Structural proteins of adenoviruses

VIROLOGY 33, 575-590 (1967)

Structural Proteins of Adenoviruses’

I. Purification and Characterization of the Adenovirus Type 2 Hexon Antigen

ULF PETTERSSON, LENNART PHILIPSON AND STEFAN HOGLUND

Division of Cell Biology, Department of Medical Microbiology and the Institute of Biochemistry,

Uppsala University, Uppsala, Sweden

Accepted July 24, 1967

Methods for partial purification of the structural proteins of adenovirus type 2 have been developed. The hexon antigen was extensively purified by a four-step procedure comprising agarose chromatography, DEAE chromatography, preparative polyacrylamide electrophoresis and sucrose gradients.

The final product was homogeneous by analytical ultracentrifugation, polyacrylamide electrophoresis, electron microscopy, and immunodiffusion.

Hexon antigen so prepared has a sedimentation coefficient of 12 S with a calculated molecular weight of 469,099 and is probably composed of subunits as revealed by electron microscopy. The monomer of hexon antigen contains only one antigenic determinant, which appears to be group specific.

Neutralizing antibodies were not induced with the purified hexon when it was iujected into rabbits.

INTRODUCTION

The structure of the adenoviruses has been extensively studied. The icosahedral arrangement of the structural proteins is supported by several investigations (Horne et al., 1959; Wilcox et al., 1963; Valentine and Pereira, 1965). The relationship between the soluble antigens associated with adenovirus reproduction and the structural proteins in the intact virion is well established (Wilcox and Ginsberg 1963a; Valentine and Pereira, 1965; Kbhler 1965).

The soluble antigens have been separated into three different structures by immuno- electrophoresis (Pereira et al., 1959) and by DEAE chromatography (Klemperer and Pereira, 1959; Philipson, 1960; Wilcox and Ginsberg, 1961). These 3 antigens have later been recognized as hexon, penton, and fiber, respectively, according to the nomenclature of Ginsberg et al. (1966). The 252 capso-

1 This work was supported by grants from the Damon Runyon Memorial Fund, the Swedish Medical Research Council, and the Swedish Can- cer Society.

meres of the virion thus consist of 240 hexon units and 12 vertex capsomeres or penton units (Valentine and Pereira, 1965). The fiber is one of the substructures of the penton.

Direct hemagglutination of the adenovirus has been ascribed to the virion (Rosen, 1960) and to a complex structure of 12 penton units (Norrby, 1966a). Indirect hemagglutination revealed in the presence of heterotypic antisera has been ascribed to the fiber and penton units of the virion (Pe- reira and de Figueiredo, 1962; Norrby, 1966b).

The aim of the present study is to obtain the structural proteins in a highly purified state, which fulfills available criteria for protein purity. A characterization of the immunological, morphological, physical, and chemical properties of these preparations may have important implications with re- spect to the biological role of the structural proteins of the virus. This report presents methods for partial purification of the soluble antigens. The hexon antigen has also

575

576 PETTERSSON, PHILIPSON, AND HijGLUND

been extensively purified and studied in detail.

MATERIALS AND METHODS

Viruses

The adenovirus type 2 prototype strain, originally obtained from Dr. Huebner, NIH, Bethesda, Maryland, was used. The proto- types of adenovirus type 5, type 7, and type 12 obtained from Dr. Svedmyr, Stock- holm, were also used in some experiments. Cell Cultures and Virus Inject&m

Virus was propagated in KB cells in spinner cultures grown in Eagle’s spinner medium (Eagle, 1959) with double amounts of amine acids, 5 % horse and 2 7, calf serum. The cells were infected at a multi- plicity of about 10 PFU/ceIl in spinner medium at a cell density of 3 to 5 X lo5 cells/ml. Two-liter batches of spinner culture were processed each time. The cells were harvested 72 hours after infection by oentrifuging and resuspending the cells in 0.05 M Tris-HCI buffer pH 7.4. After freezing and thawing 3 times or sonication for 5 minutes, the debris was removed by cnntrifugation. The supernatant was further purified by different methods. 32P- and 14C- labeled virus products were obtained by the procedure of tiberg and Philipson (1967) and Maize1 (1966)) respectively.

Exclusion Chromalography

Gel filtration on Sephadex G 100 and G 200 was performed on columns 1.5 cm X 30 cm, and for gel filtration on 4% agarose (Sepharose 4 B, Pharmacia Fine Chemicals) columns 2.5 cm X 45 cm were used. The following buffer was used for elution: 0.1 M glycine-O.01 M Tris adjusted to pH 8.0 with HCl (Tris-glycine buffer pH 8.0) containing 0.5 M NaCl, and 0.02% sodium a,zide to prevent bacterial contamination. The elution rate never exceeded 2 ml/cm2/hour. Fractions of 2 ml were collected.

