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Structural proteins of adenoviruses
VII
63, 13%147 (1973)
Structural X. Isolation
Proteins
and Topography Virion
ETNAR EVERITT, AND Deparlmerd
of Microbiology,
of Adenoviruses’
of Low Molecular of Adenovirus
Weight
Antigens
from the
Type 2
HO SUNDQUIST, ULV PETTERSSON, LESNhllT PHILIPSON
The Wullenbetg Accepled
Luboratory,
University
of C’ppsala,
Sweden
Uppsalu,
.Yovember 27, 1972
With high resolution SLSpolyacrylamide gel electrophoresis (Maizel, 1971) and a new method to extract the basic proteins, it w&9 ascertained that adenovirus type 2 contains at least 10 distinct polypcptides (II, III, IIla, IV-X) and possibly more. Five proteins (V, VI, VII, VIII, and S) were purified by selective extraction in urea at high ionic strength, and low pI1 followed by preparative polyacrylamide electrophoresis toward the cathode at ~113.4. .4ntisera, produced against proteins V, VI, and VII were used to reveal t.hat these proteins were antigenically distinct and unrelated to hexons, pentons, and fibers. The location of the polypeptides was investigated by two methods of virion degradation (Prage el al., 1970). Polypeptides V and VI1 were associated with the l>NAcont.aining core. Polypcptide VI appeared to be associat,ed with all hexons of the virion. llexons from the triangular facets of the capsid cbontained in addition to polypeptides 11 and VI, polypept,ide IX, and possibly alvo polypeptide VIII. Hexons purified from infected cells contained only polypeptide II. Polypeptide IIla was preferentially released together with the peripentonal hexons. INTROI~UCTIOS
Adenoviruses are well suited for st,udics on virion proteins since the outer capsid proteins are soluble under native condiions. SDS-polyacrvlamide gel clectrophoresis revealed a minimum of nine polypeptidcs (II-X) (Maize1 et al., 1968a; Muizel, 1971). Fiveof t.hese (II, III, IV, V, VII) areintcpral parts of hexons, pentons, (AIaizel et al., 196Sb; Laver,
fibers, or cores 1970, I’ragc and
Pettersson, 1971, Russc~ll et al., 1971). It has recently been suggclstcd t.hat the low molecular weight components of adcnoviruses may be degradst,ion products and thus that the adcnoviruscs only contain five polgpcptidcs (Pcreira and Skchel, 1971). The present study reports the polypeptide composition of ndcnovirus type 2. Ten poly1 This work waq Pupported by grants from the Swedish RIedical l
peptides have been identified and t.heir localization in the capsid has been studied by different methods to disintegrate the sdenoviriOn. ,4 hypothetical model for the topography of the polypcptides of adenovirions was constructed on t.hc basis of t.hesc results. MATEItIAI,d
@ 19i3 by Academic t’rrss, of rrpruducti~m in any form
Inc. reuwvnl.
RIKTlCOl)S
Virus
The prototype originally
adenovirus
obtained
from
Dr.
NIH, Bethesda, Alaryland, (Pettersson et al., 1967). Cell Culture
uncl I’irus
type 2 strain R. Heubncr,
was
used
Znjeclion
KB cells were grown in spinner cultures in Eagle’s spinner medium (Eagle, 1959) with 70/, calf serum. Large-scale production of unlabeled virus was performed as described previously (Pctter~son et al., 1967). The 130
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AXI)
STRUCTURAL
PROTEINS
virus was purified essentially according to the method of Green and Pina (1963), with two CsCl-gradients and a zonal rate centrifugation in sucrose gradients yielding a virus preparation with a hostcell protein contamination of less than 0.01% as previously described (Eve&t et al., 1971). Labeled virus was produced essentially according to Lonberg-Holm and Philipson (1969). When virus was labeled from zero time after infection with 14C- or 3H-amino acids (1 pCi/ml), Eagle’s spinner medium containing go the concentration of amino acids was used, except for arginine which was kept at 500 PM in order to provide a normal virus yield (Everitt et al., 1971). Purification
of Hexons, Pentons, and Fibers
The procedures have previously been described (Pettersson et al., 1967, 1968; Pettersson and Hoglund, 1969). Antisera Specific antihexon, antipenton, antifiber as well as anti-KB cell sera were prepared as described by Pettersson et al. (1968, 1969). Specific antisera against isolated low molecular weight proteins were produced in guinea pigs. Mixtures of equal volumes of a solution of the purified proteins and Freund’s complete adjuvant were injected (50 ~1) into the footpads once a week over a period of 5 weeks. Immunoassays Immunodiffusion was performed on glass slides covered with 0.8yo agarose in 0.14 M NaCl, 0.01 M Verona1 buffer, pH 7.4. Protamine sulfate (250 pg/ml) was included since only basic proteins were extracted (Conant and Barron, 1967). Quantitative determinations of hexon and penton base antigens were carried out by the Mancini technique (Mancini et al., 1965; Pettersson and Hiiglund, 1969). Gradient Centrifugation Disrupted virus preparations were analyzed on lo-25% (w/v) linear sucrose gradients in 0.002 1M TrisaHCl buffer, pH 7.5, 0.2 m&U EDTA with and without 0.15 M NaCl at f4’. The centrifugal force was
131
OF ADENOVIRUSES
calculated for the center of the tube and is given in the figure legends. Extraction of LOU Molecular Proteins
Weight Virion
Virus preparations were dialyzed for 18 hr against 3 changes of 100 volumes of 0.005 M TrisaHCl buffer, pH 8.1 at +4’. Total volume and protein content was determined and an equal volume of 10 M urea (Serva, Heidelberg, West Germany) was added, after which opalescence disappeared. After 30 min at room temperature solid sodium chloride was added to a final concentration of 2.2 M. After the salt was dissolved, 1 M citrate buffer, pH 3.1, was added to a final concentration of 0.1 M. A precipitate formed, and the viscous extract was incubated over night at +4”. After ultraccntrifugation at 110 000 g for 1.5-17 hr at +4”, the cleared supcrnatant was dialyzed against 4 M urea in 0.01 M citrate buffer, pH 3.4. Concentrated extracts were obtained by precipitation overnight at -20” with 4 volumes of acetone. After pelleting (1000 g for 10 min), the precipitate was washed twice in acetone, lyophilized, and redissolved in a small volume of 4 &! urea in 0.01 M citrate buffer, pH 3.4. Final protein concentration was 3-5 mg/ml. Polyacrylamide
Gel Electrophoresis
Analytical urea gels. Acrylamide and N , N’-methylene-bisacrylamide (Eastman Kodak, Rochester, New York) were recrystallized according to Loening (1967). Gels were made in glass tubes (5 mm internal diameter X 100 mm). The gels consisted of 7.5yo (w/v) acrylamide, 0.30y0 (w/v> N, N’methylene-bisacrylamide in 5 dl urea and 0.1 M citrate buffer, pH 3.4. Polymerization was induced by addition of N ,N ,N ,‘N’tetramethylethylcnediamine (TEMED) and of freshly prepared ammonium persulfate to 0.2y0 and O.lS%, respectively. Gels were prerun in the cold for 15 hr at 5 V/cm. The cathode and anode vials contained 5 M urea in 0.1 M citrate buffer, pH 3.4, and 0.01 M citrat,e buffer, pH 3.4, respectively. The gels were overlaid with the cathode solution. Samples (-100 ~1) were applied to the gels in 4 M urea, 0.01 M citrate buffer, pH 3.4.
