- Email: [email protected]
Structural proteins of adenoviruses
VIROLOGY
43,
123-136 (1971)
Structural
Proteins
VI. On the Antigenic ULF Department
of Microbiology,
of Adenoviruses’
Determinants
of the Hexon
PETTERSSON The Wallenberg
Laboratory,
Uppsala,
Sweden
Accepted September 19, 1970 Hexon ant.igens were purified from KB cells infected with adenovirus types 2, 3, and 5 and compared chemically and immunologically. All three types of hexons were similar in their amino acid composition and each contained 0.5-0.870 half-cystine, determined as cysteic acid after performic acid oxidation. Type- and group-specific antigenic determinants were detected by immunodiffusion on hexons from each serotype. The group-specific determinants were degraded after digestion with chymotrypsin, subtilisin, or papain, leaving a “hexon core” which retained all of its type-specific properties and which at neutral pH formed rapidly sedimenting aggregates. Trypsin removed 5-107, of the native hexon polypeptide, presumably from the C-terminal end, without affect.ing its antigenic properties. Maleic anhydride preferentially inactivated the type-specific determinants, without altering the size or the shape of the native hexon. Two populations of hexons were isolated from cells infected with adenovirus type 2. The minor of these had a lower electrophoretic mobility, but similar size and shape as the main class of hexons. Antisera against the slowly migrating population contained antibodies with 50-fold higher specific neutralizing activity (neutralizing titer/ complement fixing titer) than antisera prepared against the main population of type 2 hexons. INTRODUCTION
Adenovirus hexon antigen has been reported to contain identical subunits, each composed of one single type of polypeptide chain (Maize1 et al., 1968; Velicer and Ginsberg, 1968; Pettersson, 1970). The molecular weight of the entire hexon unit is in the range 310,000-360,000 (Franklin et al., 1970) and evidence for a complex arrangement of group- and type-specific determinants has recently been given (Norrby, 1969; Norrby and Wadell, 1969). The hexon has been considered to be an unusually resistant structure, unaffected by mild proteolysis and low pH (Allison et al., 1 This Damon Medical Society, Medical
project was supported by grants from the Runyon Memorial Fund, the Swedish Research Council, the Swedish Cancer and the Swedish Delegation for Applied Defense Research.
1960) and treatment with high concentrations of agents like formamide (Neurath et al., 1968) and urea (Short’ridge and Biddle, 1970). The present investigation describes the comparative properties of purified hexons from adenovirus types 2, 3, and 5. Proteolytic and chemical agents have been used to specifically alter or degrade different parts of the hexon molecule in order to obtain further information on the arrangement of the antigenic determinants on the hexon. MATERIALS
AND
METHODS
Cell cultures and virus infection. Adenovirus types 2, 3, and 5 were grown in 2-liter spinner cultures of KB cells as described by Pettersson and Hijglund (1969). Processing of virus and soluble antigens. The procedure of Green and Piiia (1963) was
124
PETTERSSON
used with some modifications (LonbergHolm and Philipson, 1969). Separation of antigens by DEAE chromatography. Antigens from adenovirus types 2 and 5 were separated at pH 6.5 as described previously (Pettersson et al., 1967). Antigens from adenovirus type 3 were separated at pH 8.4 as described by Norrby (1966). Exclusion chromatography. Agarose (Sepharose 6 B Pharmacia Fine Chemicals, Uppsala, Sweden), 6%, was used with 0.05 M Tris-acetate pH 8.0, containing 0.15 M NaCl as elution buffer (Pettersson and Hoglund, 1969). Rate-xonal centrifugation. Preformed linear gradients, 4.5 ml containing 5-25 % sucrose in 0.002 M Tris, pH 7.2, or 0.1 M formic acid were used in a Spinco SW 50-l rotor. The gradients were fractionated by drop collection from the bottom. Polyacrylamide gel electrophoresis. Analytical runs were made on 5 % gels in 0.05 M Tris-acetate pH 8.0 as described by Pettersson and Hoglund (1969). Preparative electrophoresis was performed at pH 9.5 in a continuous elution apparatus as described by Franklin et al. (1970). PuriJicatian of hexun antigen from types 2, S, and 5. The purification procedure of Pettersson et al. (1967), using DEAE-chromatography and preparative polyacrylamide gel electrophoresis was essentially followed with one important modification: The preparative gel electrophoresis according to Maize1 (1966) was replaced by a similar step using the continuous elution apparatus of Hjerten et al. (1965). Adenovirus type 3 antigens were separated by exclusion chromatography on 6% agarose prior to the preparative electrophoresis in order to resolve hexons and fibers which in the case of adenovirus type 3 elute close to each other after DEAE-chromatography (Norrby, 1966). Hexons so purified were homogeneous as judged by analytical polyacrylamide gel electrophoresis at pH 8.0 (Fig. 1) and at pH 9.5 (Fig. 3b) and by immunoelectrophoresis. The purity of type 2 hexons was also checked by analytical which revealed one ultracentrifugation, single homogeneous boundary both when using schlieren and UV optics. N-terminal amino acid analysis of purified type 2 hexons
using the phenylisothiocyanate-355 method showed the absence of reactive N-group (Pettersson, 1970). Enzymatic digestion. Chymotrypsin (recrystallized X 3), trypsin (TR-TPCK) and papain were purcha.sed from Worthington, New Jersey. Subtilisin (crystalline) was kindly provided by Novo Industri A/S, Denmark. All digestions were carried out at +37” in 0.05 M Tris-acetate buffer, pH 8.0. Papain was activated by the addition of cysteine to a final concentration of 0.01 M. The tryptic digestions were stopped after 1 hour by addition of 2-fold excess of trypsin inhibitor (Sigma Chemical Company, St. Louis, Missouri) and papain was inactivated after 18 hours by addition of iodoacetamide to 0.02 M final concentration. Chymotrypsin and subtilisin were inactivated by addition of 10 mM diisopropyl fluorophosphate after 60 and 180 min, respectively. In some experiment,s the samples were cooled to 0” at the end of the digestion and then directly added to sucrose gradients or chromatographic columns without specific inactivation. Maleylation. Maleic anhydride was recrystallized from chloroform prior to use. All samples were dialyzed against 0.1 M Na2HP04 before the addition of 10 mg solid maleic anhydride to 100-500 pg of hexon. The reagent was added slowly and the pH was kept constant at 9 by use of a pH-stat (Radiometer, Copenhagen, Denmark). Amino acid analysis. Samples of 0.5-2 mg were hydrolyzed as described by Pettersson et al. (1967) and analyzed on a “Biochrom” automatic amino acid analyzer. Cysteine was determined as cysteic acid after performic acid oxidation (Hirs, 1956). N-Terminal amino acid analysis. Samples of l-3 mg of hexon or marker protein (fibrogen) were analyzed by a modified Edman method using 35S-labeled phenylisothiocyanate (Laver et al., 1967; Pettersson, 1970). Peptide mapping. The method of Blomback et al. (1968) was followed. Intact or degraded hexons, 0.5-1.5 mg, were denatured by heating to 100” in 0.02 M NH4HC03, pH 8.5, for 1 minute. The denatured protein was lyophilized and resuspended in 0.05
STRUCTURAL
PROTEINS
ml 0.2 64 NH4HCOB, pH 8.5, a,nd trypsin (TR-TPCK) was added to an enzyme substrat,e ratio of 1: 100. After 18 hours the samples were lyophilized and the peptides were dissolved in distilled water. Aliquots of 3-5 ~1 were added to cellulose thin la.yer plates (20 X 20 cm) and electrophoresis was carried out at 300 V for 75 min in pyridine acetate pH 5.5. After thorough drying the plates were submitted bo chromatography in pyridine: acetic acid:n-butanol: wat,er (100: 30: 150:120). The dry plates were stained with ninhydrin. Preparation of antisera. Antisera against virions from types 2, 3, and 5 were prepared as described previously (Pettersson and HGglund, 1969). Antiserum against type-specific det’erminants on hexons from adenovirus type 2 was prepared by absorbing an antiserum against type 2 hexons with purified type 5 hexons. The efficiency of the absorption was checked by immunodiffusion against type 5 hexons. Complement Jixation was performed according to a micromethod described previously (Pettersson and Hoglund, 1969). Neutralization tests. Neutralization of virus infectivity was assayed by inhibition of plaque formation or by inhibition of fluorescent focus formation (Pettersson and HGglund, 1969). Reduction of infectivity by 1.5 log units was scored as significant neutralization. Specific neutralizing activity was measured as the neutralizing titer/complement fixing titer against purified hexons.
OF ADENOVIRUSES.
125
VI.
these hexons, while the slight differences in composition between hexons from types 2 and 5 do not permit any definite conclusions with regard to basic and dicarboxylic amino acids. Since earlier studies had indicated that hexons from both type 2 (Pettersson et al., 1967) and type 5 (Biserte et al., 1966; Boulanger et al., 1969) lack half-cyst’ine residues, this problem was reinvestigated by performing analysis of performic acid oxidized hexons. Under these conditions 0.5-0.8% half-cystine was detected in all three types of hexons (Table 1). Immunoloyicwl Hexons
Properties
of
Adenovirus
The antigenic specificities of native hexons from adenovirus t(ypes 2, 3, and 5 were investigated by use of the immunodiffusion assay. Purified hexons from the three serotypes were tested against antisera prepared toward homotypic and heterotypic hexons or virions. In all cases, spurs were seen between homotypic and heterotypic hexons indicating a relationship of partial identity (Fig. 2a), while no spurs were observed when homotypic and heterotypic antisera were compared (Fig. 2b). The presence of type-specific components on adenovirus hexons was confirmed by absorption studies; antihexon sera, absorbed with heterotypic hexons, still gave precipi-
RESULTS
Characterization
of the PuriJied Hexons
Hexon antigens from types 2, 3, and 5 were compared with regard to electrophoretic properties and amino acid composition. The three types of hexons varied slight,ly in their electrophoretic mobilities; type 2 hexons being faster and type 3 hexons slower relative to type 5 hexons (Fig. 1). With regard to amino acid composition some minor differences were observed (Table 1). Type 3 hexons exhibited a lower content of glutamic acid which may account for the lower electrophoretic mobility of
FIG. 1. Analytical polyacrylamide gel electrophoresis on 594 gels at pH 8.0 of purified hexons from adenovirus types 2 (I), 3 @), 5 (3), and a mixture of all three (4). Origin at the top and anode toward the bottom.