Ion-Exchange Chromatography

DEAE-cellulose in granular form (What- man, Microgranular DE 32) was used. Chromatography was carried out in columns 1 cm X 40 cm. A small amount of Sephadex G 25 was packed into the bottom

of the columns to improve the flow rate. Samples of 1.5-3 ml previously dialyzed against 0.0.5 114 phosphate buffer pH 6.5 were applied to the columns. The columns were subsequently washed with 25 ml of the starting buffer prior to applying a salt gradient of O-O.5 M NaCl in 0.05 M phosphate buffer pH 6.5. Fractions of 2 ml were collected.

Polyacrylamide Electrophoresis

Chemicals and bu$ers. Acrylamide and N , N’-bismethylene-acrylamide were purchased from Fluka AG, Buchs, Switzerland. The preparations were recrystallized from chloroform and acetone, respectively, before use as originally described by Loening (1967). The buffers used were 0.05 M Tris- glycine pH 9.5 for preparative and analytical electrophoresis and 0.005 M Tris-0.05 M glycine buffer pH 8.0 for analytical runs. Concentration of monomer (T) was 5 % and the concentrat’ion of cross-linker (C) was 1% in the monomer solution calculated according to Hjerten (1962). Polymeriza- tion was induced by adding 0.01 ml of p- dimethylaminoproprionitrile and 0.025 ml of 40 % fresh ammonium peroxide sulfate to 5 ml of the monomer solution.

A small amount of buffer was carefuhy layered on top of the gel to prevent air inhibition of t’he polymerization.

The samples to be studied were dialyzed against 0.01 M Tris-glycine buffer pH 9.5 or 0.001 M Tris-0.01 M glycine pH 8.0 and added sucrose to a final concentration of 7 %. The continuous system for polyacrylamide electrophoresis was used throughout (Hjerten et al., 1965).

In order to avoid interference with the moving boundary of ammonium persulfate and other impurities in the gel mat.rix, the current was applied for 1 hour in analytical gels and for 3 hours in preparative gels prior to application of the samples. The gels were stained according to Maize1 (1966).

Analytical electrophoresis was performed in glass tubes (5 mm X 75 mm) at 20 V/ cm for 1 hour at 4”. A 0.1 ml sample was applied to each gel. Autoradiography of dried gels were carried out according to a

STRUCTURAL PROTEINS OF ADENOVIRUS 577

procedure described elsewhere (Prage el al., 1967).

Preparative electrophoresis was performed in Plexiglas tubes (1 cm X 12 cm) using 25 V/cm at +4” for 3 hours. The buffer con- tainers were connected by a pump to in- crease the buffer capacity. At completion the gel was disintegrated by Maizel’s technique (Maizel, 1966). During elution the flow rate was kept at 15 ml/minute; about 50 fractions, each containing 4 ml, were collected manually. The extraction of the proteins from the disintegrated gel was improved by freezing and thawing the fractions twice and then incubating for 1 hour at 37”.

Isoelectric Separation

The method developed by Svensson (1962), based on the formation and main- tenance of a pH gradient in a sucrose gradient in an electric field, was used. A pH gradient between pH 3 and pH 6 was used for the hexon antigen. The sucrose gradient ranged from 0 to 35 % sucrose. The ampholytes were purchased from LKB In- struments Ltd, Stockholm, Sweden. The voltage ranged between 200 and 800 V, and 2 ml fractions of the gradients were collected after 48 hours. The t’echnique has been described in detail by Vesterberg and Svensson (1966).

Agarose Electrophoresis

This was carried out as described by Hjerten (1963); 0.16% of agarose in 0.1 &’ Tris-acetic acid pH 8.6 was used as support- ing media in columns (1050 X 6 mm). The electrophoresis was run for 20 hours at 406 V.

Zonal Rate Centrifugation

This was performed in linear 5-25% sucrose gradients in phosphate-buffered saline (PBS). Centrifugation was carried out in swinging-bucket rotors at 100,000 g for 17 hours. The gradients were harvested by drop collection from the bottom of the tube.

Immunodiffusion

This was performed on glass slides covered with agar in which cylindrical wells

were cut out as described previously (Philip- son, 1965). Autoradiography of the precipi- tation pattern was also described in the previous report. A modified Mancini technique (Mancini et al., 1965) described elsewhere (Prage et al., 1967) was used for quantitative assay of the hexon and penton antigens.

Preparation of Antisera

Rabbits were given 1 ml intravenously of the antigen after DEAE chromatography or after further purification, and at the same time 1 ml was injected intramuscularly in Freund’s complete adjuvant (Difco Lab., Michigan) at the start of the immunization schedule. Intramuscular injections (1 ml) were repeated weekly for 3-6 weeks, and the animals were bled 1 week after the last injection. Specific antisera against the base of the penton antigen was obtained by adsorbing the antipenton sera with fiber antigen obtained from DEAE chromatography. The specificity was tested by immunodiffusion as shown in Fig. 1. Antisera against fiber antigen was obtained by adsorbing the antifiber sera with KB-cell extracts. The antisera against the different preparations of hexon antigen were examined without further treatment.