132
EVEMTT,
SUNDQUIST,
PETTERSSON,
The potential was raised from 5 V/cm to 10 V/cm after 30-45 mm, and clectrophoresis continued for 10 hr at 4”. After electrophoresis the gels were stained and fixed for 2 hr in a solution of 0.05% Buffalo Black NBR (Allied Chemicals, Morristown, New Jersey), in 10% acetic acid, 30% methanol. The gels were destained in the same solvent and either photographed or scanned at 620 nm using a Gilford spectrophotometer Model 2400 with a linear transport accessory. Gels were soaked for 2 hours in the destaining solution supplemented with 20% glycerol and subsequently cut into 2-mm slices for determination of radioactivity. Preparative polyacrylamide gels. Preparative polyacrylamide gel electrophorcsis was performed in a continuous elution apparatus with a Pevikon-filled chamber at the lower end of the gel (HjertBn et aZ., 1965). Fractions (2 ml) were collected at, 20-min intervals. The gels (9 mm diameter X 120 mm) consisted of 7.5yo (w/v) acrylamide, 0.30% (w/v) bisacrylamide in 0.015 M ammonium formate-formic acid buffer, pH 3.4, and polymerization was induced by final concentrations of 0.1% and 0.04% of TEMED and ammonium persulfate, respectively. The gels were prerun for 20 hr at 20 V/cm at $4” with methylene blue as marker. The samples, containing 335 mg of protein and 5 X lo4 cpm of labeled extract in less than 0.8 ml of 4 IM urea in 0.01 M citrate buffer, pH 3.4, were layered onto the preparative gels. Methylene blue was used as a marker. Electrophoresis was carried out at 40 V/cm, 4” for 2rj hr. The current was approximately 8 mA. Samples from eluted fractions were assayed for radioactivity after neutralization. The pooled fractions of the main peaks were concentrated by Iyophilization and redissolved in 0.015 M formateformic acid buffer, pH 3.4. Analytical XDX-gels. The technique described by Maize1 (1971) was followed. The bisacrylamide was recrystallized as above. Acrylamide (Serva, Heidelberg, West Germany) and SDS (Schuchardt, Munich, West Germany) were used without, further purification. Acrylamide gels containing 13% or 20% acrylamide, 0.35% bisacrylamide were
AND PHILIPSON
made in 13 cm plastic tubes with an int,ernaI diameter of 6 mm. Protein samples for analysis were heated at 100” for 2 min in a solution of 0.05 M Tris-NaHzPO, , pH 7.2, 1% SDS, 10% glycerol, O.Fj% (v/v) mercaptoethanol and 0.00270 phenol red. Electrophoresis was carried out at 10 V/cm for 4 hr at room temperature. Gels were stained for 45 min in a solution of 0.2% Coomassie Brilliant Blue It 250 in 7% acetic acid, 10% methanol and destained in the same solvent. They were then photographed or scanned at 550 nm. Molecular Weight Determination The molecular weights of the isolated proteins were determined in a discontinuous SDS-polyacrylamide gel electrophoresis system by comparing their mobility with proteins of known molecular weight (Neville, 1971; Maizel, 1971). The following proteins were used as markers : bovine serum albumin (68,000), ovalbumin (43,000). L chains of IgG (23,500), ribonuclease A (13,700), cytochrome c (11,700), and insulin (5700). The molecular weights given are from Weber and Osborn (1969) except for insulin where the value was taken from Dayhoff (1969). The markers and the sample of isolated protein were mixed and analyzed on the same gel. Both 13c;I, and 20% gels were used. Protein Determination Proteins were assayed by the method of Lowry et al. (1951) with bovine serum albumin as standard. Radioisotopes and Counting Procedures Individual and mixed, 3H- or i4C-labeled amino acids as well as 3H-labeled thymidine were obtained from New England Nuclear Corp., Boston, Massachusetts. Acrylamide gel slices were either hydrdlyzed directly in Protosol (NEN, Boston, Massachusetts), or homogenized and subsequently hydrolyzed overnight at 65’ in 0.6 ml 1 M NaOH. Radioactivity in the hydrolyzates was determined with toluene- or dioxane-based scintillation fluids. Samplea, in solution were neutralized, if necessary, and counted directly in a dioxane-based counting solution (Hayes, 1963).
STRUCTURAL
PROTEINS
RESULTS
II III ma/E
Properties of PuriJed Virus Host cell protein contamination in purified virus was determined by adding about 4 X 10’ acid-insoluble counts per minute of 3H-amino acid labeled uninfected KB cells to virus material before purification. From the amount of radioactivity recovered in the purified preparations, it was estimated that less than 0.01% of the protein was derived from the host, cell (Evcritt et al., 1971). In order to exclude viral degradation by proteolysis the polypeptide pa.tterns on SDSolyacrylamide gels of virus treated in three iii iffercnt, ways were compared: Freshly pred virus and virus subjected to autoeolysis at 37” for 72 hr in 0.01 M *HCl buffer, pH 8.1, and virus purified presence of an inhibitor of proteolysis M phenylmethanesulfonyl fluoride Schulze and Colowick, 1969) were yzhd. The patterns from the three arations were observed to be identical no protein degradation products could etected (Fig. 1). Ten polypeptides desigd II, III, IIIa, IV-X were regularly 4etected (Fig. 1). Gels containing excessive t&mounts of protein contained five additional bands present in very low amounts (IVal, IVa2, Va, Via, IXa) (Fig. 2). Extraction of Virion il cid tirea
Proteins by High Salt
Several batches of purified virus were extracted with high salt in acid urea as described in Materials and Methods. This procedure extracted about 20% of the virion proteins measured by the Lowry procedure as shown in Table 1. The amount of radioactivity extracted from amino acid-labeled proteins ranged from 2Ooj, for virus labeled with a mixture of amino acids to 30yo for arginine labeled virus. No significant amounts (<0.5’%) were recovered when 3Hthymidine-lab&d virus was extracted. Analysis of Extracted Virion Proteins Proteins extracted by acid urea from adenovirions were separated by analytical urea-citrate (UC)- and SDS-polyacrylamide gels. The sediment after ultracentrifugation was dissolved in the SDS-sample solution
133
OF ADENOVIRUSES
IYal IPa P
(2)
0
FIG. 1. Analytical SDS-polyacrylamide gel electrophoresis on 13% gels of adenovirus type 2, purified according to the procedure described in Materials and Methods (I), and incubated for 72 hr at 37” (2). One virus preparation was purified in the presence of 2 mM phenylmethanesulfonyl fluoride (PMSF) (3). The polypeptides are referred t,o by Roman numerals.
and a-as also separated in SDS-polyacrylamide gels. The UC gels revealed 4 bands of proteins in the urea extract which were designated a-d as shown in Fig. 2. Band b was resolved in 2 species after preparative polyacrylamide gel-electrophoresis (Fig. 3). The five proteins in the extract migrated as polypeptides V, VI, VII, VIII, and X on SDS-gels (Fig. 2). The proteins of the sediment after ultracentrifugation, migrated as polypeptidcs II, III, IIIa, IV, IVa, and IX (Fig. 2). Small amounts of polypeptides V and VII were also present in the sediment (Fig. 2). Preparative Polyacrylamide Electrophoresis of Urea Extracted Proteins Urea extracts corresponding to 34 mg of protein were separated on acrylamide gels prepared in an ammonium formate-formic acid buffer and 60-75Yo of the input radio-
134
EVERITT,
SUNDQUIST,
PETTERSSON,
AND PHILIPSON
d
w-gel FIG. 2. Stained analytical urea-citrate-(UC-) and 13% SDS-polyacrylamide gels showing phoretic separation of adenovirus type 2 proteins extracted or precipitated in the high salt, acid ure buffer described in Materials and Methods. Freshly prepared virions were analyzed in a SDS-gel for co parison. The polypeptides in the SDS-gels were referred to by Roman numerals and the proteins on ure gels were labeled a-d.
TABLE
1
EXTRACTION OF ADENOVIRION PROTEINS HIGH SALT AND ACID UREA
Method of protein assay
WITH
Number Percentage of of exadenovirus proteins or radioac- periments tivity soluble in acid urea5
Mean Range of yield value ______ W-22 Protein (Lowry) (pg) 20 20-21 3H- or W-labeled / 20.5 amino acids 30 EH-Arginine 30 3H-Thymidine 0.4 0.3-0.5
(7) (3) (1) (3)
a Label was included from 0 to 40 hr after infection.
activity was recovered in five peaks (Fig. 3A). The pooled fractions of each of the peaks (a, bl , b , c, and d) were lyophilized and dissolved in small volumes of 0.015 M formate buffer, pH 3.4. The isolated proteins
were identified on SDS-polyacrylamide ge$ as shown in Fig. 3B. These studies demonstrated that the proteins a, bz , bl , c, and d migrated as polypeptides V, VI, VIII, VII, and X, respectively. Polypeptide VII, the arginine and alanine rich core protein (AAP), was characterized in a previous communication (Prage and Pettersson, 1971) and will not be further described here. Immunoloyical Characteristics Extracted Vi&m Protezns
of
Urea-
Urea extracted proteins were isolated by preparative polyacrylamide gel electrophoresis and were used for immunization. Antisera against proteins V and VI, which showed strong precipitation in the homologous reactions also revealed precipitation lines against electrophoretically purified hexon antigen (Fig. 4). The antihexon antibodies present in anti-V and anti-VI antisera were removed by adsorption with electrophoretically purified hexon obtained from
STRUCTURAL
PROTEINS
20
10
OF ADENOVIRUSES
30
40
FRACTION
NUMBER
50
60
FIG. 3A. Preparative polyacrylamide gel electrophoresis in 0.015 M ammonium formate-formic acid pH 3.4, of high-salt, acid urea-extracted proteins of adenovirus type 2 labeled withsH-amino acids during 0 to 40 hr post infection. The peaks corresponding to those observed in the analytical urea gel in Fig. 2 are indicated in the elution profile. The arrow indicates the methylene blue (MB) marker.
V+d
d
v+c
FIQ. 3B. Identification in 13% SDS polyacrylamide parative electrophoresis. The proteins were analyzed polypeptides as described in Materials and Methods.
infected cells. Antihexon, as w-cl1 as antipenton, antifiber, and anti-KB cell sera did not show precipitation arcs against the purified proteins V and Vl (not shown). The material used for the immunization against protein VI was also contaminated with protein VIII. Although most of the hexons were preeipitated by the high salt-urea-citrate extraction the cross reactivity between hexon dnd antisera against proteins V and VI was probably due to contaminating hexons among the extracted proteins which were incompletely separated from proteins V and VI after preparative clectrophoresis. Spe-
c
V+bl
b1+2
V+b2
V+a
a
gels of urea extracted proteins isolated by preboth separately and together with whole virion
cific antisera against proteins VIII and X were difficult to produce because insufficient amounts were obtained. Relative Amounts of Extracted Proteins in the Virion In order to determine the relative amounts and the time of synthesis of each of the proteins V, VI, VII, and X in the virus particle, virions were prepared from cells labeled for different periods before and during the infectious cycle. The virus was extracted with acid urea and analyzed on analytical UCgels or on SDS-gels without extraction.