126
PETTERSSON TABLE AMINO
ACID
COMPOSITION
Residue
Type 2c hexon
LYS His A% Asp Thr” Serb Glu Pro GUY Ala Half -Cysf Val Met Ile Leu TY~ Phe TOP
4.16 1.40 4.61 14.41 7.03 6.84 9.49 6.08 6.76 7.10 0.76 5.69 2.72 3.55 7.70 5.61 4.44 1.658
1
OF ADENOVIRUS
HEXON
ANTIGEV
Type Sd hexon
Type 3d hexon
4.60 1.72 4.95 14.46 6.95 6.30 10.04 6.10 6.34 6.95 0.79 5.26 2.73 4.40 7.80 5.60 5.05
4.02 1.65 4.92 14.52 8.25 6.80 8.31 6.14 7.43 6.82 0.48 6.02 2.95 3.85 7.29 5.46 5.01
f f + f f f Zk & zt f
0.15 0.14 0.39 0.27 0.05 0.12 0.19 0.29 0.25 0.29
+ + zk f f f ND”
0.03 0.10 0.20 0.02 0.31 0.11
zt 0.15 f 0.09 * 0.13 It 0.10 zk 0.12 zt 0.30 f 0.04 f 0.23 f 0.25 & 0.12 & 0.02 * 0.16 f 0.09 + 0.08 f 0.16 rk 0.24 f 0.06 NDB
Type 2 hexond degraded by trypsin 4.52 1.45 4.49 15.02 7.47 7.90 8.98 6.26 7.66 7.34
& zt l zk zt zt xk xt f f ND” 5.47 f 2.35 f 3.61 zk 7.69 f 5.25 zk 4.58 f ND
0.18 0.13 0.04 0.69 0.19 0.65 0.06 0.13 0.28 0.09 0.31 0.26 0.14 0.14 0.33 0.08
a Given in moles per 100. b Extrapolated to zero time hydrolysis. c 24 and 72 hour hydrolysis was performed, and the values represent the average except for Val and Ile, which were calculated from the 72 hours data only (Pettersson, 1970). The values given are the average of two determinations. d 24-hour hydrolysis only. The values are the average of two determinations. e Not determined. f Determined as cysteic acid after performic acid oxidation. The half-cystine values for type 2 and 3 hexons are the average of two determinations, and the value for type 5 hexons is from one single determination. g Determined spectrophotometrically according to Edelhoch (1967).
tation lines with homotypic hexons. A closer immunological relationship between hexons from types 2 and 5 (belonging to subgroup III) was shown by absorption of an antiserum against type 5 virions with hexons from serotype 3 (subgroup I). Such an antiserum gave precipitation lines with type 5 as well as with type 2 hexons. Isolation of a Special Class of Adenovirus Type 2 Hexons During the purification of large amounts of type 2 or type 5 hexons, it was observed that a minor population of hexons was eluted after the majority of the hexons from the preparative polyacrylamide gel and thus had a lower electrophoretic mobility (Fig. 3a). These hexons designated “special
hexons,” were aft,er this step contaminated with fast migrat,ing hexons and were further purified by recycling the fractions through the electrophoresis apparatus twice (Fig. 3~). In order to estimate the size of the “special hexons” gel chromatography on 6 % agarose (Fig. 4) as well as rate zonal centrifugation was performed with ordinary hexons as markers. Both classes of hexons eluted in identical positions after the agarose chromatography (Fig. 4) and sedimented with t,he same rates (not shown) indicating that they had similar size and shape and excluding a monomer-dimer relationship. When the two classes of hexons from serotype 2 were compared by the immunodiffusion assay no differences could be detected (not shown) but when their respective rabbit-
STRUCTURAL
PROTEINS
antisera were compared with regard to neutralization, it was observed that antiserum against the “special hexons” were approximately 50-fold more potent (Table 2). Similar results were obtained with guinea pig sera although the titers were low and do not permit as definite conclusions (Table 2). The heterogeneity among type 2 hexons was also shown by immunoelectrophoresis (Fig. 5), while electron microscopy did not
OF ADENOVIRUSES.
CPrn 30000 26000 22000
127
VI.
I
200
12000
160 14000 120 60 6000 2000
40
t
L 20
25
30
35
40
45
50
fraction cm
55
number
(b)
2600C 22000 16000
‘t
14000 10000 6000
fraction cpm
,
number
Cc)
13000 u 11000 I 9coo
/I
-
70005000
-
3000
-
1000 20
25
30
35
40
45
50
traction
FIG. 2a. Immunodiffusion of purified hexons from types 2 (2A), 3 @A), and 5 @A) against an antiserum prepared toward type 2 hexons. FIG. 2b. Immunodiffusion of purified type 2 hexons (central well) against antisera against virions from types 2 (S2), 3 (SS), and 5 (56).
55
number
FIG. 3. Elution diagrams from preparative polyacrylamide gel electrophoresis at pH 9.5 of amino acid-3H-labeled type 2 hexons: (a) Separation of hexon antigen obtained after DEAE-chromatography. Radioactivity (O--O) as well as diameters obtained by radial immunodiffusion with an antihexon serum (O--O) were measured. t-i indicates fractions from peaks I and II which were pooled and concentrated and dialyzed for recycling. (b) Recycling of material obtained from peak I. (c) Recycling of material obtained from peak II. Peak “III” corresponds to the slowly migrating hexons and was homogeneous when analyzed by analytical polyacrylamide electrophoresis.
128 CP~
PETTERSSON rg/mt
500 300 100
15
20
25
30
35
LO
45
fraction
50
after treatment with trypsin at a concentration of 1 mg/ml as judged by immunodiffusion (not shown). When, however, tryptically digested type 2 hexons were analyzed by exclusion chromatography on 6 % agarose (Fig. 6) or by rate zonal centrifugation (Fig. 7c) it was evident that 5-10% of the label from amino acid-3H-labeled hexons was recovered as low molecular weight material.
number
FIG. 4. Exclusion chromatography on 6% agarose of a mixture of purified “fast” unlabeled type 2 hexons (O--O) and purified amino acidSH-labeled “slow” type 2 hexons (O-O). The “fast” hexons were added in 50-fold excess on a weight basis and were analyzed by radial immunodiffusion. The arrow indicates the fraction corresponding to the total volume of the column.
TABLE
2
NEUTRALIZATION OF ADENOVIRUS TYPE INFECTIVITY BY DIFFERENT ANTISERA”
2
-
Antiserum
FIG. 5. Analytical polyacrylamide gel electrophoresis (left) and immunoelectrophoresis (right) of “fast” (I) and a mixture of “slow” and “fast” hexons (2) from adenovirus type 2. The antiserum was an antiserum against type 2 hexons.
against
Fast migrating type 2 hexons, rabbit Fast migrating type 2 hexons, guinea pig Slowly migrating type 2 hexons, rabbit Slowly migrating type 2 hexons, guinea pig Disrupted type 2 virions
5
2
256
cPm
/Js/ml
3500 1
20
10
1024
160
160
256
20
20
16
2560
1024
A
750
a Reduction of 1.5 log units of infectivity was scored as significant neutralization. lob-plaqueforming units or fluorescent focus units were mixed with antiserum as described in Methods. b Fluorescent focus assay. c Complement fixing titer against 4 units of purified fast or slowly migrating type 2 hexons.
between the two classes of hexons (Dr. S. Hiiglund, personal communication).
distinguish
Enzymatic Degradation of Hexon Antigen Trypsin. The antigenic specificities of hexons from types 2,3, and 5 were preserved
250 30
LO
50
60 fract’mrl
70
80 ncniber
FIG. 6. Exclusion chromatography on 6y0 agarose of a mixture of amino acid-3H-labeled type degraded with 1 mg/ml trypsin 2 hexons, (O--O) and intact unlabeled type 2 hexons (O--O). The enzyme was inhibited by trypsin inhibitor prior to the addition of a SO-fold excess of unlabeled marker. The intact hexons were traced by radial immunodiffusion. The arrow indicates the fraction corresponding to the total volume of the column. Tryptic digestion for 18 hours did not significantly change the profile.
STRUCTURAL
H3
5
cPm
PROTEINS
15
10
FRACTION
30000
NUMBER
t 20000 10000
“3
B /
t
5
cpm 30000 t
“3
CPm (
15
10 FRACTION
n
5
lo
5
10
FRACTIZ
15
NUMBER
NUMBER
OF ADENOVIRUSES.
VI.
129
peptide which was attacked by trypsin by performing N-terminal amino acid analysis. Tryptically digested hexons were purified from enzyme and released peptide by exclusion chromatography on 6 % agarose and submitted to N-terminal amino acid analysis. No free N-terminal amino acid could, however, be identified in either the undegraded hexons (Pettersson, 1970) or the digested hexons, suggesting that the peptide( released after trypsin digestion were removed from the C-terminal end of the hexon polypeptide. When hexon antigen was heated to 100” prior t,o the trypbic digestion an extensive degradation was seen (Fig. 7B). Chymotrypsin. Hexon antigen from adenovirus types 2, 3, and 5 was digested for 1 hour with chymotrypsin at a concentration of 1 mg/ml and subsequently analyzed by immunodiffusion. A reaction of partial identity was seen when digested and undigested hexons were tested against homotypic antiserum (Fig. 9 a,b), indicating loss of antigenic determinants. When these degraded hexons were compared with
20
FRACTION
NUMBER
7. Rate-zonal centrifugation (5-25s sucrose in 0.062M Tris pH 7.2; 106,060 g for 16 hours) of (A) purified amino acidJH-labeled hexons, (B) type 2 hexons degraded by 1 mg/ml trypsin after heat denaturation (lOtI”, 1 min), (C) hexons digested with 1 mg/ml trypsin, (D) type 2 hexons digested with 1 mg/ml chymotrypsin. The main peak contained antigenically degraded hexons, and the fast sedimenting peaks probably aggregates of these. FIG.
The elution profile after 6 % agarose showed a. slight shift toward the total volume of the column, thus suggesting a decrease in the Stokes’ molecular radius as compared to intact hexons (Fig. 6). A limited degradation of the hexons was also revealed by electrophoresis; the mobility of trypsin-treated hexons was lower than that of untreated hexons (Fig. 8). Amino acid analysis showed that this alteration might be due to release of peptide which have a low lysine but high glutamic acid content (Table 1). Since the degraded hexons appeared as a homogeneous preparation, it should be possible to trace the part of the hexon poly-
FIG. 8. Analytical polyacrylamide gel electrophoresis at pH 8.0 of type 2 hexons after trypsin digestion (I), a mixture of tryptically and undigested hexons (Z), and type 2 hexons after chymotrypsin digestion (3).