Complement-fixation. The micromethod of Sever (1962) was used. One unit of complement and 4 units of antigen were used in the tests determining antibody titers in a total volume of 0.125 ml; 4 units of antiserum were used when antigen titers were determined.

Hemagglutination was carried out in tubes with the samples serially diluted twofold in 0.4 ml saline. A 0.5 % suspension of rat blood cells was added in aliquots of 0.2 ml to each tube, and the pattern was read after incubation for 1 hour at 37”. Details of the technique have been reported by Rosen (1960). Indirect hemagglutination was assayed in the presence of antipenton antisera from adenovirus type 5, diluted fiftyfold.

Neutralization was carried out by mixing lo5 PFU with equal amounts of serial twofold dilutions of antisera in PBS. The mix- tures were incubated for 30 minutes at 37” and remaining infectivity assayed by

578

FIG. 1. Immunodiffusion of pure hexon (A) and DEAE preparations of penton (B) and fiber (C) antigens of adenovirus type 2. Antisera agairlst pure hexon (I), the base of the penton (2), the penton (3), and the fiber (4) were tested. (S) is a mixture of antisera against all three types of antigens.

the plaque technique in KB cells. A reduction of infectivity of 1.5 log units were considered significant. I2’eutralization at low pH was performed as described by Kjell6n (1966).

Virus infectivity was assayed by the plaque technique in KB-cells as previously described (Philipson, 1961).

Cytopathic e$ect of penton antigen. Serial twofold dilutions of the samples adjusted to isotonicity were added in O.l-ml volumes to monolayers of HeLa cells previously washed twice with PBS. After adsorption for 30 minutes at 37”, Eagle’s ME&I with 10% calf serum was added to the cultures and the results were read after 24 and 48 hours.

Electron Microscopl~

A negative contrast technique was em- ployed; 1% uranyl acetate (UAc) in 0.2 M ammonium acetate at pH 4.6, or 2% phos- photungstic acid (PTA) adjusted with ammonium hydroxide to pH 7.6 were used as contrasting agents. The samples were

applied to carbon-coated platinum disks, and the excess was drawn off with a piece of filter paper. A drop of the contrasting agent (UAc or PTA) was added to the sample, and the excess of liquid was quickly removed by a piece of filter paper. By this technique it was possible to prepare samples of particles surrounded by a very thin layer of contrasting agent (Hiiglund, 1967). The specimens were examined in the Siemens Elmiskop I at, a magnification of 80,000 using an operating voltage of 60 kV.

Stroboscopy

The micrographs of negatively contrasted adenovirus particles were enlarged photo- graphically to 1 X lo6 times. These photo- graphs of individual virus particles were placed beneath a camera and were then centered and rotated with angles eorre- sponding to 5- or 6-fold symmetry (72” or SO”) and repeatedly photographed by the Markham technique (Markham et al., 1963).


TABLE 1 PURIFICATION AND RECOVERY OF VIRIONS AND STRUCTURAL PROTEINS OF ADENOVIRUS TYPE 2

Step Method

Hexon Penton Virus in- Proteina

purification ReFzr” Fs$ ,Eery rsk t$$$ 0 factor (To) factor (%I

IA Agarose chromatography 3.1 73 2.3 55 1.7 50-100 IB Freon and RbCl 3.4 68 2.3 35 1.2 70-100 II DEAE-cellulose Hexon 10.9 80 8.7 52 5.3 -

Penton 10.1 III Acrylamide electrophoresis

and sucrose gradient Hexon 5.3 40 2.1 - - - Steps I-III for hexon antigen 179-196 22-23 46 - - -

a Protein purification refers to reduction in protein content measured by the Folin procedure in corn- parison with the immediate previous step.

b Recovery was estimated from CF or radial diffusion tests as described in Methods. Values given are the mean of 2 or 3 experiments. The purification factor was obtained by multiplying the fraction recovered with the protein purification factor.

Chemical Analysis

Amino acid analysis. Samples of 0.25-0.5 mg of hexon antigen and l-2 mg of KB- cell proteins were hydrolyzed in 6 N HCl in sealed evacuated tubes at 110” in an oil bath. Analysis of acid hydrolyzates was carried out according to the procedure of Moore et al. (1958) using Beckman/Spinco model 120 and 120 B amino acid analyzers (Spackman 1960). Tryptophan was det,er- mined spectrophotometrically according to Beaven and Holiday (1952).

Protein. Protein concentration was determined by the Folin procedure after dialysis of samples against PBS (Lowry et al., 1951).

Carbohydrate Analysis

A. Neutral sugars. The analysis was made by paper chromatography in a butanol:pyridine: water (6:4:3) system. Prior to this procedure the sample was subjected to hydrolysis, neutralization, and ion exchange chromatography as described by Lundblad (1967). Glucose was determined quantita- tively with the aid of glucose oxidase (Hjelm and De Verdier, 1963).