136
EVERITT,
SUNDQUIST,
PETTERSSON,
AND
PHILIPSON
FIG. 4. Immunodiffusion of purified proteins V and VI after preparative polyacrylamide gel electrophoresis. The center well of panel I contained unadsorbed anti-V guinea pig serum (A) and the center well of panel II an anti-V-serum adsorbed with electrophoretically purified hexons (A’). The anti-VIserum in the center well of panel III was mltreated (B) and that in the center well of panel IV was adsorbed with hexon in the same way as the anti-V-serum (R’). The sera were tested against purified hexon (l), penton (2), fiber (3), protein VII extracted with sulfuric acid (4AP) (Prage el ul., 1970) (a), protein V (5), protein VI ((i), protein VII (7), and a KH cell extract (8). The amount of hexon antigen added to the wells in all panels was 10 pg whereas proteins V, VI, atld VII were present in 2.0 pg per well in panels I and II and 1.6 pg per well in panels III and IV.
TJC-gels revealed that protein V and VI contained approximately 25% each of the radioactivity extracted by acid urea, whereas protein VII (AAP) accounted for about 50% of the total extract (Table 2). These figures were in good agreement with the data obtained from the SDS-acrylamido gels (Table 2). The distribution of label between the four extracted proteins V, VI, VII, and X did not change with different periods of labeling, and none of the extracted proteins, wit#h the
possible exception of protein X, appeared to be derived from prelabeled host-cell material (Table 2). The relative proportion of t’he total protein of the virus recovered as protein V, VI,, VII and X was also determined. SDS- and UC-gels showed that polypeptides V, VI, and VII accounted for about 5, fi-6, and 10-120Jq of the total virus protcin content, respecr tively, whereas polypeptide X accounted fob only 0.24.374 (Table 2).
STRUCTURAL
PROTEINS TABLE
2
RELATIVE: AMOUNTS OF BASIC PROTICINS IN THE: AIXNOVIRUS
Method
UC-gels0 SDS-gels UC-gels UC-gels UC-gels
137
OF ADENOVIRUSES
TYPI.: 2
Specific 1 Percent radioactivity of total urea Percent radioactivity of total Label period post infection activity of la- extracted radioactivity in protein / radioactivity of virions in protein beled virions (hr) (cpm/1012 I ~_ __~ _____~~ particles) ~ V x VII ?< v ( VI , VII VP ~ ~~ --~- ,-~ 3.5 x 040 3.5 x 040 ’ 5.3 x o-13 1.9 x 18-40 3.9 x Prelabeled cells’: -48 to -24
105 ’ 22.5 105 23.0 104 27.1 105 20.3 103 23.4
24.3 26.0 22.1 25.0 28.7
i 51.6 51.0 48.8 52.7 i 44.2
1.6 4.5d 1 4.9d ~ 10.3d 0.3d 11.7 0.2 1.0 5.3 6.0 ~ 2.0 ~ -6 ~ 2.0,-:[3.7 -I -
~
~
~
1
~
0 The figures are the mean of two separate experiments where 3H and 14Camino acid labels were used. b The protein band VI was contaminated with protein VIII under the conditions used. c The cells were labeled for 24 hr, starting 48 hr before infection, and were washed and chased for 24 hr in normal growth medium prior to infection. d Based on the assumption that 20y0 of the total proteins was extracted by this method. e Not done.
Molecular Weight Determination of Proteins V-X in Adenovirus Type 2 The molecular weights of polypeptides V, VI, VII, VIII, IX and X, were determined on SDS-gels with six markers of known molecular weight. Their molecular weights ranged from 6500 to 50,000 as shown in Table 3. Io?.enti$catiun of Low Molecular Weight Antigens after Sequential Degradation of the Virion The method for sequential degradation of adenovirions as described by Prage et al. (1970) (Table 4) was used to study the location of all polypeptides in the virion. 3H-amino acids labeled virus preparations were dialyzed overnight at +4’ against 0.005 M Tris-maleate buffer, pH 6.3, 1 mM EDTA in order to remove the pentons (Prage et al., 1970). The supernatant (A) after centrifugation at 50,000 g for 30 min was removed and analyzed by SDS-polyacrylamide electrophoresis and immunodiffusion. Immunodiffusion of supernatant (A) (Table 4) showed the presence of pentons and low amounts of hexon (Fig. 5B). SDSels showed the presence of penton base t polypeptide III), fiber (polypeptide IV), small amounts of hexon (polypeptide II), as
TABLE
3
MOLECULAR WEIGHT OF ISOLATED PROTEINS ESTIMATED FROM MOBILITY IN SDSACRYLAMIDF, GELS
Protein
Approximate molecular weight”
V VI VII VIII IX x
48 500 24 000 18 500 13 000 12 000 6 500
-__ a Average of four determinations. The molecular weight of proteins V-VII were determined in 13% and proteins VIII-X in 200/, analytical SDS polyacrylamide gels.
well as low amounts of polypeptides VI and X (Fig. 5A). Radial immunodiffusion showed that all pentons were released, but only around 2% of the total hexons were recovered in this fraction. The pentonless virions in the pellet from the previous step were dissolved in 0.01 M Tris-aceta.te buffer, pH 8.0, and further incubated at 25” for 3 hr and centrifuged in the cold at 50,000 g for 30 min. This procedure is supposed to release the peripentonal hexons (Prage et al., 1970). The supernatant (B) was removed and analyzed by SDS-gels,
135
l:VI~:IWT,
SUNl)(JUIST,
PETTl:HSSO~,
AND PIIILIPSOI%
TABL15 4 SCIIEMF; _..-.
--.-
oli
S~:overwI~L
LIISINTKORATIOX -.... .-
---
Method
0~
\'II~I~Ns~ -..--.~
.-
1:taction -.--
Characterization -
Dialysis against: 0.005 M Ttis-maleate buffet pH 6.3, 1 m:M EI)TA at 4”, 18-20 ht Ulttacenttifugation 50 000 g/30 min r
-.
-
----
--
t
I
Supetnatant (A) contains mainly pentons and fibers
j SDS-gel
I Immunodiffueioq See Fig. 5
Sediment, redissolved in: 0.01 ‘II ‘Es-IIAc buffet pH 8.0 kept at 25”, for 2-3 ht Tntracenttifugation 50 000 g/30 min ..-..-, r .- .-. - --
Supctnatant. (B) contains mainly petipentonnl hexons
. Sediment., redissolved in 0.01 M glycine 0.001 M Ttis.IICl buffet pI1 7.2, O.Ol$; Ttiton X-100; frozen and thawed 16 t.imes at -20” and 37”, respectively Ulttacenttifugation 50 Wo g/30 min -
I-
_
-,
Supetnatant, (C) contains mainly the hexor~s at thr facets of the icosnhedton LSediment (1)) contains mainly the cots structure
I 1
SDS-gel Immunodiffusion SW Fig. 6
(‘SDS-gel 1 Immunodiffusion I UC-gel” lLSeeFig. 7 SDS-gel i) UC-gel (See Fig. 8 -.-. .-
a This scheme was described in a previous communication (Ptagc eL (11., 1970). * SDS-gel refers to analytical polyactylamide gels in sodium dodecyl sulfate. UCgel lytical polyactylamidc gels in acid urea. Both methods ate described in Materials md
refers to anaMethods.
immunodiffusion, and UC-gels after UIYYLoxt.raction. tidial immunodiffusion showed that about 20(& of the hctxo~~s wcrc rcleased, presumably thfl peripontonal hexons, which theoretically would amount to 25% of
(H). Small amounts of polypcptidcs V and VII wrc also prcwnt in this fraction. UCgels shuwctd the prcsencc of proteins VI and X and tract: amounts of proteins V :md VII
the total 1~x01~s in the virion (I’ragc et al., 1970). Immunodiffusion only rcvealcd the but protein VI could not prcsenct: of llcxo~ls be dtttwted probably bccausc of insuflicicnt amounts. SDS-g& rcwalcd l~~~on~ (II), traces of pcnton bases (III), and fibers (IV), and also polvpctptides VI, III+ and X (Fig. 6). Polypcptidr IIIa was difficult to quantitatc, since: it was poorly rcsolwd from
The pellet from the previous stcbp ww resuspcnd(~d in 0.01 :1f glycinc 0.001 ‘11 Tris. HCl buffer, pH 7.2, 0.01% Triton X-100 and frozen and thawed 16 times in order to break the rcmaindcr of the capsid and rclcasct tht> cow (Pragc ef al., 1970). After c:entrifugnt.ion 50 000 q for 30 min thtl supcrnutant (C) was analyzed by nnalvtiwl PDSgels, immunodiffusion, and Ii6gels :lfter acid urea ext,raction. Radial immunodiffusion of the super-
polypeptidt:
IV in l$jo
~~1s. Knowing
the
ratio b&wcen polypcptidcs III and IV in isolat.c:d pcntons, WCcould, however, wtimatc the approximate amount of IIIn in the mixed peak of IIIa and IV cve11 whl pcantons were prcscnt. Since thcrc was more polypcptide II1 than IV in pent,ons, polypcptidc IITa must, bc prcwnt in ~up(~rrl:~t:Lnt
(Ilot.
show11).
nat.ant, (C) rc>vcsled that
this fraction
c*on-
taincd the, bulk of hcxons (75-SO~j) presumably originating from the triangular facets of the virion (l’ragc et al.. 1970). SDSncrylnmidc gc.1 (,lclctrophorcsis sho\vc,d a prominent peak of polypt:pt.id(~ 1I :md smaller
STRUCTURAL
PROTEINS
139
OF ADENOVIRUSES
peaks of polypeptides IIIa, VI, VIII, IX, against sera toward protein VI and hexon and X (Fig. 7A). Acid urea extracted two (Fig. 7C). proteins bz (VI) and bl (VIII) as judged by The DNA containing sediment (D) Table electrophoresis on UC-gels (Fig. 7B). Im- 4) was extracted with acid urea or treated munodiffusion showed precipitation arcs with SDS before analyses on UC- or SDSgels.