FIG. 9. (a) Immunodiffusion of type 2 hcxons ($A), type 2 hexons after chymotrypsin digestion @AC), type 3 hexons ($A), and type 5 hexons (6.4). Autiserrun against type 2 hexons in the central well. (b) Immunodiffusion of type 3 hexons after chymotryptic digestion (SAC) and intact hexons from types 2, 3, and 5. Antiserum against type 3 virions in the central well. (c) Immunodiffusion of type 2 hexons after chymotrypsin digestion (central well) against antisera against type 2 hexons (S.S), type 3 virions (SS) and type 5 virions (S5). (d) Immunodiffusion of intact type 2 hexons @A), type 2 hexons after digestion with 1 mg/ml chymotrypsin @AC) and with 0.1 mg/ml papain @Al’) and type 2 hexons digested with 1 mg/ml subtilisin @AS). Antiserum against type 2 hexons in central well. (e) Type 2 hcxons after digestion with papain (ZAP) with chymotrypsin @AC) and with 1 mg/ml subtilisin @AS), intact type 2 hexons (2A) and type 5 hexons @A) tested against an anti-type 2 hexon serum absorbed with type 5 hexons. 130
STRUCTURAL
PROTEINS
OF ADENOVIRUSES.
VI.
131
hexons from other types (i.e., types 3 and 5) gated into larger units which sedimented faster than intact hexons. a reaction of nonidentity was observed considerably (Fig. 9a,b) and when degraded and unde- These aggregates could be dissociated by graded type 2 hexons were tested against an performing the centrifugation in the presanti-hexon serum, containing antibodies only ence of 0.1 M formic acid (Fig. 10a). against type-specific parts of the hexon (an CPm, anti-adenovirus type 2 hexon serum, ab- H3 1 sorbed with type 5 hexons) a reaction of identity was seen (Fig. 9e). These findings suggest that the chymotryptic digestion selectively removed group-specific determinants from the hexon antigen, although when digested hexons were tested against 10 20 FRACTION NUMBER heterotypic antisera a slight precipitation line was seen (Fig. 9c) revealing that some residual group specificity remained after digest’ion. The extent of the chymotryptic digestion was evaluated by sucrose gradient centrifugation (Fig. 7~). Around 20 % of the counts from amino acid-3H-labeled hexons remained near the top of the gradient after FRACTION NUMBER 10 20 centrifugation of the digested hexons. The remaining antigenic portions of the hexons cpm showed a tendency to aggregate since some Ii3 material with faster sedimentation rates than intact hexons was observed (Fig. 7~). 2000 C TOP Such aggregates were also demonstrated when digested hexons were submitted to gel 1000 chromatography on 6% agarose. The digested hexons eluted as a broad peak corFRACTION NUMBER 10 20 cc-m responding to a K,, value (Laurent and Killander, 1964) of 0.39-0.42 while intact hexons under the same conditions had a KAv value of 0.43. Electron microscopy of the digested hexons showed structures with a morphology similar to that of intact hexons, but they usually appeared as aggregates of several units (Dr. S. Hoglund, 10 20 FRACTION NUMBER personal communication). FIG. 10. Rate-zonal centrifugation in 5-25s Papain. Papain at a concentration of 0.1 sucrose. Gradients A and B were centrifuged at 100,000 g for 16 hours and gradients B and C at mg/ml in the presence of 10 mM cysteine and at pH 8.0 digested adenovirus type 2 200,000 g for 5 hours. (A) 3H-amino acid labeled type 2 hexons after papain digestion. Separation hexons extensively, leaving a “hexon core” was done in gradients containing 0.1 M formic which was still ant,igenic (Fig. 9d and lob). acid. (B) The same material as in (A) but separaWhen tested by immunodiffusion, it was tion carried out in gradients containing 0.002 M apparent that these “hexon cores” retained Tris, pH 7.2. (C) Amino acidJH-labeled type 2 their antigenicity t’o the same extent as hexons digested with 1 mg/ml subtilisin. The hexons digested with chymotrypsin, since a gradients contain 0.1 A4 formic acid. (U) The same reaction of identit,y was observed bet’ween material as in (C), but separation carried out in hexons digested by these two enzymes gradients containing 0.002 M Tris, pH 7.2. The arrows in (A) and (C) . indicate the oositions of (Fig. 9d). When analyzed by sucrose gra- undigested hexons run on formic acid containing dient centrifugation it was shown that the gradients, and (!--I) indicates fractions which “hexon core” after papain digestion aggre- were precipitated by antihexon serum.
132
PETTERSSON
More than 50% of the label from amino acid-3H-labeled hexons was recovered on top of the sucrose gradients after centrifugation of papain digested material (Fig. lob). Subtilisin. Subtilisin at a concentration of 0.1 mg/ml digested adenovirus type 2 hexons to a similar extent as does papain, also leaving a LLhexon core.” Immunodiffusion analysis showed that these “cores” were antigenically altered to the same extent as were hexons after degradation with papain or chymotrypsin (not shown). When a lo-fold higher enzyme concentration (1 mg/ml) was used, a more extensive loss of antigenic specificity was seen, demonstrated as a spur between material obtained after subtilisin and chymotrypsin treatment (Fig. 9d). When these hexons were tested against a strictly type-specific antihexon serum a reaction of identity was observed even between intact hexons and these extensively digested hexons, suggesting that the type-specific portions remained unaltered (Fig. 9e). When, however, subtilisin degraded material was tested against heterotypic sera, faint precipitation lines were still detected, indicating the presence of some remaining group-specific determinant (not shown). When hexons degraded by subtilisin were analyzed on sucrose gradients the subtilisin “cores” were shown to form fast-sedimenting aggregates, which also were dissociable under acid conditions (Fig. lOc,d). Peptide Mapping 2 Hexons
peptides showed that the degraded hexons lacked 15-20 peptides, most of which were comparatively hydrophilic. Alteration of the Hexon Antigenic Specijicities after Treatment with Maleic Anhydride Maleylation of native type 2 hexons altered them into electrophoretically fast migrating structures (Fig. 11). This change was considered to be due to blocking of the lysine E-amino groups rather than to dissociation, since gel chromatography (not shown) and sucrose gradient centrifugation (Fig. 12) did not show any drastic changes in size or shape. When maleylated hexons were compared to untreated hexons by immunodiffusion, it was evident that maleylation was accompanied by changes in antigenic properties which could be described as follows: When treated and untreated type 2 hexons were tested against sera containing antibodies against type 5 or type 2 hexons a pattern of partial identity was observed, while when tested against serum containing antibodies against type 3 hexons a pattern of identity was seen (Fig. 13a). When maleylated hexons were tested against an antiserum
of Intact or Degraded Type
Peptide mapping was performed in an attempt to find some possible explanation for the resistance of some parts of the hexon molecule towards proteolytic degrsdation. Maps of the tryptic peptides from heat denatured adenovirus type 2 hexons showed 40-50 ninhydrin-positive peptides, many of these having hydrophobic properties (Pettersson, 1970). For comparison, type 2 hexons were degraded with subtilisin (1 mg/ml) and the “core” was purified by rate zonal centrifugation (200,000 g for 5 hr), dialyzed, lyophilized, and finally digested with trypsin after heat denaturation. Mapping of these
FIG. 11. Analytical polyacrylamide gel electrophoresis at pH 8.0 of type 2 hexons (9) type 2 hexons after maleylation (f), and a mixture of both (2).
STRUCTURAL
PROTEINS cpm H3
!
10
20
le.00
FRACTION
NUMBER
12. Rate-zonal centrifugation (5-25s sucrose; 0.602 M Tris, pH 7.2, 160,660 g for 16 hours) of 3H-labeled type 2 hexons after maleylation (O--O). Untreated hexons (O---O) were run on a separate gradient, since the maleylated protein showed a tendency to interact with the marker protein. FIG.
containing antibodies only against typespecific hexon components, no precipitation line was obtained (Fig. 13b). These findings suggest that the parts preferent’ially altered by maleic anhydride were those corresponding to type- and subgroup-specific portions of the hexon.
OF ADENOVIRUSES.
VI.
133
hexons against antihexon sera from different types. This technique is not so sensitive to minor variat’ions in antigenic composition (Fig. 2b). A minor population of hexons (“special hexons”) with a lower electrophoretic mobility than the main class of hexons was also isolated from cells infected with type 2 or type 5 adenoviruses. The two classes of hexons were of similar size and shape as demonstrated by sucrose gradient centrifugation or exclusion chromatography. Antigenitally they were indistinguisha.ble when conventional assays such as immunodiff usion were employed, but when antisera against the two populations of hexons were compared, it was shown that sera against the slowly migrating type contained approximately 50-fold higher specific neutralizing activity (neutralizing titer/complement fixing titer) (Table 2). Such factors might explain, in part, the controversy regarding the neutralizing capacity of hexon antisera (Wilcox and Ginsberg, 1963; Kasel et al.,
DISCUSSION antigen has been purified from infected with three different serotypes of adenovirus and compared chemically and immunologically. The amino acid compositions of hexons from unrelated types, namely, type 2 belonging to Rosen’s subgroup III (Rosen, 1960) and type 3 belonging to subgroup I, were remarkably similar. Contrary to earlier findings (Biserte et al., 1966; Pettersson et al., 1967; Boulanger et al., 1969), half-cystine was detected in hexons from all three serotypes with techHexon KB-cells
niques
which
quantitatively
preserve
this
amino acid during hydrolysis. Recently Neurath and co-workers (1970) demonstrated the presence of half-cystine in type 7 hexons after labeling of viral components with cystineJ*C. In the present study our purified hexon preparations were shown to contain type-specific antigenic determinants (Fig. 2a) which is in conflict with our previously reported data (Pettersson et al., 1967). This discrepancy may be explained by variations in the techniques used to demonstrate antigenic differences. In our previous communication we tested type 2
FIG. 13. (a) Immunodiffusion of maleylated hexons (2AM) and untreated hexons from adenovirus type 2 (ZA) against antisera toward: type 2
hexons (S2), type 3 virions (SS), type 5 virions (86). (b) Immunodiffusion of maleylated type 2 hexons in the central well against an anti-type 3 virion serum (SS) and an anti-type 2 hexon serum, absorbed with excess of type 5 hexons (S-6).