B. Hexoseamines. After hydrolysis the analysis was made according to Stoffyn and Jeanloz (1954) and by chromatography in a pyridine : ethyl acetate : water: acetic acid system (5 : 5 : 3 : 1) (Lundblad, 1967).

Analytical ultr~centr~jugat~~. Analytical

ultracentrifugation was performed in a Model E Spinco instrument using a capillary type synthetic boundary cell at 20” and a speed of 59,780 rpm. Schlieren optics were used for registration. The diffusion coefficient was determined in the same type of cell with interference optics and at a speed of 4908 rpm.

RESULTS

Separation of Virions from tural Protein

Soluble Struc-

The primary step in the purification of the structural proteins was the separation of virions from the structural units. Two methods were investigated.

Method A. The extract from infected KB-cells was first extracted by Freon and subsequently layered on top of rubidium chloride with a density of 1.40 according to Green and P&a (1963) and centrifuged at 45,000 g for 45 minutes. The virions were layered in a sharp band at the interface, and the soluble units were recovered from the zone above the virion band. The purification factor was assayed by measuring CF-antigen titers against specific antisera for the respective antigens and by a modified Mancini technique. As seen in Table 1, the purification factor for these steps were 2.3 for the hexon and 1.2 for the penton

580 PETTERSSON, PHILIPSON, AND HOGLUND

FIG. 2. Agarose exclusion chromatography of a cell extract from KB cells infected by adenovirus type 2. Percent transmission (-) was recorded at 254 ~QJ. The hexon (X-X), the penton (O-O), and the fiber (A-A) were monitored by immunodiffusion. Infectivity (-), direct HA (O-O), and indirect HA (+-+) were also recorded.

structures. The recovery was 68% for the hexon and 35 % for the penton.

Method B. Agarose chromatography was attempted as an alternative procedure for separation of virions from soluble units. The procedure used is reported in Materials and Methods. Figure 2 shows the elution diagram of crude cell extracts on 4% agarose columns. The exclusion limit of 4% agarose correspond to a molecular weight of about S X lo6 for spherical particles. The intact virion and thus infectivity elutes as ex- pected in the void volume. The penton antigen elutes with a peak at 5&60%, the hexon 6&65% and the fiber at 65-70% of the bed volume, approximately.

Direct hemagglutination was detectsed only in the peak corresponding to the virions; however, a structural complex containing the 12 vertex capsomeres as described by Norrby (1966a) would also elute at the void volume in this system. Indirect hemagglutination was demonstrated in the peak eluting at the void volume and in the area corresponding to the penton antigen.

When methods A and B were compared with regard to purification achieved, it appears that the purification of hexon units was the same with the two procedures, but the recovery of penton units was slightly higher with agarose chromatography as seen in Table 1. The discrepancy could

probably be explained by disintegration of penton units into fibers in method A. This interpretation is supported by a higher yield of fiber units when the latter procedure was used.

Since there is a significant overlapping between hexon, penton, and fiber units on agarose chromatography, all fractions showing immunological activity were collected in a single pool for further purification.

The virions from the first peak in agarose chromatography or RbCl centrifugation were purified further by centrifugation in CsCl isodensity gradients according to Green and P&a (1963) and the recovery of infectivity (50-100 %) was the same irrespec- tive of whether Freon-RbCl-CsCl or agarose-Freon-CsCl purification was used.

Separation of Soluble Antigens by Chroma- tography on DEAE-Cellulose

The modified technique for DEAE chromatography reported in Materials and Methods gives an improved separation of fiber and penton antigens. Figure 3 shows a typical elution diagram after DEAE chromatography. The fiber antigen is eluted in the first peak, which eluted before the salt

FIG. 3. DEAE-cellulose chromatography of the soluble antigens after Freon treatment and centrifugation on a cushion of rubidium chloride. Opti- cal density at 280 rnp (---) was recorded as well as indirect HA (+---f). The hexon (X-X), the penton (O-O) and the fiber (A-A) were monitored by immunodiffusion. Peaks of indirect HA were detected only under the areas of the fiber and the penton antigens, but low titers were also demonstrated at the peak of the hexon


FIG. 4. Analytical polyacrylamide electrophoresis at pH 8.0 of fiber (left) and penton antigen (right) obtained from DEAE-cellulose.

gradient is applied. The penton cannot be detected in this peak. Antigenic material from t’he host cells was, however, abundant in the first peak as revealed by immunodiffusion against anti KB-cells sera. The heterogeneity of the fiber material at this step is revealed by analytical polyacrylamide electrophoresis as seen in Fig. 4.