I
A
n nIa/IP:
m xl
!
1-
II
X IL/
i;
!
00
FIG. 5A. Analytical
13y0 SDS-polyacrylamide gel pattern of material released in supernatant (A) (Table 4) of adenovirus type 2. The gels were traced densitometrically as described in Materials and Methods.
FIG. 5B. Immunodiffusion of supernatant (A) from adenovirus type 2 (Table 4) in the center well and specific antisera toward hexon (2), penton (3), fiber (4), protein V (5), protein VI (6), and frozen and thawed virions (1) in the peripheral wells.
L d 5
Enl
10
FIG. 6A. Analytical 13’% SDS-polyacrylamide gel pattern of material released into supernatant (B) (Table 4) of adenovirus type 2, shown by densitometric tracing as described in Materials and Methods.
FIG. 6B. Immunodiffusion of supernatant (B) from adenovirus type 2 (Table 4) in the center well against specific antisera toward hexon (2), penton (3)) fiber (4)) protein V (5), protein VI (6), and frozen and thawed virions (1) in the peripheral wells.
140
EVERITT,
SUNDQUIST,
PETTERSSON,
Analytical UC-gels showed the presence of protein a (V) and protein c (VII) (Fig. SB). SDS-gels showed prominent peaks of polypeptides II, V and VII and trace amounts of polypeptide VI (Fig. SA). The presence df hexons in the core fraction was probably due to an unspecific adsorption of hexons to the cores. Since polypeptide VI was detect.ed in all fractions which contained hexons, we wanted to establish if these proteins were attached to each other. Therefore supernatants (B) and (C) were analyzed by centrifugation in sucrose gradients with and without 0.15 M NaCl as described in Materials and Rlethods. The fractions corresponding to the hexon peak and the top fract’ions were precipit’ated with 10% trichloroacetic acid
0
5
cm
10
FIG. 7A. Densitometric tracing of analytical 13y0 SDS-polyacrylamide gels showing separation of material released into supernatant (C) (Table 4) of adenovirus type 2.
b2
AA bl
,-0
5
cm
FIG. 7B. Analytical UC-polyacrylamide gel separation of high-salt, acid urea-extracted material from supernatant (C) of adenovirus type 2 (Table 4).
AND
PHILIPSON
FIG. 7C. Immunodiffusion of supernatant (C) from adenovirus type 2 (Table 4) in the center well and specific antisera toward hexon (2), penton (3), fiber (4), protein V (5)) protein VI (6), and frozen and thawed virions (1) in the peripheral wells.
and analyzed on SDS-gels. The stained gels were traced with a densitometer and the location of the peak of polypeptide VI was determined (Fig. 9). The results showed that polypeptide VI was attached to the hexon unit after fractionation of supernatant (B) in gradients containing no salt, while polypeptide VI was recovered at the top of the gradient in the presence of 0.15 M of NaCl. The same result was obtained for supernatant (C) (not shown). Polypeptides VIII and IX were not detectable in individual fractions of the hexon peak after sucrose gradient centrifugation, probably due to insufficient concentrations. When all fractions of the hexon peak after salt free sucrose gradient centrifugation of supernatant (C) were pooled and analyzed on SDS-gel, polypeptides II, VI, and VIII were detected suggesting that both polypeptide VI and VIII may be associated with the hexons of the triangular facets, since polypeptide VIII was not observed in supernatant (B) (Fig. 6A). On the other hand, polypeptides IX and X were observed in supernatant (C) free from the hexons at sucrose gradient centrifugation without salt, which may suggest speci,-
STRUCTURAL
PROTEINS
ii&y to the association of polypeptides VIII and II. Polypeptides VI, VIII and IX could not be detected in hexons purified from the
141
OF ADENOVIRUSES
zoo-
A
A
1
III A P
looL 25
bottom
cm
.-
B C
b
frscrmn
number
30 top
FIG. 9. Sucrose gradient centrifugation of the material released in supernatant (B) from adenovirus type 2 (Table 4). Gradients without NaCl (panel A) and with 0.15 M NaCI (panel B) were made as described in Materials and Methods and centrifuged in the SW 40 rotor at 198,000 g for 14 hr at 4’. --, Continuously recorded optical density at 280 nm. O-O, The peak area of the hexon polypeptide (II) in analytical 13% SDS-polyacrylamide gels densitometrically traced at 620 nm scaled from zero to 3 OD due to the large amounts of protein applied to the gels. O-----O, The peak of the polypeptide VI in analytical 13% SDS-polyacrylamide gels densitometrically traced at 550 nm and scaled from zero to 1 OD.
a excess pool of structural proteins in infected cells by the procedure of Pettersson et al. (1967). Disruption
/L 0
5
cm
FIG. 8. Analytical 137?eSDS- and UC-polycrylamide gel patterns (panels A and B, respecively) of material from the sediment (D) (Table ) of adenovirus type 2, shown by densitometric I racing as described in Materials and Methods. he sediment was taken up in SDS-gel sample uffer or extracted with high salt in acid urea nd subjected to electrophoresis as described in i aterials and Methods.
of Viriom
with Pyridine
Pyridine disrupts adenovirions into t#hree fract.ions, which may be separated on sucrose gradient.s (Fig. 10). The fast sedimenting peak (a) contains the adenovirus core, the second (p) contains aggregates of nine hexons, and the third and slowest sedimenting peak (y) contains hexons and pentons presumably from the peripentonal region (Prage et al., 1970). The three peaks were analyzed by immunodiffusion and SDS-gel and UC-gel electrophoresis after acid urea extraction. The core fractions ((Y) gave faint precipi-
142
EVERITT,
SUNDQUIST,
PETTERSSON,
tation arcs with sera against proteins V and VII (not shown). The second peak (p) gave precipitation lines with antiserum against protein VI and antiserum against virions, but smprisingly no precipitation lines with antihexon serum (Pig. 12C). The arc against antivirion serum shows identity with the anti-VI precipitation line (not shown). The
AND
PHILIPSON C
1
B
a
FIG. 11B. Analytical UC-gel electrophoresis after high salt, acid urea extraction of material in peak (u) of Fig. 10.
FRACTION
NUMBER
A
FIG. 10. Rate sonal salt free sucrose gradient centrifugation (at 90,000 g for 150 min in the SW 27 rotor at 4’) of W-amino acid and 3H-thymidine labeled adenovirus type 2 virions disrupted by 10% pyridine (Prage et al., 1970). The gradients were made as described in Materials and Methods. Three fractions were collected: peak (or) at the bottom, peak (0) in the intermediate position, peak (y) near the top of the gradient. The horizontal bars indicate fractions pooled. P denotes the radioactivity in the pellet of the tube. Sedimentation is from right to left.
Ix
A
A-
c 0
5
cm
lo
FIG. 12A. Analytical 1376 SDS-polyacrylamide gel pattern of material in peak (a) of Fig. 10. The gel was traced densitometrically as described in Materials and Methods.
m
B
a
/./ 5
, cm
10
FIG. 11A. Analytical 13y0 SDS-polyacrylamide gel pattern of material in peak (01) of Fig. 10. The gel was traced densitometrically as described in Materials and Methods.
P
0
5
cm
FIG. 12B. Analytical UC-gel electrophoresis after high-salt, acid urea extraction of material in peak (0) of Fig. 10.
STRUCTURAL
PROTEINS-OF
143
ADENOVIRUSES
B
b 0
Jc 5
cm
FIG. 13B. Analytical UC-gel electrophoresis after high salt, acid urea extraction of material in peak (7) of Fig. 10.
FIG. 12C. Immunodiffusion pattern of material in peak (0) (the center well) surrounded by specific antisera against hexon (2), penton (3), fiber (4), protein V (5), protein VI (6), and frozen and thawed virions (1) in the peripheral wells.
m
B 3
A
FIG. 13C. Immunodiffusion pattern of material in peak (7) (the center well) surrounded by specific antisera against hexon (2), penton (3), fiber (4), protein V (5), protein VI (6), and frozen and thawed virions (1) in the peripheral wells.