134
PETTERSSON
1966; Pettersson et al., 1967; Kjellen and Pereira, 1968; Norrby, 1969), since some of these investigators have used purification methods which would not separate the two classes of hexons. The relationship bet’ween the (‘special hexons” and the hexons of the mature virion is not yet clear and several possibilities could be considered: the virion might a priori be suspected to contain two classes of hexons, since the hexons in peripentonal positions have different surroundings than the hexons on the faces of the adenovirus icosahedron. Hexons of peripentonal origin have recently been isolated after dialysis of virions against a Trismaleate buffer at pH 6.4 (Prage et al., 1970). These hexons are, however, not electrophoretically identical to t#he “special hexons.” Aberrant hexons could also be present elsewhere in the adenovirus icosahedron; the “special hexons” could, for example, be precursors to the aggregates of nine hexons, which often are obtained when adenovirus particles are disintegrated (Smith et al., 1965; Laver et al., 1968; Maize1 et al., 1968; Prage et al., 1970). The recent findings of separate populations of hexons with different isoelectric points from type 5 adenovirions dissociated with 8 M urea (Shortridge and Biddle, 1970) do imply the existence of more than one class of hexon in the mature virion as well. The difference on the polypeptide level between the two classes of hexons could either be related to the presence of an additional peptide on the “special hexons” or to conformational differences. A special case of the former alternative would be the presence of a large polypeptide in “special hexons” which during maturation is split into a small peptide plus an “ordinary hexon” which both are incorporated in the mature virus particle. The possibility of creating artificial hexons during the purification procedure, due to losses of peptides or conformational changes, is also conceivable and could not be excluded from available experimental data. At,tempts have been made to convert the two classes into one under denaturing conditions but urea up to 7 M concentration did not eliminate the difference in their electrophoretic properties (unpublished). Further experiment,al data
are obviously required on t’he relationship between the hexons, synt’hesized in excess in adenovirus infect.ed cells, and the hexons of the mature virion, before the importance of hexon antigen for adenovirus neutralization may be established. Previous reports have indicated that native adenovirus hexons are unaffected by the proteolytic activity of trypsin, chymotrypsin, pepsin, and papain (Allison et al., 1960; Norrby, 196s). The possibility of accomplishing a limited proteolytic degradation was reinvestigated in the present study, since it was considered useful for further structural studies on adenovirus hexon to have access to smaller hexon fragments. Trypsin was found to split off 5-10 % of the hexon polypeptide, probably from the Cterminal end, without changing the antigenie properties of t’he hexon. By the use of subtilisin, chymotrypsin or papain it was possible to obtain a limited degradation which in all cases preserved a “core structure” which had lost part of its group-specific properties but had retained all its typespecific properties (Table 3), and which at neutral pH readily formed fast sedimenting aggregates. TABLE ANTIGENIC
MODIFICATION
3 OF TYPE
Part of the hexon PoikplPModifying
agent
Trypsin, 1 mg/ml Chymotrypsin, 1 m&ml Papain, 1 mg/ml Subtilisin, 0.1 mg/m 1 Subtilisin, 1 mg/ml Maleic anhydride
released as low molecular weight material (%I
2 HEXONS
1[nactiva- I nactivation of tion of typegroup specific specific deterdeterminants’” I ninants”
5-10 15-25
None None
>50 >50 >50 0
None None None +++t
None ++
Q The change in antigenic determinants were classified on an arbitrary scale (None -+++i-), based on immunological differences as revealed by immunodiffusion. b Corresponds to the subgroup specific part of the type 2 hexon.
STRUCTURAL
PROTEINS
Subtilisin at high concentrations (1 mg/ml) removed further specificities from the group-specific portion (Table 3) indicating a complex arrangement of the antigenic determinants of hexons, which previously has been demonstrated in cross-absorption studies (Norrby, 1969; Norrby and Wadell, 1969). The hexon structure t#hus appears to be divided into two entities, one corresponding to the type-specific portion characterized by compact structure and resistant to enzymatic degradat’ion even by enzymes with broad specificities like papain and subtilisin, and the other containing groupspecific determinants which may be digested by several proteolytic enzymes. The recent findings of Norrby et al. (1969), that only type specific antigenic determinants are exposed outside t’he type 3 virion would indicate that adenoviruses have an enzyme resistant surface structure, which could be advantageous for the survival of the infectious particle. Peptide mapping showed that the isolated “hexon core” after subtilisin digestion was enriched in hydrophobic sequences. Such sequences may provide the basis for a compact tertiary struct,ure which could account for the resistance towards proteolytic degradat,ion in these parts of the hexon polypeptide, and also for the tendency of “hexon cores” to form fast sedimenting aggregates. Treatment with maleic anhydride preferentially altered type-specific parts of the hexons (Table 3; Fig. 13a,b). This was unexpected since those portions which were available for proteolysis also should be preferentially susceptible to maleylation. However, substances like maleic anhydride might have a selective affinity on the hydrophobic regions in proteins presumably present in abundance in the “hexon core.” ACKNOWLEDGMENT The author is indebted to Professor L. Philipson for helpful criticism and discussion, to Professor J. Sjiiquist and B. Blomback for supplying laboratory facilities for N-terminal analysis and peptide mapping and to Dr. S. Hoglund for aid with electron microscopic examinations. The skillful technical assistance by Miss S. Rosen, Miss H. Hessle, Mrs. E. Hjertson, and Mrs. S. Siiderman is also gratefully acknowledged.
OF ADENOVIRUSES.
VI.
135
REFERENCES ALLISON, A. C., PERUUR~, H. G., and FSRTHING, C. P. (1960). Investigation of adenovirus antigens by agar gel diffusion techniques. Virology 10, 316-328. BISERTE, G., S.~M.~ILLE:, J., D.~UTREVAUX, M., BOULBNGER, P., S~UTI&R~:, P., RING&L, J., and !J%‘.~ROCQUI~, R. (196F). Composition en acides amines de l’antigene de structure A de l’adenovirus 5. C. R. Acad. Sci. Ser. D 263, 1648-1649. BLOMI&K, M., BLOMHHCK, B., M:LMMF:N, E., and Puasnn, A. S. (1968). Fibrinogen Detroit-a molecular defect in the N-terminal disulphide knot of human Fibrinogen? Xa/ure (London) 218, 134-137. BOULANGEH, P. A., FLIMb:NCOuRT, P., and BISEHTE, G. (1969). Isolation and comparative chemical study of structure proteins of the adenovirus 2 and 5: hexon and fiber antigens. Eur. J. Biochem. 10, 116-131. EDELHOCH, H. (1967). Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6, 1948-1954. EDMAN, P., and SJ~QUIST, J. (1956). Identification and semiquantitative determination of 3phenyl-2-thiohydantoins. Acta Chem. &and. 10, 1507-1509. FRaNKLIN, R.M., PETTERSSON, U., &ICRVALL, K., STRANDBERG, B., and PHILIPSON, L. (1979). Structural proteins of adenoviruses. V. On the size and structure of the adenovirus type 2 hexon. J. Mol. Biol. in press. GREEN, M., and PIRI, M. (1963). Biochemical studies on the adenovirus multiplication. IV. Isolation, purification, and chemical analysis of adenovirus. Virology 20, 199-207. HIRS, C. H. W. (1956). The oxidation of ribonuclease with performic acid. J. Biol. Chem. 219, 611-621. HJI"RT&N, S., JERST~:DT, S., and TIS~LIUS, A. (1965). Electrophoretic “particle sieving” in polyacrylamide gels as applied to ribosomes. Anal. Biochem. 11, 211-218. Kase~, J. A., ALFORD, R. H., LEHRICH, J. It., BANI(S, P. A., HUMOR, M., and KNIGHT, V. (1966). Adenovirus soluble antigens for human immunization. A progress report. Amer. Rev. Respirut. Dis. 94, 168-174. KJ)>LL&N, L., and PEREIRA, H. G. (1968). Role of adenovirus antigens in the induction of virus neutralizing antibodies. J. Gen. Viral. 2, 177185. LAURENT, T. C., and KILLANDER, J. (1964). A theory of gelfiltration and its experimental verification. J. Chromutog. 14, 317-330. LBVER, W. G., SURIANO, R., and GREEN, M. (1967). Adenovirus proteins. II. N-terminal amino acid analysis. J. Viral. 1, 723-728.
136
PETTERSSON
L.~vER, W., PERF,IRA, H. G., RUSSELL, W. C., and VALENTINE, R. (1968). Isolation of an internal component from adenovirus type 5. J. Mol. Biol. 37, 379-386. LONRXRG-HOLM, K., and PHILIPSON, L. (1969). Early events of virus-cell interaction in an adenovirus system. J. Viral. 4, 323-338. MAIZEL, J. V., JR. (1966). Acrylamide-gel electropherograms by mechanical fractionation: radioactive adenovirus proteins. Science 151, 988-990. MAIZEL, J. V., JR., WHITE, D. O., and SCHARFF, M. D. (1968). The polypeptides of adenovirus I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12. Virology 36, 115-125. NEURATH, A. H., RURIN, A. B., and STASNY, J. T. (1968). Cleavage by formamide of intercapsomer bonds in adenovirus type 4 and 7 virions and hemagglutinins. J. Viral. 2, 1086-1095. NEURATH, A. R., RUBIN, B. A., WIENER, F. P., and HARTZELL, It. W. (1970). Evidence for the presence of half-cystine residues in the capsid of adenovirus type 7. Fed. Ew-. Biochem. Sot. Lett. 7, 114-118. NORRBY, E. (1966). The relationship between the soluble antigens and the virion of type 3. II. Identification and characterization of an incomplete hemagglutinin. Virology 30, 608-617. NORRBY, E. (1968). Biological significance of structural adenovirus components. Curr. Top. Microbial. Immunol. 43, 143. NORRBY, E. (1969). The relationship between the soluble antigens and the virion of adenovirus type 3. IV. Immunological characteristics. Virology 37, 565-576. NORRBY, E., and WADF,LL, G. (1969). Immunological relationships between hexons of certain human adenoviruses. J. Vi’irol. 4, 663-670. NORRBY, E., MARUSYK, H., and HAMMARsKJ~LD,
M.-L. (1969). The relationship between the soluble antigens and the virion of adenovirus type 3. V. Identification of antigen specificities available at the surface of virions. Virology 38, 477-482. PETTERSSON, U. (1970). Structural proteins of adenoviruses. VII. Studies on the subunit arrangement of the hexon. Thesis, Upsala University. PETTERSSON, U., and H~QLUND, S. (1969). Structural proteins of adenoviruses. III. Purification and characterization of the adenovirus type 2 penton antigen. Virology 39,90-106. PETTERSSON, U., PHILIPSON, L.,and H~GLUND,~. (1967). Structural proteins of adenoviruses. 1. Purification and characterization of adenovirus type 2 hexon antigen. Virology 33, 575690. PRAGE, L., PETTERSSON, U., HBGLUND, S., LONBERG-H• LM, K., and PHILIPSON, L. (1970). Structural proteins of adenoviruses. IV. Sequential degradation of adenovirus type 2 &ions. Virology 42, 341-368. ROSEN, L. (1960). A hemagglutination-inhibition technique for typing adenoviruses. Amer. J. Hyg. 71, 120-128. SHORTRIDGE, K. F., and BIDDLE, F. (1970). The proteins of adenovirus type 5. Arch. Ges. Virusjorsch. 29, l-24. SMITH, K. O., GEHLE, W. D., and TROUSDhLE, M. D. (1965). Architechture of the adenovirus capsid. J. Bacterial. 90, 25&261. VELICER, L., and GINSBERG, H. (1968). Cytoplasmic synthesis of type 5 adenovirus capsid proteins. Proc. Nat. Acad. Sci. U. S. 61, 12641271. WILCOX, W. C., and GINSBERG, H. S. (1963). Production of specific neutralizing antibody with soluble antigens of type 5 adenovirus Proc. Sot. Exp. Med. 114, 37-42.