The penton antigen elutes in the middle area after the gradient is introduced in the columns. Fiber antigen was irregularly revealed in this area, probably due to the rapid decay of the penton structure. When the peak of the penton antigen after DEAE chromatography was collected and studied by analytical polyacrylamide electrophoresis, 7-10 bands were detected as shown in Fig. 4. Peaks of indirect hemagglutination were detected in the area under the fiber and the penton antigen, but the hemagglu- tinating activity of the penton antigen showed a great tendency to tail with low titers into the peak of the hexon antigen as seen in Fig. 3. The hexon antigen elutes in t.he third peak (Fig. 3). The peak of immunological activity appears to coincide with

the peak of UV-adsorbing material. The hexon recovered from the ion-exchange chromatography was contaminated with penton antigen as revealed in some cases by cytopathic effects in low titers (X-W) and indirect hemagglutination.

Analytical polyacrylamicle electrophoresis of hexun antigen. Analysis of the hexon material from DEAE chromatography on polyacrylamide electrophoresis revealed 6-S bands as shown in Fig. 5. It was, however, observed that electrophoresis of the hexon antigen requires precautions with regard to purification and prerun of the gels as described in Materials and Methods, since the hexon antigen and the impurities will other- wise migrate with the front in gel electrophoresis, as also shown in Fig. 5. When 32P- or 14C-labeled material was used the hexon peak from ion-exchange chromatography contained high activity of the respec-

FIG. 5. Analytical polyacrylamide electrophoresis at pH 8.0 of hexon antigen after DEAE-cellulose chromatography on gels of different purity. Identical samples were applied to both gels and the runs were made at the same time. Recrystal- lization of monomer and prerun were omitted in Gel 1, but carried out according to Methods for Gel 2. The broad heavy stained band in Gel 2 corresponds to the hexon antigen.


FIG. 6. Autoradiography of I%- and 32P-labeled hexon antigen from DEAE-cellulose after analytical polyacrylamide electrophoresis at pH 8.0. 1, 1% pattern;$, protein pattern; and 9, 3*P pattern. The heavy stained band in the W pattern corresponds to the position of the hexon antigen.

tive isotopes but autoradiography of the polyacrylamide after electrophoresis revealed that the 32P label was confined to impurities and that not all of the 14C-labeled proteins were recovered in the hexon antigen bands, as demonstrated in Fig. 6.

Purification of the Hexon Antigen by Pre- parative Polyacrylamide Electrophoresis

Preparative polyacrylamide electrophoresis combined with mechanical sectioning of the gel according to Maize1 (1966) was attempted for further purification of the hexon antigen. The procedure is described in detail in Materials and Methods. The hexon antigen was eluted in 2-6 fractions in different experiments, when the antigen was monitored by immunodiffusion. The purification achieved with regard to protein in this step is shown in Table 1. When this material was analyzed by analytical electrophoresis only one band was seen, as shown in Fig. 7. For comparison, the hexon preparation after DEAE chromatography is also included in the same figure as well as a partially purified penton antigen preparation. The band corresponding to the penton structure is present in the hexon preparation after DEAE chromatography, but not after the subsequent preparative electrophoresis.

Analytical ultracentrifugation revealed, however, that the final product after preparative electrophoresis contained nonpro- tein contaminants with low sedimentation coefficients. Since these contaminants were

not present after ion-exchange chromatography, they were supposedly partially cross- linked polyacrylamide polymers. These impurities were, however, too large in size to be removed from the hexon antigen by gel filtration on Sephadex. Zonal rate sedimentation on linear sucrose gradients from 5 to 25 % as described in Materials and Methods removed the polyacrylamide impurities.

The final preparation of hexon antigen from adenovirus type 2 after a 180-fold purification from cell sap with regard to protein showed the following characteristics: only a single band on polyacrylamide electrophoresis, only a single symmetrical peak by analytical ultracentrifugation, homogeneous in the electron microscope, and immunologically pure by several criteria as shown below. The yield of hexon antigen was in the order of 0.25-0.50 mg from a 2- liter spinner culture.

Alternative Procedures to Purify Hexon Antigens

In order to find alternative methods of purifying the hexon antigen subsequent to

FIG. 7. Analytical polyacrylamide electrophoresis at pH 8.0 of partially purified penton antigen (1)) hexon antigen from DEAE-cellulose (2), and pure hexon antigen (3).

583

FIG. 8. Immunodiffusion of pure hexon antigen (I), hexon antigen from DEAE-cellulose @), and an extract from infected KB cells (3) against A., an antiserum, produced with adenovirus infected KB cells (S 1) and against B., antiserum produced with pure hexon antigen (S 2).

DEAE chromatography, the following methods were studied.

Method A. Gel filtration on Sephadex G 200 or G 100 contributed little to the purification of the hexon antigen since this antigen as well as the other structural units of adenoviruses elute in or close to the void volume. The fact. that fiber antigen also appears close to the void volume may be due to the elongated structure of this antigen although the assumed molecular weight (- 80,000) (Kiihler, 1965) would infer that this unit penetrated the gel matrix. Neither gel type gave additional purification with reference to analytical polyacrylamide electrophoresis.