YII
0
5
cm
10
FIG. 13A. Analytical
13% SDS-polyacrylamide gel pattern of material in peak (y) of Fig. 10. The gel was traced densitometrically as described in Materials and Methods.
antigenic determinants of the hexons therefore seemed to be masked by protein VI or protein IX and thus were unaccessible to antibodies specified by the hexon. The top fraction (y) gave specific precipitation lines with antisera against hexons, pentons, fibers and protein VI (Fig. 13C). SDS-gels and UC-gels after urea extrac-
tion clearly showed that the core contained polypeptides V and VII (Fig. 11). Fractions of the intermediate peak (the groups of nine hexons) were shown to contain a large amount of polypeptide II and polypeptides VI and IX on SDS-gels (Fig. 12A), and protein bz was recovered after yrea extraction (Fig. 12B). To rule out that polypeptides VI and IX contaminated peak 6 from the top of the gradient the fractions from this peak were pooled, dialyzed against 0.002 M TriseHCl buffer, pH 7.5, 0.2 mM EDTA, and recentrifuged in sucrose gradi-
144
EVERITT,
SUNDQUIST,
PETTERSSON,
ents containing no salt. SDS-gels showed that polypeptides II, VI, and IX were present in the same relative proportion after recentrifugation as in peak (p) (not shown). At recentrifugation in 0.15 M NaCl polypeptide VI but not IX disappeared from the groups of nine hexons. The top fraction (y) contained polypeptides II, III, IV, VI, VIII, and X (Fig. 13A). Polypeptide IIIa is probably present as well, since peak IIIa/IV
GROUPS OF NINE
AND
PHILIPSON
is higher than would be expected if only polypeptides from the penton were present. Trace amounts of polypeptides V and VII were also present on the top of the gradient. DISCUSSION
The polypeptide pattern of adenovirus type 2 has been studied by SDS-disc-gel electrophoresis according to Maize1 (1971). This system has better resolution than the
HEXONS II
MAGNIFIED IN
YT
lx
II
ma
FIG. 14. An hypothetical model of the location of isolated proteins in the virus particle of adenovirus type 2. The molar ratio between the internal basic proteins VII and V was 5 (Table 5), which is maintained in the schematic representation. Protein VII may cover about 50% of all phosphates of the DNA (Prage and Pettersson, 1971). The molar ratio of protein VI to hexons was 1.8. To account for the masked antigenic specificities of the hexon in the group of nine structures (Fig. 12C) protein VI may be located both at the inner and outer surface of the hexons. Peripentonal hexons also possess protein VI. The acidic, low molecular weight protein IX is probably the cementing substance between the hexons, since it is the only polypeptide, except for protein VI, associated with groups of nine hexons after pyridine treatment (Fig. 12A). Protein VIII associated with the facet hexons after sequential disintegration and showing basic properties (Fig. 7A) may reside at the inner surface of the triangular facets to neutralize the DNAphosphate groups. Polypeptide IIIa may be associated with material in the peripentonal region of the virion (Fig. 6A). The localization of protein X is unclear at present. (A) A schematic view of a vertical section of adenovirus type 2. The different proteins are indicated by their Roman numerals, according to Fig. 1. Magnified views of groups of nine hexons and the peripentonal region are given in B, C, and D. (B) A vertical section througha group oi nine hexons structure showing the location of proteins VI, VIII, and IX. (C) A horizontal view from the outside of the group of nine hexons showing the location of proteins VI and IX. (D) A magnification of the peripentonal region showing the proteins II, III, IIIa, IV, and VI.
STRUCTURAL
PROTEINS
method of Maize1 et al. (1968a) and 10 distinct polypeptides designated II, III, IIIa, IV-X according to the nomenclature proposed by Maize1 et al. (1968a), and Maize1 (1971) have been resolved. When excessive amounts of protein were analyzed, four additional bands were seen: IVa, Va, Via, and IXa (Fig. 2). Polypeptides IVal, IVa2, Va, Via, and IXa constitute each less than 0.1% of the total mass of protein in the virion based on radioactivity, which means that only a few copies of each of these peptides would be present per virus particle. They may therefore not represent unique polypeptides from the virion. Pereira and Skehel (1971) have suggested that the small polypeptides VIII, IX, and X of adenovirus type 5 are derived from the hexons and pentons because of proteolytic degradation during storage of the virus. We have compared gel patterns from virus prepared in the presence of a protease inhibitor and virus that was stored for 72 hr at 37’. Our results do not lend support to their hypothesis (Fig. 1). High-salt acid urea extraction of freshly prepared virions preferentially released polypeptides V, VI, VII, VIII, and X as revealed both by SDS- and analytical urea gels (Fig. 2). These proteins could be separated and purified by preparative polyacrylamide electrophoresis, and the relative amount of each of these in the adenovirions was determined (Table 2). Our estimated amounts agree with the figures reported by Maize1 et al. (196813) assuming that the gels described by Maize1 et al. (1968a) did not resolve polypeptides VI from VII and VIII from IX. Antisera produced against the proteins V, VI, and VII showed that all three antigens were dist’inct and different from the hexons, fibers, and penton bases (Fig. 4). The location of the adenovirus polypeptides was studied by analysis of fractions which were obtained after different stages of capsid degradation. The two methods described by Prage et al. (1970) were used, and they are outlined in Table 4. Both methods revealed that the adenovirus core contains two proteins corresponding to polypeptides V and VII (Fig. S and 11). Polypeptide IX seems to be the cementing polypeptide between the hexons, which
145
OF ADENOVIRUSES
originate from the facets of the adenovirus capsid since it is present in the aggregates of nine hexons which are released by pyridine (Fig. 12A). This polypeptide was released from the hexons when the capsid was disintegrated into single capsomers by freezing and thawing. Polypeptide VIII was recovered in association with hexons after freezing and thawing, but was separate from the groups of nine hexons after degradation with pyridine. Possibly this polypeptide is linked to the hexons in the capsid, but is detached by pyridine. Polypeptide VI appears to be associated with all hexons present in the capsid since (i) all fractions from disintegrated virus which contained hexons also contained polypeptide VI ; (ii) polypeptide VI has an affinity to hexons since it cosediments with hexons in sucrose gradients without salt irrespective of whether the hexons were obtained by freezing and thawing or by pyridine treatment. The location of polypeptide IIIa is difficult to determine, since this polypeptide is incompletely resolved from polypeptide IV on 13% SDS-gels. It appears, however, that polypeptide IIIa is preferentially released after pyridine treatment and together with the peripentonal hexons after dialysis against Tris-maleate buffer, pH 6.3. We therefore TABLE MOLAR
PROPORTION OF MINOR IN THE VIRION~
Protein6
V VI VII X
5
Molecules/virion
180 420 1070 50
POLYPEPTIDES
Molecules/hexon capsomerc 1.8 -
a The values are obtained from the radioactivity data given in Tables l-3 together with the fact that 10’2 physical particles of adenovirus type 2 correspond to 0.28 mg of protein (Prage et az., 1970). b Corresponding figures could not be obtained from protein VIII and IX, since these proteins have not been purified. c The number of morphological hexon units per virion is 240, and there are 3 molecules of polypeptide II per hexon (Valentine and Pereira, 1965; Maize1 et al., 1968,).
