soluble proteins, These data are not surprising owing to the cores, top components and the structure of the virion. complex morphology of the native molecule. A VirologyR. 36:126-136. possible model would be that the subunits are 14. Martin, G., and B. N. Ames. 1961. A method for

identical polypeptide polypepte arranged that the the two two identical arranged so that determining the sedimentation behavior of enzymes: application to protein mixtures. J. Biol. Chem. chains form the shaft of the molecule and the 236:1372-1379. remaining chain forms the terminal knob. If this is the case, then the type-specific antigen 15. Moore, S., D. H. Spackman, and W. H. Stein. 1958. polystyChromatography of amino acids on sulfonated should be localized in one polypeptide chain. rene resins-an improved system. Anal. Chem. 30:1185-1190. The findings by Ishibashi and Maizel (5) that the fiber is a glycoprotein with only two of the 16. Norrby, E., and P. Skaaret. 1967. The relationship between soluble antigens and the virion of adenovirus polypeptide chains glucosylated is consistent type 3. III. Immunological identification of fiber antione of of the which one model in in which with thze the proposed proposed model with the gen and isolated vertex capsomere antigen. Virology 32:489-502. chains is chemically distinct from the other two. so

ACKNOWLEDGMENTS We sincerely thank Kathlyn Coll for her outstanding technical aid and Joseph Higgs for his expert assistance in propogation of cells. We are indebted to Nino Incardona, Florida State University, for assistance and helpful criticisms of the ultracentrifugation analyses. This investigation was supported by Public Health Service research grants AI-05731 and AI-0360, and training grant AI-203, from the National Institute of Allergy and Infectious Diseases, and by research contract DADA-17-70-C-0121 from the U.S. Army Medical Research and Development Commandh Department of the Army. LITERATURE CITED 1. Bello, L. J., and H. S. Ginsberg. 1969. Relationship between deoxyribonucleic acid-like ribonucleic acid synthesis and inhibition of host protein synthesis in type 5 adenovirus-infected KB cells. J. Virol. 3:106-113. 2. Bencze, W. L., and K. Schmid. 1957. Determination of

17. Pereira, H. G., and M. V. T. De Figueiredo. 1962. Mechanism of hemagglutination by adenovirus types 1, and 6. Virology 18:1-8. 2, 4P 18 Pettersson, U., L. Philipson, and S. Hglundr 1968 S Structural croteins of adenoviruses. II. Purification and characterization of the adenovirus type 2 fiber antigen. Virology 35:204-215. 19. Schachman, H. K. 1957. Ultracentrifugation, diffusion and viscometry, p. 32. In S. P. Colowick and N. 0. Kaplan, (ed.), Methods in enzymology, vol. 4. Academic Press Inc., New York. 20- Sundquist, B., U. Pettersson, L. Thelander, and L. Philipson. 1973. Structural proteins of adenoviruses. IX. Molecular weight and subunit composition of adenovirus type 2 fiber. Virology 51:252-256. 21. Svensson, H. 1962. Isoelectric fractionation, analysis and characterization of ampholytes in natural pH gradients. III. Description of an apparatus for electrolysis in column stabilized by density gradients and direct determination of isoelectric points. Arch. Biochem. Biophys. Suppl. 1:132-138.

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Characterization of type 5 adenovirus fiber protein.

Type 5 adenovirus fiber protein was purified and subjected to chemical characterization. Equilibrium sedimentation ultracentrifugation analysis indica...
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