Method B. Isoelectric separation as described in Materials and Methods was also tried for preparative separation and also for determination of the isoelectric point of the hexon antigen. However, the hexon antigen was found to be insoluble at the isoelectric point somewhere in the range pH 4.6-5.1. This method, thus was not suitable for preparative separation of the hexon antigen. The preparation recovered after isoelectric separation still showed 3 or 4 bands after analytical polyacrylamide electrophoresis.

Method C. Agarose electrophoresis. Hexon antigen after DEAE chromatography was separated by preparative agarose electrophoresis as described in Materials and Meth- ods, The hexon antigen material recovered after electrophoresis still contained 4 or 5 bands by analytical polyacrylamide electrophoresis, but the band corresponding to the penton antigen was not present.

It thus appears that only the outlined

purification steps involving agarose chromatography-DEAE chromatography-preparative polyacrylamide electrophoresis followed by sucrose gradients will give a hexon preparation fulfilling the criteria of protein purity described above.

Characteristics of the Hexon Antigen

A. Immunological properties. (1) Immu- nodi$usion. The pure hexon antigen from adenovirus type 2 gave only a single pre- cipitation line with antisera against infected KB cells, and antisera produced against pure hexon antigen revealed only a single line when tested against extracts from infected KB cells as shown in Fig. 8. No cross reactivity between the hexon and pento 1 antigens could be demonstrated (Fig. I).

Furthermore, the pure hexon antigen appears to be strictly group specific when tested with antisera against hexon antigens from adenovirus type 5, 7, and 12 as shown in Fig. 9. DEAE preparations were used for producing these sera. Only one immunological specificity was revealed. When hexon was recovered from 32P-labeled cells, no 32P could be detected in the specific pre- cipitate by the autoradiographic technique.

(2) Complement fixation. When antisera prepared against DEAE-purified hexon was compared with antisera against pure hexon in the CF test, it was demonstrated that the former sera contained low titers of antibodies against both the penton and the fiber antigens, but in the antisera produced against pure hexon antigen, antifiber and antipenton antibodies could not be detected,


FIG. 9. Immunodiffusion for detection of additional immunologic specificities of the pure hexon (A). The following antisera were tested: antihexon type 2 (2) and (4), antihexon type 5 (f), antihexon type 7 (S), antihexon type 12 (5).

TABLE 2

CROSS REACTIVITY OF ANTIHEXON ANTISERA BY COMPLEMENT FIXATION

Reciprocal CF titer against TABLE 3

Penton Fiber Pure antigen antigen fet-

from from att;i& DEAE DEAE ex-

cellu- cellu- tracta

Rabbit antisera against

lose lose

Pure hexon antigen

Hexon antigen from DEAE cellulose

256 <2 <2 <: 2

256 8 4 <2

a The KB-cell extract was obtained from cell packs (4 X lo7 cells/ml) by sonication or Freon treatment.

as seen in Ta’_le 2. Neither could antibodies

against KB-cells components be demonstrated in these antisera.

(3) Neutralizatim. It has been claimed repeatedly that purified preparations of hexon antigen can give rise to neutralizing antibodies against the respective adenovirus types (Wilcox and Ginsberg, 196313; Kasel, et al., 1966). The neutralizing capacity of antisera prepared against DEAE-purified and pure hexon antigen was therefore inves- t#igated. The former antiserum neutralized 1.5 log units of infectivity at a reciprocal titer of 128, while the latter neutralized only 1 log unit when tested undiluted; at a

twofold dilution, no significant neutralization was observed, as shown in Table 3. Both antisera showed the same antibody titers in the CF test against hexon antigen. Since it was recently suggested (Kjelkn, 1966) that heterotypic antisera could neu- tralize virus infectivity at low pH, the antihexon sera were also studied with regard to this property according to the technique of KjelMn (1966). Table 4 shows that antiserum against pure hexon failed to neu- tralize adenovirus type 2, even at low pH.

It thus appears that whatever the cause for the heterotypic neutralization at low pH, common antigenic determinants in the hexon antigen are probably not involved.

B. Electron microscopy. The highly purified hexon antigen was &died by electron microscopy with uranyl acetate as the contrasting agent. Figure 10 shows that essen- tially no aggregate could be okserved, and

NEUTRALIZATION OF ADENOVIRUS TYPE 2 INFECTIVITY BY DIFFERENT

ANTIHEXON SERA

Rabbit antisera againsta

Hexon antigen from DEAE cellulose

Pure hexon antigen

128 256

<2 256

a Three different antisera were examined; the mean values are given.

TABLE 4

NEUTRALIZATION OF ADENOVIRUS TYPE 2 INFECTIVITY AT PH 2.5 WITH DIFFERENT

ANTIHEXON ANTISERA

Rabbit antisera pH 2.5 pH 7.5 againsta (PFU/ml) (PFU/ml)

Hexon antigen from <l x 102 3.5 x 102 DEAE cellulose

Pure hexon antigen 1.4 x 10’ 1.6 X lo6 None 1.0 x 106 2.0 x 106

a The antisera were tested at a twofold dilution. Three different antisera were tested with the same virus dose; the mean values are given.