146
EVERITT,
SUNDQUIST,
PETTERSSON,
propose that polypeptide IIIa is located in the peripentonal region. Polypeptide X was recovered in several fractions after sequential degradation and its location in the capsid is unknown. The topography of minor polypeptides within the virion was also studied by Maize1 et al. (1968b), and they reported a similar, but less detailed pattern than ours. The higher resolving power of the SDS-disc-gels (Maizel, 1971) and the urea extraction procedure allowed a better separation and enabled us to purify some of the minor polypeptides of the adenovirion. Our results are summarized in a hypothetical model of the adenovirus particle (Fig. 14). ACKNOWLEDGMENTS The superb technical assistance of Miss Solveig Rosen and Miss Ingela Hiibinette is gratefully acknowledged. We also want to thank Mr. Hannu Ukkonnen for drawing the adenovirus model. REFERENCES CONANT, R. M., and BARRON, A. L. (1967). Enhanced diffusion of enterovirus antigens in agar in the presence of protamine. virology 33, 547-549. DAYHOFF, M. 0. (Ed.) (1969). “Atlas of Protein Sequence and Structure,” Vol. 4, p. D-164. National Biochemical Research Foundation, Silver Spring, Maryland. EAGLE, H. (1959). Amino acid metabolism in mammalian cell cultures. Science 130, 432437. EVERITT, E., SUNDQUIST, B., and PHILIPSON, L. (1971). Mechanism of the arginine requirement for adenovirus synthesis. I. Synthesis of structural proteins. J. Viral. 5, 742-753. GREEN, M., and PIR.I, M. (1963). Biochemical studies on the adenovirus multiplication. IV. Isolation, purification and chemical analysis of adenovirus. Virology 20, 199-207. Hauss, F. N. (1963). Solutes and solvents for liquid scintillation counting. Packard Tech. Bull. No. I, revised. Packard Instrument Company, Downers Grove, Illinois. HJ$:RTI~N, S., JERSTEDT, S., and TISELIUS, A. (1965). Electrophoretic “particle sieving” in polyacrylamide gels as applied to ribosomes. Anal. Biochem. 11, 211-218. Lavr:~, W. G. (1970). Isolation of an argininerich protein from particles of adenovirus type 2. Virology 41, 488-500. LOENING, U. E. (1967). The fractionation of high molecular weight ribonucleic acid by poly-
AND
PHILIPSON
acrylamide gel electrophoresis. Biochem. J. 192, 25-257. LONBERG-HOLM, K., and PHILIPSON, L. (1969). Early events of virus-cell interaction in adenovirus system. J. Viral. 4, 323338. LOWRY, 0. H., ROSEBROUGH, N. J., F~RR, A. F., and RANDALL, R. J. (1951). Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MAIZEL, J. V., JR. (1971). Polyacrylamide gel electrophoresis of viral proteins. In “Methods in Virology” (K. Maramorosch and H. Koprowski, eds.), Vol. V, pp. 179-246. Academic Press, New York. MAIZEL, J. V., JR., WHITE, D. O., and SCHARFF, M.D. (1968a). The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12. Virology 36, 1X-125. MAIZEL, J. V., JR., WHITE, D. O., and SCHARFF, M.D. (1968b). The polypeptides of adenovirus. II. Soluble proteins, cores, top components, and the structure of the virion. 1Ti’irology 36, 126-136. MANCINI, G., CARBONAHB, A. O., and HEREMAKS, J. F. (1965). Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2, 23&254. NEVILLE, D. N., JR. (1971). Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J. Biol. Chem. 246, 63284334. PEREIRB, H. G., and SKEHEL, J. J. (1971). Spontaneous and tryptic degradation of virus particles and structural components of adenoviruses. J. Gen. Viral. 12, 13-24. PETTERSSON, U. (1971). Structural proteins of adenoviruses. VI. On the antigenic determinants of the hexon. Virology 43, 123-136. PETTERSSON, U., and H~GLUND, 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~GLUNU, S. (1967). Structural proteins of adenoviruses. I. Purification and characterization of adenovirus type 2 hexon antigen. Virology 33, 575590. PETTERSSON, U., PHILIPSON, L., and H~GLUND, S. (1968). Structural proteins of adenoviruses. II. Purification and characterization of adenovirus type 2 fiber antigen. Virology 35, 204-215. PRAGE, L., and PETTERSSON, U. (1971). Structural proteins of adenoviruses. VII. Purification and properties of an arginine-rich core protein from adenovirus type 2 and type 3. Virology 45, 364-373.
STRUCTURAL
PROTEINS
PRAQE, L., PETTERSSON, U., H~GLUND, S., LONBERG-HOLM, K., and PHILIPSON, L. (1970).
Structural proteins of adenoviruses. IV. Sequential degradation of the adenovirus type 2 virion. Virology 42, 341358. RUSSELL, W. C., MCINTOSH, K., and SKEHEL, J. J. (1971). The preparation and properties of adenovirus cores. J. Gen. Vi’irol. 11, 3546. SCHULZE, I. T., and COLOWICK, S. P. (1969). The modification of yeast hexokinases by pro-
OF ADENOVIRUSES
147
teases and its relationship to the dissociation of hexokinase into subunits. J. Biol. Chem. 244, 2396-2316. VALENTINE, R. C., and PEREIRA, H. G. (1965). Antigens and structure of the adenovirus. J. Mol. Biol. 13, 13-20. WEBER, K., and OSBORN, M. (1969). The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 44064412.
63, 13%147 (1973)
Structural X. Isolation
Proteins
and Topography Virion
ETNAR EVERITT, AND Deparlmerd
of Microbiology,
of Adenoviruses’
of Low Molecular of Adenovirus
Weight
Antigens
from the
Type 2
HO SUNDQUIST, ULV PETTERSSON, LESNhllT PHILIPSON
The Wullenbetg Accepled
Luboratory,
University
of C’ppsala,
Sweden
Uppsalu,
.Yovember 27, 1972
With high resolution SLSpolyacrylamide gel electrophoresis (Maizel, 1971) and a new method to extract the basic proteins, it w&9 ascertained that adenovirus type 2 contains at least 10 distinct polypcptides (II, III, IIla, IV-X) and possibly more. Five proteins (V, VI, VII, VIII, and S) were purified by selective extraction in urea at high ionic strength, and low pI1 followed by preparative polyacrylamide electrophoresis toward the cathode at ~113.4. .4ntisera, produced against proteins V, VI, and VII were used to reveal t.hat these proteins were antigenically distinct and unrelated to hexons, pentons, and fibers. The location of the polypeptides was investigated by two methods of virion degradation (Prage el al., 1970). Polypeptides V and VI1 were associated with the l>NAcont.aining core. Polypcptide VI appeared to be associat,ed with all hexons of the virion. llexons from the triangular facets of the capsid cbontained in addition to polypeptides 11 and VI, polypept,ide IX, and possibly alvo polypeptide VIII. Hexons purified from infected cells contained only polypeptide II. Polypeptide IIla was preferentially released together with the peripentonal hexons. INTROI~UCTIOS
Adenoviruses are well suited for st,udics on virion proteins since the outer capsid proteins are soluble under native condiions. SDS-polyacrvlamide gel clectrophoresis revealed a minimum of nine polypeptidcs (II-X) (Maize1 et al., 1968a; Muizel, 1971). Fiveof t.hese (II, III, IV, V, VII) areintcpral parts of hexons, pentons, (AIaizel et al., 196Sb; Laver,
fibers, or cores 1970, I’ragc and
Pettersson, 1971, Russc~ll et al., 1971). It has recently been suggclstcd t.hat the low molecular weight components of adcnoviruses may be degradst,ion products and thus that the adcnoviruscs only contain five polgpcptidcs (Pcreira and Skchel, 1971). The present study reports the polypeptide composition of ndcnovirus type 2. Ten poly1 This work waq Pupported by grants from the Swedish RIedical l
peptides have been identified and t.heir localization in the capsid has been studied by different methods to disintegrate the sdenoviriOn. ,4 hypothetical model for the topography of the polypcptides of adenovirions was constructed on t.hc basis of t.hesc results. MATEItIAI,d
@ 19i3 by Academic t’rrss, of rrpruducti~m in any form
Inc. reuwvnl.
RIKTlCOl)S
Virus
The prototype originally
adenovirus
obtained
from
Dr.
NIH, Bethesda, Alaryland, (Pettersson et al., 1967). Cell Culture
uncl I’irus
type 2 strain R. Heubncr,
was
used
Znjeclion
KB cells were grown in spinner cultures in Eagle’s spinner medium (Eagle, 1959) with 70/, calf serum. Large-scale production of unlabeled virus was performed as described previously (Pctter~son et al., 1967). The 130
Copyright All rights
AXI)
STRUCTURAL
PROTEINS
virus was purified essentially according to the method of Green and Pina (1963), with two CsCl-gradients and a zonal rate centrifugation in sucrose gradients yielding a virus preparation with a hostcell protein contamination of less than 0.01% as previously described (Eve&t et al., 1971). Labeled virus was produced essentially according to Lonberg-Holm and Philipson (1969). When virus was labeled from zero time after infection with 14C- or 3H-amino acids (1 pCi/ml), Eagle’s spinner medium containing go the concentration of amino acids was used, except for arginine which was kept at 500 PM in order to provide a normal virus yield (Everitt et al., 1971). Purification
of Hexons, Pentons, and Fibers
The procedures have previously been described (Pettersson et al., 1967, 1968; Pettersson and Hoglund, 1969). Antisera Specific antihexon, antipenton, antifiber as well as anti-KB cell sera were prepared as described by Pettersson et al. (1968, 1969). Specific antisera against isolated low molecular weight proteins were produced in guinea pigs. Mixtures of equal volumes of a solution of the purified proteins and Freund’s complete adjuvant were injected (50 ~1) into the footpads once a week over a period of 5 weeks. Immunoassays Immunodiffusion was performed on glass slides covered with 0.8yo agarose in 0.14 M NaCl, 0.01 M Verona1 buffer, pH 7.4. Protamine sulfate (250 pg/ml) was included since only basic proteins were extracted (Conant and Barron, 1967). Quantitative determinations of hexon and penton base antigens were carried out by the Mancini technique (Mancini et al., 1965; Pettersson and Hiiglund, 1969). Gradient Centrifugation Disrupted virus preparations were analyzed on lo-25% (w/v) linear sucrose gradients in 0.002 1M TrisaHCl buffer, pH 7.5, 0.2 m&U EDTA with and without 0.15 M NaCl at f4’. The centrifugal force was
131
OF ADENOVIRUSES
calculated for the center of the tube and is given in the figure legends. Extraction of LOU Molecular Proteins
Weight Virion
Virus preparations were dialyzed for 18 hr against 3 changes of 100 volumes of 0.005 M TrisaHCl buffer, pH 8.1 at +4’. Total volume and protein content was determined and an equal volume of 10 M urea (Serva, Heidelberg, West Germany) was added, after which opalescence disappeared. After 30 min at room temperature solid sodium chloride was added to a final concentration of 2.2 M. After the salt was dissolved, 1 M citrate buffer, pH 3.1, was added to a final concentration of 0.1 M. A precipitate formed, and the viscous extract was incubated over night at +4”. After ultraccntrifugation at 110 000 g for 1.5-17 hr at +4”, the cleared supcrnatant was dialyzed against 4 M urea in 0.01 M citrate buffer, pH 3.4. Concentrated extracts were obtained by precipitation overnight at -20” with 4 volumes of acetone. After pelleting (1000 g for 10 min), the precipitate was washed twice in acetone, lyophilized, and redissolved in a small volume of 4 &! urea in 0.01 M citrate buffer, pH 3.4. Final protein concentration was 3-5 mg/ml. Polyacrylamide
Gel Electrophoresis
Analytical urea gels. Acrylamide and N , N’-methylene-bisacrylamide (Eastman Kodak, Rochester, New York) were recrystallized according to Loening (1967). Gels were made in glass tubes (5 mm internal diameter X 100 mm). The gels consisted of 7.5yo (w/v) acrylamide, 0.30y0 (w/v> N, N’methylene-bisacrylamide in 5 dl urea and 0.1 M citrate buffer, pH 3.4. Polymerization was induced by addition of N ,N ,N ,‘N’tetramethylethylcnediamine (TEMED) and of freshly prepared ammonium persulfate to 0.2y0 and O.lS%, respectively. Gels were prerun in the cold for 15 hr at 5 V/cm. The cathode and anode vials contained 5 M urea in 0.1 M citrate buffer, pH 3.4, and 0.01 M citrat,e buffer, pH 3.4, respectively. The gels were overlaid with the cathode solution. Samples (-100 ~1) were applied to the gels in 4 M urea, 0.01 M citrate buffer, pH 3.4.