43,
123-136 (1971)
Structural
Proteins
VI. On the Antigenic ULF Department
of Microbiology,
of Adenoviruses’
Determinants
of the Hexon
PETTERSSON The Wallenberg
Laboratory,
Uppsala,
Sweden
Accepted September 19, 1970 Hexon ant.igens were purified from KB cells infected with adenovirus types 2, 3, and 5 and compared chemically and immunologically. All three types of hexons were similar in their amino acid composition and each contained 0.5-0.870 half-cystine, determined as cysteic acid after performic acid oxidation. Type- and group-specific antigenic determinants were detected by immunodiffusion on hexons from each serotype. The group-specific determinants were degraded after digestion with chymotrypsin, subtilisin, or papain, leaving a “hexon core” which retained all of its type-specific properties and which at neutral pH formed rapidly sedimenting aggregates. Trypsin removed 5-107, of the native hexon polypeptide, presumably from the C-terminal end, without affect.ing its antigenic properties. Maleic anhydride preferentially inactivated the type-specific determinants, without altering the size or the shape of the native hexon. Two populations of hexons were isolated from cells infected with adenovirus type 2. The minor of these had a lower electrophoretic mobility, but similar size and shape as the main class of hexons. Antisera against the slowly migrating population contained antibodies with 50-fold higher specific neutralizing activity (neutralizing titer/ complement fixing titer) than antisera prepared against the main population of type 2 hexons. INTRODUCTION
Adenovirus hexon antigen has been reported to contain identical subunits, each composed of one single type of polypeptide chain (Maize1 et al., 1968; Velicer and Ginsberg, 1968; Pettersson, 1970). The molecular weight of the entire hexon unit is in the range 310,000-360,000 (Franklin et al., 1970) and evidence for a complex arrangement of group- and type-specific determinants has recently been given (Norrby, 1969; Norrby and Wadell, 1969). The hexon has been considered to be an unusually resistant structure, unaffected by mild proteolysis and low pH (Allison et al., 1 This Damon Medical Society, Medical
project was supported by grants from the Runyon Memorial Fund, the Swedish Research Council, the Swedish Cancer and the Swedish Delegation for Applied Defense Research.
1960) and treatment with high concentrations of agents like formamide (Neurath et al., 1968) and urea (Short’ridge and Biddle, 1970). The present investigation describes the comparative properties of purified hexons from adenovirus types 2, 3, and 5. Proteolytic and chemical agents have been used to specifically alter or degrade different parts of the hexon molecule in order to obtain further information on the arrangement of the antigenic determinants on the hexon. MATERIALS
AND
METHODS
Cell cultures and virus infection. Adenovirus types 2, 3, and 5 were grown in 2-liter spinner cultures of KB cells as described by Pettersson and Hijglund (1969). Processing of virus and soluble antigens. The procedure of Green and Piiia (1963) was
124
PETTERSSON
used with some modifications (LonbergHolm and Philipson, 1969). Separation of antigens by DEAE chromatography. Antigens from adenovirus types 2 and 5 were separated at pH 6.5 as described previously (Pettersson et al., 1967). Antigens from adenovirus type 3 were separated at pH 8.4 as described by Norrby (1966). Exclusion chromatography. Agarose (Sepharose 6 B Pharmacia Fine Chemicals, Uppsala, Sweden), 6%, was used with 0.05 M Tris-acetate pH 8.0, containing 0.15 M NaCl as elution buffer (Pettersson and Hoglund, 1969). Rate-xonal centrifugation. Preformed linear gradients, 4.5 ml containing 5-25 % sucrose in 0.002 M Tris, pH 7.2, or 0.1 M formic acid were used in a Spinco SW 50-l rotor. The gradients were fractionated by drop collection from the bottom. Polyacrylamide gel electrophoresis. Analytical runs were made on 5 % gels in 0.05 M Tris-acetate pH 8.0 as described by Pettersson and Hoglund (1969). Preparative electrophoresis was performed at pH 9.5 in a continuous elution apparatus as described by Franklin et al. (1970). PuriJicatian of hexun antigen from types 2, S, and 5. The purification procedure of Pettersson et al. (1967), using DEAE-chromatography and preparative polyacrylamide gel electrophoresis was essentially followed with one important modification: The preparative gel electrophoresis according to Maize1 (1966) was replaced by a similar step using the continuous elution apparatus of Hjerten et al. (1965). Adenovirus type 3 antigens were separated by exclusion chromatography on 6% agarose prior to the preparative electrophoresis in order to resolve hexons and fibers which in the case of adenovirus type 3 elute close to each other after DEAE-chromatography (Norrby, 1966). Hexons so purified were homogeneous as judged by analytical polyacrylamide gel electrophoresis at pH 8.0 (Fig. 1) and at pH 9.5 (Fig. 3b) and by immunoelectrophoresis. The purity of type 2 hexons was also checked by analytical which revealed one ultracentrifugation, single homogeneous boundary both when using schlieren and UV optics. N-terminal amino acid analysis of purified type 2 hexons
using the phenylisothiocyanate-355 method showed the absence of reactive N-group (Pettersson, 1970). Enzymatic digestion. Chymotrypsin (recrystallized X 3), trypsin (TR-TPCK) and papain were purcha.sed from Worthington, New Jersey. Subtilisin (crystalline) was kindly provided by Novo Industri A/S, Denmark. All digestions were carried out at +37” in 0.05 M Tris-acetate buffer, pH 8.0. Papain was activated by the addition of cysteine to a final concentration of 0.01 M. The tryptic digestions were stopped after 1 hour by addition of 2-fold excess of trypsin inhibitor (Sigma Chemical Company, St. Louis, Missouri) and papain was inactivated after 18 hours by addition of iodoacetamide to 0.02 M final concentration. Chymotrypsin and subtilisin were inactivated by addition of 10 mM diisopropyl fluorophosphate after 60 and 180 min, respectively. In some experiment,s the samples were cooled to 0” at the end of the digestion and then directly added to sucrose gradients or chromatographic columns without specific inactivation. Maleylation. Maleic anhydride was recrystallized from chloroform prior to use. All samples were dialyzed against 0.1 M Na2HP04 before the addition of 10 mg solid maleic anhydride to 100-500 pg of hexon. The reagent was added slowly and the pH was kept constant at 9 by use of a pH-stat (Radiometer, Copenhagen, Denmark). Amino acid analysis. Samples of 0.5-2 mg were hydrolyzed as described by Pettersson et al. (1967) and analyzed on a “Biochrom” automatic amino acid analyzer. Cysteine was determined as cysteic acid after performic acid oxidation (Hirs, 1956). N-Terminal amino acid analysis. Samples of l-3 mg of hexon or marker protein (fibrogen) were analyzed by a modified Edman method using 35S-labeled phenylisothiocyanate (Laver et al., 1967; Pettersson, 1970). Peptide mapping. The method of Blomback et al. (1968) was followed. Intact or degraded hexons, 0.5-1.5 mg, were denatured by heating to 100” in 0.02 M NH4HC03, pH 8.5, for 1 minute. The denatured protein was lyophilized and resuspended in 0.05
STRUCTURAL
PROTEINS
ml 0.2 64 NH4HCOB, pH 8.5, a,nd trypsin (TR-TPCK) was added to an enzyme substrat,e ratio of 1: 100. After 18 hours the samples were lyophilized and the peptides were dissolved in distilled water. Aliquots of 3-5 ~1 were added to cellulose thin la.yer plates (20 X 20 cm) and electrophoresis was carried out at 300 V for 75 min in pyridine acetate pH 5.5. After thorough drying the plates were submitted bo chromatography in pyridine: acetic acid:n-butanol: wat,er (100: 30: 150:120). The dry plates were stained with ninhydrin. Preparation of antisera. Antisera against virions from types 2, 3, and 5 were prepared as described previously (Pettersson and HGglund, 1969). Antiserum against type-specific det’erminants on hexons from adenovirus type 2 was prepared by absorbing an antiserum against type 2 hexons with purified type 5 hexons. The efficiency of the absorption was checked by immunodiffusion against type 5 hexons. Complement Jixation was performed according to a micromethod described previously (Pettersson and Hoglund, 1969). Neutralization tests. Neutralization of virus infectivity was assayed by inhibition of plaque formation or by inhibition of fluorescent focus formation (Pettersson and HGglund, 1969). Reduction of infectivity by 1.5 log units was scored as significant neutralization. Specific neutralizing activity was measured as the neutralizing titer/complement fixing titer against purified hexons.
OF ADENOVIRUSES.
125
VI.
these hexons, while the slight differences in composition between hexons from types 2 and 5 do not permit any definite conclusions with regard to basic and dicarboxylic amino acids. Since earlier studies had indicated that hexons from both type 2 (Pettersson et al., 1967) and type 5 (Biserte et al., 1966; Boulanger et al., 1969) lack half-cyst’ine residues, this problem was reinvestigated by performing analysis of performic acid oxidized hexons. Under these conditions 0.5-0.8% half-cystine was detected in all three types of hexons (Table 1). Immunoloyicwl Hexons
Properties
of
Adenovirus
The antigenic specificities of native hexons from adenovirus t(ypes 2, 3, and 5 were investigated by use of the immunodiffusion assay. Purified hexons from the three serotypes were tested against antisera prepared toward homotypic and heterotypic hexons or virions. In all cases, spurs were seen between homotypic and heterotypic hexons indicating a relationship of partial identity (Fig. 2a), while no spurs were observed when homotypic and heterotypic antisera were compared (Fig. 2b). The presence of type-specific components on adenovirus hexons was confirmed by absorption studies; antihexon sera, absorbed with heterotypic hexons, still gave precipi-
RESULTS
Characterization
of the PuriJied Hexons
Hexon antigens from types 2, 3, and 5 were compared with regard to electrophoretic properties and amino acid composition. The three types of hexons varied slight,ly in their electrophoretic mobilities; type 2 hexons being faster and type 3 hexons slower relative to type 5 hexons (Fig. 1). With regard to amino acid composition some minor differences were observed (Table 1). Type 3 hexons exhibited a lower content of glutamic acid which may account for the lower electrophoretic mobility of
FIG. 1. Analytical polyacrylamide gel electrophoresis on 594 gels at pH 8.0 of purified hexons from adenovirus types 2 (I), 3 @), 5 (3), and a mixture of all three (4). Origin at the top and anode toward the bottom.