STRUCTURAL PROTEINS OF’ ADENOVIRUS 585

FIG. 10. (a) Adenovirus type 2 particles contrasted with uranyl acetate. The hexon capsomers are clearly resolved.

FIG. 10. (b) Pure hexon preparation contrasted with UAc. Note the central hole in the structure.

the preparation appears structurally homo- between 80 and 110 A, with a mean diam- geneous. A polygonal structure with a eter of 95 A as calculated from prints of central hole is clearly resolved on the micro- 322 particles. The central hole was approxi- graph. The diameter of the hexon varied mately 25 A in width.

586 PETTERSSON, PHILIPSON, AND HiSGLUND

FIG. 11. (a) A virus particle showing 6-fold symmetry. The arrows indicate some of the fibers with knobs. PTA-contrasted.

FIG. 11. (b) Stroboscopy picture obtained by 6-fold rotation of the virus particle in (a). Note enhancement of fiber structure and connection between vertex and fiber.

The detailed structure of the hexon appears, however, complex and it cannot be classified as a sphere or a hollow cylinder. When purified intact adenovirus particles were contrasted with the same technique, a similar structure could be resolved as also shown in Fig. 10; however, the diameter of the hexon in the virus particles seemed to be somewhat smaller. Thus the identity of the

soluble hexon antigen appears to have been established.

To verify that intact adenovirus type 2 particles show the same arrangement of the penton and fiber antigens as previously reported for t,ype 5 and type 3 (Valentine and Pereira, 1965; Norrby, 1966a), intact particles were contrasted with phospho- tungstic acid at neutral pH. Figure 11


shows a particle with sixfold symmetry, where the fiber can be discerned in at least 4 or 5 vertices. The length of the fiber is 250-300 A and the width is approximately 15 A. The peripheral knob had a diameter of around 40 A. It was shown with the aid of stroboscopy (Fig. 11) that the fibers originated from the vertex capsomeres. Only sixfold rotation enhanced the fiber structure at the vertex, Eut it should be emphasized that the knob in the periphery was obscured with this technique, possibly indicating that part of the fiber is flexible.

C. Analytical ultracentrifuyation. The hexon preparation was homogeneous by the criterion of analytical ultracentrifugation. The sedimentation constant for the hexon antigen was 12.2 & 0.2 S in three different preparations, and the diffusion coefficient was estimated to be 2.6 X lo-’ cm2/sec. The molecular weight of the hexon structure was estimated to be 400,000 when a value of 0.720 was used for the partial specific volume, which was calculated from the amino acid composition.

D. Amino acid analysis. The amino acid compositions of adenovirus type 2 hexon antigen, and KB-cell proteins extractabe by Freon are shown in Table 5. The protein preparations were dialyzed against dist.illed water prior to acid hydrolysis. Large differ- ences in composition were found between KB-cell proteins on one hand and adenovirus type 2 hexon antigen on the other. In contrast the amino acid composition of the hexon antigen was similar to that of the intact virus, reported by Polasa and Green (1967), but the lower arginine values for the hexon antigen are noteworthy. The variation in percentage for 4 different preparations of hexon amounted to &0.2-0.4% on an average for the amino acids.

E. Carbohydrate analysis. Glucose could not be detected in the hexon preparations when tested with glucose oxidase after hydrolysis. This infers a glucose content of <0.5% on a protein weight basis. Other neutral sugars and hexoseamines were also not observed, which infers ~0.5% on a protein weight Easis. It is thus concluded that the carbohydrate moiety of the hexon antigen, if any, must be less t,han 2000 molecular weight, which only corresponds

TABLE 5

AMINO ACID COMPOSITION OFADENOVIRUSTYPE 2 HEXON ANTIGEN AND KB CELL PROTEINS Ex- TRACTABLE BYFREOS COMPARED WITH THATOF ADENOVIRUS TYPE 2

LYS 4.4 7.6 4.4 His 1.7 2.1 1.6

Arg 4.7 4.8 7.9

Asp 14.4 10.0 11.8 Thr* 7.3 5.3 6.9 Serb 7.2 6.3 6.7 Glu 9.7 12.0 9.0 Pro 6 .6 4.9 7.2

GUY 7.8 8.3 7.8 Ala 7.5 8.0 9.0 Val 5.4 6.5 6.1 Met 2.3 2.1 2.3 Ile 3.4 5.1 3.4 Leu 7.5 8.1 7.4

Tyr 5.0 2.7 4.4 Phe 4.3 3.6 3.8 Trpc 0.9 1.2 1.2 Half-cys Not detectable 1.5 0.3

Ad- enovirus type 2

Amino acid Hexon antigen” KB cells (Polasa and

Green, 1967)

a The result is obtained from the average of four different preparations and given in moles per 100 moles of amino acid.

* Extrapolated to zero time hydrolysis. c Measured spectrophotometrically.

to l-2 sugar residues per subunit, assuming 5-6 subunits within the hexon antigen.