132
EVEMTT,
SUNDQUIST,
PETTERSSON,
The potential was raised from 5 V/cm to 10 V/cm after 30-45 mm, and clectrophoresis continued for 10 hr at 4”. After electrophoresis the gels were stained and fixed for 2 hr in a solution of 0.05% Buffalo Black NBR (Allied Chemicals, Morristown, New Jersey), in 10% acetic acid, 30% methanol. The gels were destained in the same solvent and either photographed or scanned at 620 nm using a Gilford spectrophotometer Model 2400 with a linear transport accessory. Gels were soaked for 2 hours in the destaining solution supplemented with 20% glycerol and subsequently cut into 2-mm slices for determination of radioactivity. Preparative polyacrylamide gels. Preparative polyacrylamide gel electrophorcsis was performed in a continuous elution apparatus with a Pevikon-filled chamber at the lower end of the gel (HjertBn et aZ., 1965). Fractions (2 ml) were collected at, 20-min intervals. The gels (9 mm diameter X 120 mm) consisted of 7.5yo (w/v) acrylamide, 0.30% (w/v) bisacrylamide in 0.015 M ammonium formate-formic acid buffer, pH 3.4, and polymerization was induced by final concentrations of 0.1% and 0.04% of TEMED and ammonium persulfate, respectively. The gels were prerun for 20 hr at 20 V/cm at $4” with methylene blue as marker. The samples, containing 335 mg of protein and 5 X lo4 cpm of labeled extract in less than 0.8 ml of 4 IM urea in 0.01 M citrate buffer, pH 3.4, were layered onto the preparative gels. Methylene blue was used as a marker. Electrophoresis was carried out at 40 V/cm, 4” for 2rj hr. The current was approximately 8 mA. Samples from eluted fractions were assayed for radioactivity after neutralization. The pooled fractions of the main peaks were concentrated by Iyophilization and redissolved in 0.015 M formateformic acid buffer, pH 3.4. Analytical XDX-gels. The technique described by Maize1 (1971) was followed. The bisacrylamide was recrystallized as above. Acrylamide (Serva, Heidelberg, West Germany) and SDS (Schuchardt, Munich, West Germany) were used without, further purification. Acrylamide gels containing 13% or 20% acrylamide, 0.35% bisacrylamide were
AND PHILIPSON
made in 13 cm plastic tubes with an int,ernaI diameter of 6 mm. Protein samples for analysis were heated at 100” for 2 min in a solution of 0.05 M Tris-NaHzPO, , pH 7.2, 1% SDS, 10% glycerol, O.Fj% (v/v) mercaptoethanol and 0.00270 phenol red. Electrophoresis was carried out at 10 V/cm for 4 hr at room temperature. Gels were stained for 45 min in a solution of 0.2% Coomassie Brilliant Blue It 250 in 7% acetic acid, 10% methanol and destained in the same solvent. They were then photographed or scanned at 550 nm. Molecular Weight Determination The molecular weights of the isolated proteins were determined in a discontinuous SDS-polyacrylamide gel electrophoresis system by comparing their mobility with proteins of known molecular weight (Neville, 1971; Maizel, 1971). The following proteins were used as markers : bovine serum albumin (68,000), ovalbumin (43,000). L chains of IgG (23,500), ribonuclease A (13,700), cytochrome c (11,700), and insulin (5700). The molecular weights given are from Weber and Osborn (1969) except for insulin where the value was taken from Dayhoff (1969). The markers and the sample of isolated protein were mixed and analyzed on the same gel. Both 13c;I, and 20% gels were used. Protein Determination Proteins were assayed by the method of Lowry et al. (1951) with bovine serum albumin as standard. Radioisotopes and Counting Procedures Individual and mixed, 3H- or i4C-labeled amino acids as well as 3H-labeled thymidine were obtained from New England Nuclear Corp., Boston, Massachusetts. Acrylamide gel slices were either hydrdlyzed directly in Protosol (NEN, Boston, Massachusetts), or homogenized and subsequently hydrolyzed overnight at 65’ in 0.6 ml 1 M NaOH. Radioactivity in the hydrolyzates was determined with toluene- or dioxane-based scintillation fluids. Samplea, in solution were neutralized, if necessary, and counted directly in a dioxane-based counting solution (Hayes, 1963).
STRUCTURAL
PROTEINS
RESULTS
II III ma/E
Properties of PuriJed Virus Host cell protein contamination in purified virus was determined by adding about 4 X 10’ acid-insoluble counts per minute of 3H-amino acid labeled uninfected KB cells to virus material before purification. From the amount of radioactivity recovered in the purified preparations, it was estimated that less than 0.01% of the protein was derived from the host, cell (Evcritt et al., 1971). In order to exclude viral degradation by proteolysis the polypeptide pa.tterns on SDSolyacrylamide gels of virus treated in three iii iffercnt, ways were compared: Freshly pred virus and virus subjected to autoeolysis at 37” for 72 hr in 0.01 M *HCl buffer, pH 8.1, and virus purified presence of an inhibitor of proteolysis M phenylmethanesulfonyl fluoride Schulze and Colowick, 1969) were yzhd. The patterns from the three arations were observed to be identical no protein degradation products could etected (Fig. 1). Ten polypeptides desigd II, III, IIIa, IV-X were regularly 4etected (Fig. 1). Gels containing excessive t&mounts of protein contained five additional bands present in very low amounts (IVal, IVa2, Va, Via, IXa) (Fig. 2). Extraction of Virion il cid tirea
Proteins by High Salt
Several batches of purified virus were extracted with high salt in acid urea as described in Materials and Methods. This procedure extracted about 20% of the virion proteins measured by the Lowry procedure as shown in Table 1. The amount of radioactivity extracted from amino acid-labeled proteins ranged from 2Ooj, for virus labeled with a mixture of amino acids to 30yo for arginine labeled virus. No significant amounts (<0.5’%) were recovered when 3Hthymidine-lab&d virus was extracted. Analysis of Extracted Virion Proteins Proteins extracted by acid urea from adenovirions were separated by analytical urea-citrate (UC)- and SDS-polyacrylamide gels. The sediment after ultracentrifugation was dissolved in the SDS-sample solution
133
OF ADENOVIRUSES
IYal IPa P
(2)
0
FIG. 1. Analytical SDS-polyacrylamide gel electrophoresis on 13% gels of adenovirus type 2, purified according to the procedure described in Materials and Methods (I), and incubated for 72 hr at 37” (2). One virus preparation was purified in the presence of 2 mM phenylmethanesulfonyl fluoride (PMSF) (3). The polypeptides are referred t,o by Roman numerals.
and a-as also separated in SDS-polyacrylamide gels. The UC gels revealed 4 bands of proteins in the urea extract which were designated a-d as shown in Fig. 2. Band b was resolved in 2 species after preparative polyacrylamide gel-electrophoresis (Fig. 3). The five proteins in the extract migrated as polypeptides V, VI, VII, VIII, and X on SDS-gels (Fig. 2). The proteins of the sediment after ultracentrifugation, migrated as polypeptidcs II, III, IIIa, IV, IVa, and IX (Fig. 2). Small amounts of polypeptides V and VII were also present in the sediment (Fig. 2). Preparative Polyacrylamide Electrophoresis of Urea Extracted Proteins Urea extracts corresponding to 34 mg of protein were separated on acrylamide gels prepared in an ammonium formate-formic acid buffer and 60-75Yo of the input radio-
134
EVERITT,
SUNDQUIST,
PETTERSSON,
AND PHILIPSON
d
w-gel FIG. 2. Stained analytical urea-citrate-(UC-) and 13% SDS-polyacrylamide gels showing phoretic separation of adenovirus type 2 proteins extracted or precipitated in the high salt, acid ure buffer described in Materials and Methods. Freshly prepared virions were analyzed in a SDS-gel for co parison. The polypeptides in the SDS-gels were referred to by Roman numerals and the proteins on ure gels were labeled a-d.