126
PETTERSSON TABLE AMINO
ACID
COMPOSITION
Residue
Type 2c hexon
LYS His A% Asp Thr” Serb Glu Pro GUY Ala Half -Cysf Val Met Ile Leu TY~ Phe TOP
4.16 1.40 4.61 14.41 7.03 6.84 9.49 6.08 6.76 7.10 0.76 5.69 2.72 3.55 7.70 5.61 4.44 1.658
1
OF ADENOVIRUS
HEXON
ANTIGEV
Type Sd hexon
Type 3d hexon
4.60 1.72 4.95 14.46 6.95 6.30 10.04 6.10 6.34 6.95 0.79 5.26 2.73 4.40 7.80 5.60 5.05
4.02 1.65 4.92 14.52 8.25 6.80 8.31 6.14 7.43 6.82 0.48 6.02 2.95 3.85 7.29 5.46 5.01
f f + f f f Zk & zt f
0.15 0.14 0.39 0.27 0.05 0.12 0.19 0.29 0.25 0.29
+ + zk f f f ND”
0.03 0.10 0.20 0.02 0.31 0.11
zt 0.15 f 0.09 * 0.13 It 0.10 zk 0.12 zt 0.30 f 0.04 f 0.23 f 0.25 & 0.12 & 0.02 * 0.16 f 0.09 + 0.08 f 0.16 rk 0.24 f 0.06 NDB
Type 2 hexond degraded by trypsin 4.52 1.45 4.49 15.02 7.47 7.90 8.98 6.26 7.66 7.34
& zt l zk zt zt xk xt f f ND” 5.47 f 2.35 f 3.61 zk 7.69 f 5.25 zk 4.58 f ND
0.18 0.13 0.04 0.69 0.19 0.65 0.06 0.13 0.28 0.09 0.31 0.26 0.14 0.14 0.33 0.08
a Given in moles per 100. b Extrapolated to zero time hydrolysis. c 24 and 72 hour hydrolysis was performed, and the values represent the average except for Val and Ile, which were calculated from the 72 hours data only (Pettersson, 1970). The values given are the average of two determinations. d 24-hour hydrolysis only. The values are the average of two determinations. e Not determined. f Determined as cysteic acid after performic acid oxidation. The half-cystine values for type 2 and 3 hexons are the average of two determinations, and the value for type 5 hexons is from one single determination. g Determined spectrophotometrically according to Edelhoch (1967).
tation lines with homotypic hexons. A closer immunological relationship between hexons from types 2 and 5 (belonging to subgroup III) was shown by absorption of an antiserum against type 5 virions with hexons from serotype 3 (subgroup I). Such an antiserum gave precipitation lines with type 5 as well as with type 2 hexons. Isolation of a Special Class of Adenovirus Type 2 Hexons During the purification of large amounts of type 2 or type 5 hexons, it was observed that a minor population of hexons was eluted after the majority of the hexons from the preparative polyacrylamide gel and thus had a lower electrophoretic mobility (Fig. 3a). These hexons designated “special
hexons,” were aft,er this step contaminated with fast migrat,ing hexons and were further purified by recycling the fractions through the electrophoresis apparatus twice (Fig. 3~). In order to estimate the size of the “special hexons” gel chromatography on 6 % agarose (Fig. 4) as well as rate zonal centrifugation was performed with ordinary hexons as markers. Both classes of hexons eluted in identical positions after the agarose chromatography (Fig. 4) and sedimented with t,he same rates (not shown) indicating that they had similar size and shape and excluding a monomer-dimer relationship. When the two classes of hexons from serotype 2 were compared by the immunodiffusion assay no differences could be detected (not shown) but when their respective rabbit-
STRUCTURAL
PROTEINS
antisera were compared with regard to neutralization, it was observed that antiserum against the “special hexons” were approximately 50-fold more potent (Table 2). Similar results were obtained with guinea pig sera although the titers were low and do not permit as definite conclusions (Table 2). The heterogeneity among type 2 hexons was also shown by immunoelectrophoresis (Fig. 5), while electron microscopy did not
OF ADENOVIRUSES.
CPrn 30000 26000 22000
127
VI.
I
200
12000
160 14000 120 60 6000 2000
40
t
L 20
25
30
35
40
45
50
fraction cm
55
number
(b)
2600C 22000 16000
‘t
14000 10000 6000
fraction cpm
,
number
Cc)
13000 u 11000 I 9coo
/I
-
70005000
-
3000
-
1000 20
25
30
35
40
45
50
traction
FIG. 2a. Immunodiffusion of purified hexons from types 2 (2A), 3 @A), and 5 @A) against an antiserum prepared toward type 2 hexons. FIG. 2b. Immunodiffusion of purified type 2 hexons (central well) against antisera against virions from types 2 (S2), 3 (SS), and 5 (56).
55
number
FIG. 3. Elution diagrams from preparative polyacrylamide gel electrophoresis at pH 9.5 of amino acid-3H-labeled type 2 hexons: (a) Separation of hexon antigen obtained after DEAE-chromatography. Radioactivity (O--O) as well as diameters obtained by radial immunodiffusion with an antihexon serum (O--O) were measured. t-i indicates fractions from peaks I and II which were pooled and concentrated and dialyzed for recycling. (b) Recycling of material obtained from peak I. (c) Recycling of material obtained from peak II. Peak “III” corresponds to the slowly migrating hexons and was homogeneous when analyzed by analytical polyacrylamide electrophoresis.
128 CP~
PETTERSSON rg/mt
500 300 100
15
20
25
30
35
LO
45
fraction
50
after treatment with trypsin at a concentration of 1 mg/ml as judged by immunodiffusion (not shown). When, however, tryptically digested type 2 hexons were analyzed by exclusion chromatography on 6 % agarose (Fig. 6) or by rate zonal centrifugation (Fig. 7c) it was evident that 5-10% of the label from amino acid-3H-labeled hexons was recovered as low molecular weight material.
number
FIG. 4. Exclusion chromatography on 6% agarose of a mixture of purified “fast” unlabeled type 2 hexons (O--O) and purified amino acidSH-labeled “slow” type 2 hexons (O-O). The “fast” hexons were added in 50-fold excess on a weight basis and were analyzed by radial immunodiffusion. The arrow indicates the fraction corresponding to the total volume of the column.
TABLE
2
NEUTRALIZATION OF ADENOVIRUS TYPE INFECTIVITY BY DIFFERENT ANTISERA”
2
-
Antiserum
FIG. 5. Analytical polyacrylamide gel electrophoresis (left) and immunoelectrophoresis (right) of “fast” (I) and a mixture of “slow” and “fast” hexons (2) from adenovirus type 2. The antiserum was an antiserum against type 2 hexons.
against
Fast migrating type 2 hexons, rabbit Fast migrating type 2 hexons, guinea pig Slowly migrating type 2 hexons, rabbit Slowly migrating type 2 hexons, guinea pig Disrupted type 2 virions
5
2
256
cPm
/Js/ml
3500 1
20
10
1024
160
160
256
20
20
16
2560
1024
A
750
a Reduction of 1.5 log units of infectivity was scored as significant neutralization. lob-plaqueforming units or fluorescent focus units were mixed with antiserum as described in Methods. b Fluorescent focus assay. c Complement fixing titer against 4 units of purified fast or slowly migrating type 2 hexons.
between the two classes of hexons (Dr. S. Hiiglund, personal communication).
distinguish
Enzymatic Degradation of Hexon Antigen Trypsin. The antigenic specificities of hexons from types 2,3, and 5 were preserved
250 30
LO
50
60 fract’mrl
70
80 ncniber
FIG. 6. Exclusion chromatography on 6y0 agarose of a mixture of amino acid-3H-labeled type degraded with 1 mg/ml trypsin 2 hexons, (O--O) and intact unlabeled type 2 hexons (O--O). The enzyme was inhibited by trypsin inhibitor prior to the addition of a SO-fold excess of unlabeled marker. The intact hexons were traced by radial immunodiffusion. The arrow indicates the fraction corresponding to the total volume of the column. Tryptic digestion for 18 hours did not significantly change the profile.
STRUCTURAL
H3
5
cPm
PROTEINS
15
10
FRACTION
30000
NUMBER
t 20000 10000
“3
B /
t
5
cpm 30000 t
“3
CPm (
15
10 FRACTION
n
5
lo
5
10
FRACTIZ
15
NUMBER
NUMBER
OF ADENOVIRUSES.
VI.
129
peptide which was attacked by trypsin by performing N-terminal amino acid analysis. Tryptically digested hexons were purified from enzyme and released peptide by exclusion chromatography on 6 % agarose and submitted to N-terminal amino acid analysis. No free N-terminal amino acid could, however, be identified in either the undegraded hexons (Pettersson, 1970) or the digested hexons, suggesting that the peptide( released after trypsin digestion were removed from the C-terminal end of the hexon polypeptide. When hexon antigen was heated to 100” prior t,o the trypbic digestion an extensive degradation was seen (Fig. 7B). Chymotrypsin. Hexon antigen from adenovirus types 2, 3, and 5 was digested for 1 hour with chymotrypsin at a concentration of 1 mg/ml and subsequently analyzed by immunodiffusion. A reaction of partial identity was seen when digested and undigested hexons were tested against homotypic antiserum (Fig. 9 a,b), indicating loss of antigenic determinants. When these degraded hexons were compared with
20
FRACTION
NUMBER
7. Rate-zonal centrifugation (5-25s sucrose in 0.062M Tris pH 7.2; 106,060 g for 16 hours) of (A) purified amino acidJH-labeled hexons, (B) type 2 hexons degraded by 1 mg/ml trypsin after heat denaturation (lOtI”, 1 min), (C) hexons digested with 1 mg/ml trypsin, (D) type 2 hexons digested with 1 mg/ml chymotrypsin. The main peak contained antigenically degraded hexons, and the fast sedimenting peaks probably aggregates of these. FIG.
The elution profile after 6 % agarose showed a. slight shift toward the total volume of the column, thus suggesting a decrease in the Stokes’ molecular radius as compared to intact hexons (Fig. 6). A limited degradation of the hexons was also revealed by electrophoresis; the mobility of trypsin-treated hexons was lower than that of untreated hexons (Fig. 8). Amino acid analysis showed that this alteration might be due to release of peptide which have a low lysine but high glutamic acid content (Table 1). Since the degraded hexons appeared as a homogeneous preparation, it should be possible to trace the part of the hexon poly-
FIG. 8. Analytical polyacrylamide gel electrophoresis at pH 8.0 of type 2 hexons after trypsin digestion (I), a mixture of tryptically and undigested hexons (Z), and type 2 hexons after chymotrypsin digestion (3).