DISCUSSION

The present study reports methods for partial purification of the structural proteins of adenovirus as well as means of obtaining an extensively purified product of the hexon antigen. A four-step procedure involving agarose chromatography, DEAE cellulose chromatography, preparative polyacrylamide electrophoresis, and sucrose gradients yielded a pure hexon antigen judged by the following criteria: analytical ultracentrifugation, electron microscopy, polyacrylamide electrophoresis, and immunodiff usion.

The pitfall of acrylamide electrophoresis revealed in this study should be empha-

PETTERSSON, PHILIPSON, AND HOGLUND

sized. It appears likely that proteins with a comparatively high negative charge may adhere to the catalysts used for polymerization or other impurities and move as a single band at the front of the migration zone. Such drawbacks may give erroneous results concerning protein purity with this technique. Similar effects may have played a role in the study of Kijhler (1965); where he reported on pure hexon antigens from adenovirus, which however have different properties than our preparation. In spite of the precautions, the acrylamide electrophoresis pattern varied slightly for proteins with a higher negative charge t’han the hexon antigen. This may be due to varying degrees of complexing with nucleic acid fragments (Fig. 6).

The high resolving power of the acrylamide electrophoresis appears necessary to separate the hexon antigen from minor contaminants. The conclusion is based on the incomplete separat,ion, achieved by agarose electrophoresis and isoelectric separation, which both separate on similar principles. ,411 the contaminants were labeled with 14C amino acids as shown by acrylamide electrophoresis of 14C-labeled hexon antigen (Fig. 6). Two bands probably correspond to the monomer and an aggregate of the hexon antigen (unpublished).

The hexon antigen appeared in the electron microscope as a somewhat complex structure, probably composed of a number of subunits. The hexon might perhaps be described as a hollow cylinder (Dales, 1962), although this represents a simplifica- tion of the structure, but it has been established that it does not constitute a sphere as previously proposed by Valentine and Pereira (1965). The dimension of the tightly packed hexon in the virus particle is difficult to estimate, but it appears that it has a somewhat smaller diameter than the purified hexon in solution. This could possibly be due to a swelling of the free hexon particles. Contrasting with UAc resolved the hexon units better than when PTA was used, but the fibers could be visualized only on PTA- contrasted particles.

The arrangement of the fiber antigen at the vertices of the intact virion was in this study extended to adenovirus type 2. Simi-

lar findings have previously been reported for adenovirus type 5 and type 3 (Valentine and Pereira, 1965; Norrby 1966a). The fiber appears, however, thinner in type 2 than in type 5. Furthermore stroboscopy of the virus particle suggests that the structure of the base plate of the penton from which the fiber extends might be different from that of the adjacent hexon units.

The isolated hexon antigen is immunologically pure and does not give rise to neutralizing antibodies when injected into rabbits. Furt,hermore only a single antigenic determinant could be observed when the preparations were tested with antihexon antisera against other adenovirus types. This is in contrast to the findings of K6hler (1965), who found a reaction of partial identity between the different hexons. It has also been claimed repeatedly (Wilcox and Ginsberg, 196313; Kasel et al., 1966) that purified hexon antigen elicit the production of neutralizing antibody. The discrepancy could possibly be explained by the different degree of purity of the hexon since DEAE chromatography was used as the last step in previous studies. The presence of trace amounts of penton antigen in the hexon preparations purified by this method (Fig. 7) may explain this discrepancy. Alterna- tively, aggregates of hexon antigen which were removed in the present study may furnish an additional antigenic specificity not present in the monomer solution. Such an antigenic shift between subunits and intact virions has been reported for the poliovirus system (Scharff and Levintow, 1963).

The amino acid composition of the hexon antigen from adenovirus type 2 is similar to that reported for int’act virus by Polasa and Green (1967) but is significantly different from that reported by Biserte et al. (1966) for adenovirus type 5 hexon antigen. How- ever, since the purity of the preparation of the latter investigators is hard to assess, a direct comparison cannot be made at present. The lack of detectable half-cystine in both adenovirus t,ype 2 and type 5 hexon ant.igen is, however, notewort.hy. Carbohy- drate does not appear to be an integral part of the hexon antigen.

The significantly lower arginine cont& in the hexon compared to the intact virions


may indicate the presence of a fairly abundant protein inside the virion rich in this amino acid, since the hexon antigen consti- tutes about 95% of the proteins in the structural outer shell observed in the electron microscope. Evidence for an internal component has been obtained in electron microscopic studies (Epstein, et al. 1960; Horne, 1963) and in studies of adenovirus eclipse (Philipson, 1967).

ACKNOWLEDGMENTS

We are indebted to Drs. Pertoft, Eaker, Lund- blad, and Hjerten for aid with analytical ultra- eentrifugation, amino acid analysis, carbohydrate determination, and agarose electrophoresis, respectively.

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Structural proteins of adenoviruses