FIG. 9. (a) Immunodiffusion of type 2 hcxons ($A), type 2 hexons after chymotrypsin digestion @AC), type 3 hexons ($A), and type 5 hexons (6.4). Autiserrun against type 2 hexons in the central well. (b) Immunodiffusion of type 3 hexons after chymotryptic digestion (SAC) and intact hexons from types 2, 3, and 5. Antiserum against type 3 virions in the central well. (c) Immunodiffusion of type 2 hexons after chymotrypsin digestion (central well) against antisera against type 2 hexons (S.S), type 3 virions (SS) and type 5 virions (S5). (d) Immunodiffusion of intact type 2 hexons @A), type 2 hexons after digestion with 1 mg/ml chymotrypsin @AC) and with 0.1 mg/ml papain @Al’) and type 2 hexons digested with 1 mg/ml subtilisin @AS). Antiserum against type 2 hexons in central well. (e) Type 2 hcxons after digestion with papain (ZAP) with chymotrypsin @AC) and with 1 mg/ml subtilisin @AS), intact type 2 hexons (2A) and type 5 hexons @A) tested against an anti-type 2 hexon serum absorbed with type 5 hexons. 130
STRUCTURAL
PROTEINS
OF ADENOVIRUSES.
VI.
131
hexons from other types (i.e., types 3 and 5) gated into larger units which sedimented faster than intact hexons. a reaction of nonidentity was observed considerably (Fig. 9a,b) and when degraded and unde- These aggregates could be dissociated by graded type 2 hexons were tested against an performing the centrifugation in the presanti-hexon serum, containing antibodies only ence of 0.1 M formic acid (Fig. 10a). against type-specific parts of the hexon (an CPm, anti-adenovirus type 2 hexon serum, ab- H3 1 sorbed with type 5 hexons) a reaction of identity was seen (Fig. 9e). These findings suggest that the chymotryptic digestion selectively removed group-specific determinants from the hexon antigen, although when digested hexons were tested against 10 20 FRACTION NUMBER heterotypic antisera a slight precipitation line was seen (Fig. 9c) revealing that some residual group specificity remained after digest’ion. The extent of the chymotryptic digestion was evaluated by sucrose gradient centrifugation (Fig. 7~). Around 20 % of the counts from amino acid-3H-labeled hexons remained near the top of the gradient after FRACTION NUMBER 10 20 centrifugation of the digested hexons. The remaining antigenic portions of the hexons cpm showed a tendency to aggregate since some Ii3 material with faster sedimentation rates than intact hexons was observed (Fig. 7~). 2000 C TOP Such aggregates were also demonstrated when digested hexons were submitted to gel 1000 chromatography on 6% agarose. The digested hexons eluted as a broad peak corFRACTION NUMBER 10 20 cc-m responding to a K,, value (Laurent and Killander, 1964) of 0.39-0.42 while intact hexons under the same conditions had a KAv value of 0.43. Electron microscopy of the digested hexons showed structures with a morphology similar to that of intact hexons, but they usually appeared as aggregates of several units (Dr. S. Hoglund, 10 20 FRACTION NUMBER personal communication). FIG. 10. Rate-zonal centrifugation in 5-25s Papain. Papain at a concentration of 0.1 sucrose. Gradients A and B were centrifuged at 100,000 g for 16 hours and gradients B and C at mg/ml in the presence of 10 mM cysteine and at pH 8.0 digested adenovirus type 2 200,000 g for 5 hours. (A) 3H-amino acid labeled type 2 hexons after papain digestion. Separation hexons extensively, leaving a “hexon core” was done in gradients containing 0.1 M formic which was still ant,igenic (Fig. 9d and lob). acid. (B) The same material as in (A) but separaWhen tested by immunodiffusion, it was tion carried out in gradients containing 0.002 M apparent that these “hexon cores” retained Tris, pH 7.2. (C) Amino acidJH-labeled type 2 their antigenicity t’o the same extent as hexons digested with 1 mg/ml subtilisin. The hexons digested with chymotrypsin, since a gradients contain 0.1 A4 formic acid. (U) The same reaction of identit,y was observed bet’ween material as in (C), but separation carried out in hexons digested by these two enzymes gradients containing 0.002 M Tris, pH 7.2. The arrows in (A) and (C) . indicate the oositions of (Fig. 9d). When analyzed by sucrose gra- undigested hexons run on formic acid containing dient centrifugation it was shown that the gradients, and (!--I) indicates fractions which “hexon core” after papain digestion aggre- were precipitated by antihexon serum.
132
PETTERSSON
More than 50% of the label from amino acid-3H-labeled hexons was recovered on top of the sucrose gradients after centrifugation of papain digested material (Fig. lob). Subtilisin. Subtilisin at a concentration of 0.1 mg/ml digested adenovirus type 2 hexons to a similar extent as does papain, also leaving a LLhexon core.” Immunodiffusion analysis showed that these “cores” were antigenically altered to the same extent as were hexons after degradation with papain or chymotrypsin (not shown). When a lo-fold higher enzyme concentration (1 mg/ml) was used, a more extensive loss of antigenic specificity was seen, demonstrated as a spur between material obtained after subtilisin and chymotrypsin treatment (Fig. 9d). When these hexons were tested against a strictly type-specific antihexon serum a reaction of identity was observed even between intact hexons and these extensively digested hexons, suggesting that the type-specific portions remained unaltered (Fig. 9e). When, however, subtilisin degraded material was tested against heterotypic sera, faint precipitation lines were still detected, indicating the presence of some remaining group-specific determinant (not shown). When hexons degraded by subtilisin were analyzed on sucrose gradients the subtilisin “cores” were shown to form fast-sedimenting aggregates, which also were dissociable under acid conditions (Fig. lOc,d). Peptide Mapping 2 Hexons
peptides showed that the degraded hexons lacked 15-20 peptides, most of which were comparatively hydrophilic. Alteration of the Hexon Antigenic Specijicities after Treatment with Maleic Anhydride Maleylation of native type 2 hexons altered them into electrophoretically fast migrating structures (Fig. 11). This change was considered to be due to blocking of the lysine E-amino groups rather than to dissociation, since gel chromatography (not shown) and sucrose gradient centrifugation (Fig. 12) did not show any drastic changes in size or shape. When maleylated hexons were compared to untreated hexons by immunodiffusion, it was evident that maleylation was accompanied by changes in antigenic properties which could be described as follows: When treated and untreated type 2 hexons were tested against sera containing antibodies against type 5 or type 2 hexons a pattern of partial identity was observed, while when tested against serum containing antibodies against type 3 hexons a pattern of identity was seen (Fig. 13a). When maleylated hexons were tested against an antiserum
of Intact or Degraded Type
Peptide mapping was performed in an attempt to find some possible explanation for the resistance of some parts of the hexon molecule towards proteolytic degrsdation. Maps of the tryptic peptides from heat denatured adenovirus type 2 hexons showed 40-50 ninhydrin-positive peptides, many of these having hydrophobic properties (Pettersson, 1970). For comparison, type 2 hexons were degraded with subtilisin (1 mg/ml) and the “core” was purified by rate zonal centrifugation (200,000 g for 5 hr), dialyzed, lyophilized, and finally digested with trypsin after heat denaturation. Mapping of these
FIG. 11. Analytical polyacrylamide gel electrophoresis at pH 8.0 of type 2 hexons (9) type 2 hexons after maleylation (f), and a mixture of both (2).
STRUCTURAL
PROTEINS cpm H3
!
10
20
le.00
FRACTION
NUMBER
12. Rate-zonal centrifugation (5-25s sucrose; 0.602 M Tris, pH 7.2, 160,660 g for 16 hours) of 3H-labeled type 2 hexons after maleylation (O--O). Untreated hexons (O---O) were run on a separate gradient, since the maleylated protein showed a tendency to interact with the marker protein. FIG.
containing antibodies only against typespecific hexon components, no precipitation line was obtained (Fig. 13b). These findings suggest that the parts preferent’ially altered by maleic anhydride were those corresponding to type- and subgroup-specific portions of the hexon.
OF ADENOVIRUSES.
VI.
133
hexons against antihexon sera from different types. This technique is not so sensitive to minor variat’ions in antigenic composition (Fig. 2b). A minor population of hexons (“special hexons”) with a lower electrophoretic mobility than the main class of hexons was also isolated from cells infected with type 2 or type 5 adenoviruses. The two classes of hexons were of similar size and shape as demonstrated by sucrose gradient centrifugation or exclusion chromatography. Antigenitally they were indistinguisha.ble when conventional assays such as immunodiff usion were employed, but when antisera against the two populations of hexons were compared, it was shown that sera against the slowly migrating type contained approximately 50-fold higher specific neutralizing activity (neutralizing titer/complement fixing titer) (Table 2). Such factors might explain, in part, the controversy regarding the neutralizing capacity of hexon antisera (Wilcox and Ginsberg, 1963; Kasel et al.,
DISCUSSION antigen has been purified from infected with three different serotypes of adenovirus and compared chemically and immunologically. The amino acid compositions of hexons from unrelated types, namely, type 2 belonging to Rosen’s subgroup III (Rosen, 1960) and type 3 belonging to subgroup I, were remarkably similar. Contrary to earlier findings (Biserte et al., 1966; Pettersson et al., 1967; Boulanger et al., 1969), half-cystine was detected in hexons from all three serotypes with techHexon KB-cells
niques
which
quantitatively
preserve
this
amino acid during hydrolysis. Recently Neurath and co-workers (1970) demonstrated the presence of half-cystine in type 7 hexons after labeling of viral components with cystineJ*C. In the present study our purified hexon preparations were shown to contain type-specific antigenic determinants (Fig. 2a) which is in conflict with our previously reported data (Pettersson et al., 1967). This discrepancy may be explained by variations in the techniques used to demonstrate antigenic differences. In our previous communication we tested type 2
FIG. 13. (a) Immunodiffusion of maleylated hexons (2AM) and untreated hexons from adenovirus type 2 (ZA) against antisera toward: type 2
hexons (S2), type 3 virions (SS), type 5 virions (86). (b) Immunodiffusion of maleylated type 2 hexons in the central well against an anti-type 3 virion serum (SS) and an anti-type 2 hexon serum, absorbed with excess of type 5 hexons (S-6).
134
PETTERSSON
1966; Pettersson et al., 1967; Kjellen and Pereira, 1968; Norrby, 1969), since some of these investigators have used purification methods which would not separate the two classes of hexons. The relationship bet’ween the (‘special hexons” and the hexons of the mature virion is not yet clear and several possibilities could be considered: the virion might a priori be suspected to contain two classes of hexons, since the hexons in peripentonal positions have different surroundings than the hexons on the faces of the adenovirus icosahedron. Hexons of peripentonal origin have recently been isolated after dialysis of virions against a Trismaleate buffer at pH 6.4 (Prage et al., 1970). These hexons are, however, not electrophoretically identical to t#he “special hexons.” Aberrant hexons could also be present elsewhere in the adenovirus icosahedron; the “special hexons” could, for example, be precursors to the aggregates of nine hexons, which often are obtained when adenovirus particles are disintegrated (Smith et al., 1965; Laver et al., 1968; Maize1 et al., 1968; Prage et al., 1970). The recent findings of separate populations of hexons with different isoelectric points from type 5 adenovirions dissociated with 8 M urea (Shortridge and Biddle, 1970) do imply the existence of more than one class of hexon in the mature virion as well. The difference on the polypeptide level between the two classes of hexons could either be related to the presence of an additional peptide on the “special hexons” or to conformational differences. A special case of the former alternative would be the presence of a large polypeptide in “special hexons” which during maturation is split into a small peptide plus an “ordinary hexon” which both are incorporated in the mature virus particle. The possibility of creating artificial hexons during the purification procedure, due to losses of peptides or conformational changes, is also conceivable and could not be excluded from available experimental data. At,tempts have been made to convert the two classes into one under denaturing conditions but urea up to 7 M concentration did not eliminate the difference in their electrophoretic properties (unpublished). Further experiment,al data
are obviously required on t’he relationship between the hexons, synt’hesized in excess in adenovirus infect.ed cells, and the hexons of the mature virion, before the importance of hexon antigen for adenovirus neutralization may be established. Previous reports have indicated that native adenovirus hexons are unaffected by the proteolytic activity of trypsin, chymotrypsin, pepsin, and papain (Allison et al., 1960; Norrby, 196s). The possibility of accomplishing a limited proteolytic degradation was reinvestigated in the present study, since it was considered useful for further structural studies on adenovirus hexon to have access to smaller hexon fragments. Trypsin was found to split off 5-10 % of the hexon polypeptide, probably from the Cterminal end, without changing the antigenie properties of t’he hexon. By the use of subtilisin, chymotrypsin or papain it was possible to obtain a limited degradation which in all cases preserved a “core structure” which had lost part of its group-specific properties but had retained all its typespecific properties (Table 3), and which at neutral pH readily formed fast sedimenting aggregates. TABLE ANTIGENIC
MODIFICATION
3 OF TYPE
Part of the hexon PoikplPModifying
agent
Trypsin, 1 mg/ml Chymotrypsin, 1 m&ml Papain, 1 mg/ml Subtilisin, 0.1 mg/m 1 Subtilisin, 1 mg/ml Maleic anhydride
released as low molecular weight material (%I
2 HEXONS
1[nactiva- I nactivation of tion of typegroup specific specific deterdeterminants’” I ninants”
5-10 15-25
None None
>50 >50 >50 0
None None None +++t
None ++
Q The change in antigenic determinants were classified on an arbitrary scale (None -+++i-), based on immunological differences as revealed by immunodiffusion. b Corresponds to the subgroup specific part of the type 2 hexon.
STRUCTURAL
PROTEINS
Subtilisin at high concentrations (1 mg/ml) removed further specificities from the group-specific portion (Table 3) indicating a complex arrangement of the antigenic determinants of hexons, which previously has been demonstrated in cross-absorption studies (Norrby, 1969; Norrby and Wadell, 1969). The hexon structure t#hus appears to be divided into two entities, one corresponding to the type-specific portion characterized by compact structure and resistant to enzymatic degradat’ion even by enzymes with broad specificities like papain and subtilisin, and the other containing groupspecific determinants which may be digested by several proteolytic enzymes. The recent findings of Norrby et al. (1969), that only type specific antigenic determinants are exposed outside t’he type 3 virion would indicate that adenoviruses have an enzyme resistant surface structure, which could be advantageous for the survival of the infectious particle. Peptide mapping showed that the isolated “hexon core” after subtilisin digestion was enriched in hydrophobic sequences. Such sequences may provide the basis for a compact tertiary struct,ure which could account for the resistance towards proteolytic degradat,ion in these parts of the hexon polypeptide, and also for the tendency of “hexon cores” to form fast sedimenting aggregates. Treatment with maleic anhydride preferentially altered type-specific parts of the hexons (Table 3; Fig. 13a,b). This was unexpected since those portions which were available for proteolysis also should be preferentially susceptible to maleylation. However, substances like maleic anhydride might have a selective affinity on the hydrophobic regions in proteins presumably present in abundance in the “hexon core.” ACKNOWLEDGMENT The author is indebted to Professor L. Philipson for helpful criticism and discussion, to Professor J. Sjiiquist and B. Blomback for supplying laboratory facilities for N-terminal analysis and peptide mapping and to Dr. S. Hoglund for aid with electron microscopic examinations. The skillful technical assistance by Miss S. Rosen, Miss H. Hessle, Mrs. E. Hjertson, and Mrs. S. Siiderman is also gratefully acknowledged.
OF ADENOVIRUSES.
VI.
135
REFERENCES ALLISON, A. C., PERUUR~, H. G., and FSRTHING, C. P. (1960). Investigation of adenovirus antigens by agar gel diffusion techniques. Virology 10, 316-328. BISERTE, G., S.~M.~ILLE:, J., D.~UTREVAUX, M., BOULBNGER, P., S~UTI&R~:, P., RING&L, J., and !J%‘.~ROCQUI~, R. (196F). Composition en acides amines de l’antigene de structure A de l’adenovirus 5. C. R. Acad. Sci. Ser. D 263, 1648-1649. BLOMI&K, M., BLOMHHCK, B., M:LMMF:N, E., and Puasnn, A. S. (1968). Fibrinogen Detroit-a molecular defect in the N-terminal disulphide knot of human Fibrinogen? Xa/ure (London) 218, 134-137. BOULANGEH, P. A., FLIMb:NCOuRT, P., and BISEHTE, G. (1969). Isolation and comparative chemical study of structure proteins of the adenovirus 2 and 5: hexon and fiber antigens. Eur. J. Biochem. 10, 116-131. EDELHOCH, H. (1967). Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6, 1948-1954. EDMAN, P., and SJ~QUIST, J. (1956). Identification and semiquantitative determination of 3phenyl-2-thiohydantoins. Acta Chem. &and. 10, 1507-1509. FRaNKLIN, R.M., PETTERSSON, U., &ICRVALL, K., STRANDBERG, B., and PHILIPSON, L. (1979). Structural proteins of adenoviruses. V. On the size and structure of the adenovirus type 2 hexon. J. Mol. Biol. in press. GREEN, M., and PIRI, M. (1963). Biochemical studies on the adenovirus multiplication. IV. Isolation, purification, and chemical analysis of adenovirus. Virology 20, 199-207. HIRS, C. H. W. (1956). The oxidation of ribonuclease with performic acid. J. Biol. Chem. 219, 611-621. HJI"RT&N, S., JERST~:DT, S., and TIS~LIUS, A. (1965). Electrophoretic “particle sieving” in polyacrylamide gels as applied to ribosomes. Anal. Biochem. 11, 211-218. Kase~, J. A., ALFORD, R. H., LEHRICH, J. It., BANI(S, P. A., HUMOR, M., and KNIGHT, V. (1966). Adenovirus soluble antigens for human immunization. A progress report. Amer. Rev. Respirut. Dis. 94, 168-174. KJ)>LL&N, L., and PEREIRA, H. G. (1968). Role of adenovirus antigens in the induction of virus neutralizing antibodies. J. Gen. Viral. 2, 177185. LAURENT, T. C., and KILLANDER, J. (1964). A theory of gelfiltration and its experimental verification. J. Chromutog. 14, 317-330. LBVER, W. G., SURIANO, R., and GREEN, M. (1967). Adenovirus proteins. II. N-terminal amino acid analysis. J. Viral. 1, 723-728.
136
PETTERSSON
L.~vER, W., PERF,IRA, H. G., RUSSELL, W. C., and VALENTINE, R. (1968). Isolation of an internal component from adenovirus type 5. J. Mol. Biol. 37, 379-386. LONRXRG-HOLM, K., and PHILIPSON, L. (1969). Early events of virus-cell interaction in an adenovirus system. J. Viral. 4, 323-338. MAIZEL, J. V., JR. (1966). Acrylamide-gel electropherograms by mechanical fractionation: radioactive adenovirus proteins. Science 151, 988-990. MAIZEL, J. V., JR., WHITE, D. O., and SCHARFF, M. D. (1968). The polypeptides of adenovirus I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12. Virology 36, 115-125. NEURATH, A. H., RURIN, A. B., and STASNY, J. T. (1968). Cleavage by formamide of intercapsomer bonds in adenovirus type 4 and 7 virions and hemagglutinins. J. Viral. 2, 1086-1095. NEURATH, A. R., RUBIN, B. A., WIENER, F. P., and HARTZELL, It. W. (1970). Evidence for the presence of half-cystine residues in the capsid of adenovirus type 7. Fed. Ew-. Biochem. Sot. Lett. 7, 114-118. NORRBY, E. (1966). The relationship between the soluble antigens and the virion of type 3. II. Identification and characterization of an incomplete hemagglutinin. Virology 30, 608-617. NORRBY, E. (1968). Biological significance of structural adenovirus components. Curr. Top. Microbial. Immunol. 43, 143. NORRBY, E. (1969). The relationship between the soluble antigens and the virion of adenovirus type 3. IV. Immunological characteristics. Virology 37, 565-576. NORRBY, E., and WADF,LL, G. (1969). Immunological relationships between hexons of certain human adenoviruses. J. Vi’irol. 4, 663-670. NORRBY, E., MARUSYK, H., and HAMMARsKJ~LD,
M.-L. (1969). The relationship between the soluble antigens and the virion of adenovirus type 3. V. Identification of antigen specificities available at the surface of virions. Virology 38, 477-482. PETTERSSON, U. (1970). Structural proteins of adenoviruses. VII. Studies on the subunit arrangement of the hexon. Thesis, Upsala University. PETTERSSON, U., and H~QLUND, S. (1969). Structural proteins of adenoviruses. III. Purification and characterization of the adenovirus type 2 penton antigen. Virology 39,90-106. PETTERSSON, U., PHILIPSON, L.,and H~GLUND,~. (1967). Structural proteins of adenoviruses. 1. Purification and characterization of adenovirus type 2 hexon antigen. Virology 33, 575690. PRAGE, L., PETTERSSON, U., HBGLUND, S., LONBERG-H• LM, K., and PHILIPSON, L. (1970). Structural proteins of adenoviruses. IV. Sequential degradation of adenovirus type 2 &ions. Virology 42, 341-368. ROSEN, L. (1960). A hemagglutination-inhibition technique for typing adenoviruses. Amer. J. Hyg. 71, 120-128. SHORTRIDGE, K. F., and BIDDLE, F. (1970). The proteins of adenovirus type 5. Arch. Ges. Virusjorsch. 29, l-24. SMITH, K. O., GEHLE, W. D., and TROUSDhLE, M. D. (1965). Architechture of the adenovirus capsid. J. Bacterial. 90, 25&261. VELICER, L., and GINSBERG, H. (1968). Cytoplasmic synthesis of type 5 adenovirus capsid proteins. Proc. Nat. Acad. Sci. U. S. 61, 12641271. WILCOX, W. C., and GINSBERG, H. S. (1963). Production of specific neutralizing antibody with soluble antigens of type 5 adenovirus Proc. Sot. Exp. Med. 114, 37-42.