TABLE
1
EXTRACTION OF ADENOVIRION PROTEINS HIGH SALT AND ACID UREA
Method of protein assay
WITH
Number Percentage of of exadenovirus proteins or radioac- periments tivity soluble in acid urea5
Mean Range of yield value ______ W-22 Protein (Lowry) (pg) 20 20-21 3H- or W-labeled / 20.5 amino acids 30 EH-Arginine 30 3H-Thymidine 0.4 0.3-0.5
(7) (3) (1) (3)
a Label was included from 0 to 40 hr after infection.
activity was recovered in five peaks (Fig. 3A). The pooled fractions of each of the peaks (a, bl , b , c, and d) were lyophilized and dissolved in small volumes of 0.015 M formate buffer, pH 3.4. The isolated proteins
were identified on SDS-polyacrylamide ge$ as shown in Fig. 3B. These studies demonstrated that the proteins a, bz , bl , c, and d migrated as polypeptides V, VI, VIII, VII, and X, respectively. Polypeptide VII, the arginine and alanine rich core protein (AAP), was characterized in a previous communication (Prage and Pettersson, 1971) and will not be further described here. Immunoloyical Characteristics Extracted Vi&m Protezns
of
Urea-
Urea extracted proteins were isolated by preparative polyacrylamide gel electrophoresis and were used for immunization. Antisera against proteins V and VI, which showed strong precipitation in the homologous reactions also revealed precipitation lines against electrophoretically purified hexon antigen (Fig. 4). The antihexon antibodies present in anti-V and anti-VI antisera were removed by adsorption with electrophoretically purified hexon obtained from
STRUCTURAL
PROTEINS
20
10
OF ADENOVIRUSES
30
40
FRACTION
NUMBER
50
60
FIG. 3A. Preparative polyacrylamide gel electrophoresis in 0.015 M ammonium formate-formic acid pH 3.4, of high-salt, acid urea-extracted proteins of adenovirus type 2 labeled withsH-amino acids during 0 to 40 hr post infection. The peaks corresponding to those observed in the analytical urea gel in Fig. 2 are indicated in the elution profile. The arrow indicates the methylene blue (MB) marker.
V+d
d
v+c
FIQ. 3B. Identification in 13% SDS polyacrylamide parative electrophoresis. The proteins were analyzed polypeptides as described in Materials and Methods.
infected cells. Antihexon, as w-cl1 as antipenton, antifiber, and anti-KB cell sera did not show precipitation arcs against the purified proteins V and Vl (not shown). The material used for the immunization against protein VI was also contaminated with protein VIII. Although most of the hexons were preeipitated by the high salt-urea-citrate extraction the cross reactivity between hexon dnd antisera against proteins V and VI was probably due to contaminating hexons among the extracted proteins which were incompletely separated from proteins V and VI after preparative clectrophoresis. Spe-
c
V+bl
b1+2
V+b2
V+a
a
gels of urea extracted proteins isolated by preboth separately and together with whole virion
cific antisera against proteins VIII and X were difficult to produce because insufficient amounts were obtained. Relative Amounts of Extracted Proteins in the Virion In order to determine the relative amounts and the time of synthesis of each of the proteins V, VI, VII, and X in the virus particle, virions were prepared from cells labeled for different periods before and during the infectious cycle. The virus was extracted with acid urea and analyzed on analytical UCgels or on SDS-gels without extraction.
136
EVERITT,
SUNDQUIST,
PETTERSSON,
AND
PHILIPSON
FIG. 4. Immunodiffusion of purified proteins V and VI after preparative polyacrylamide gel electrophoresis. The center well of panel I contained unadsorbed anti-V guinea pig serum (A) and the center well of panel II an anti-V-serum adsorbed with electrophoretically purified hexons (A’). The anti-VIserum in the center well of panel III was mltreated (B) and that in the center well of panel IV was adsorbed with hexon in the same way as the anti-V-serum (R’). The sera were tested against purified hexon (l), penton (2), fiber (3), protein VII extracted with sulfuric acid (4AP) (Prage el ul., 1970) (a), protein V (5), protein VI ((i), protein VII (7), and a KH cell extract (8). The amount of hexon antigen added to the wells in all panels was 10 pg whereas proteins V, VI, atld VII were present in 2.0 pg per well in panels I and II and 1.6 pg per well in panels III and IV.
TJC-gels revealed that protein V and VI contained approximately 25% each of the radioactivity extracted by acid urea, whereas protein VII (AAP) accounted for about 50% of the total extract (Table 2). These figures were in good agreement with the data obtained from the SDS-acrylamido gels (Table 2). The distribution of label between the four extracted proteins V, VI, VII, and X did not change with different periods of labeling, and none of the extracted proteins, wit#h the
possible exception of protein X, appeared to be derived from prelabeled host-cell material (Table 2). The relative proportion of t’he total protein of the virus recovered as protein V, VI,, VII and X was also determined. SDS- and UC-gels showed that polypeptides V, VI, and VII accounted for about 5, fi-6, and 10-120Jq of the total virus protcin content, respecr tively, whereas polypeptide X accounted fob only 0.24.374 (Table 2).
STRUCTURAL
PROTEINS TABLE
2
RELATIVE: AMOUNTS OF BASIC PROTICINS IN THE: AIXNOVIRUS
Method
UC-gels0 SDS-gels UC-gels UC-gels UC-gels
137
OF ADENOVIRUSES
TYPI.: 2
Specific 1 Percent radioactivity of total urea Percent radioactivity of total Label period post infection activity of la- extracted radioactivity in protein / radioactivity of virions in protein beled virions (hr) (cpm/1012 I ~_ __~ _____~~ particles) ~ V x VII ?< v ( VI , VII VP ~ ~~ --~- ,-~ 3.5 x 040 3.5 x 040 ’ 5.3 x o-13 1.9 x 18-40 3.9 x Prelabeled cells’: -48 to -24
105 ’ 22.5 105 23.0 104 27.1 105 20.3 103 23.4
24.3 26.0 22.1 25.0 28.7
i 51.6 51.0 48.8 52.7 i 44.2
1.6 4.5d 1 4.9d ~ 10.3d 0.3d 11.7 0.2 1.0 5.3 6.0 ~ 2.0 ~ -6 ~ 2.0,-:[3.7 -I -
~
~
~
1
~
0 The figures are the mean of two separate experiments where 3H and 14Camino acid labels were used. b The protein band VI was contaminated with protein VIII under the conditions used. c The cells were labeled for 24 hr, starting 48 hr before infection, and were washed and chased for 24 hr in normal growth medium prior to infection. d Based on the assumption that 20y0 of the total proteins was extracted by this method. e Not done.
Molecular Weight Determination of Proteins V-X in Adenovirus Type 2 The molecular weights of polypeptides V, VI, VII, VIII, IX and X, were determined on SDS-gels with six markers of known molecular weight. Their molecular weights ranged from 6500 to 50,000 as shown in Table 3. Io?.enti$catiun of Low Molecular Weight Antigens after Sequential Degradation of the Virion The method for sequential degradation of adenovirions as described by Prage et al. (1970) (Table 4) was used to study the location of all polypeptides in the virion. 3H-amino acids labeled virus preparations were dialyzed overnight at +4’ against 0.005 M Tris-maleate buffer, pH 6.3, 1 mM EDTA in order to remove the pentons (Prage et al., 1970). The supernatant (A) after centrifugation at 50,000 g for 30 min was removed and analyzed by SDS-polyacrylamide electrophoresis and immunodiffusion. Immunodiffusion of supernatant (A) (Table 4) showed the presence of pentons and low amounts of hexon (Fig. 5B). SDSels showed the presence of penton base t polypeptide III), fiber (polypeptide IV), small amounts of hexon (polypeptide II), as
TABLE
3
MOLECULAR WEIGHT OF ISOLATED PROTEINS ESTIMATED FROM MOBILITY IN SDSACRYLAMIDF, GELS
Protein
Approximate molecular weight”
V VI VII VIII IX x
48 500 24 000 18 500 13 000 12 000 6 500
-__ a Average of four determinations. The molecular weight of proteins V-VII were determined in 13% and proteins VIII-X in 200/, analytical SDS polyacrylamide gels.
well as low amounts of polypeptides VI and X (Fig. 5A). Radial immunodiffusion showed that all pentons were released, but only around 2% of the total hexons were recovered in this fraction. The pentonless virions in the pellet from the previous step were dissolved in 0.01 M Tris-aceta.te buffer, pH 8.0, and further incubated at 25” for 3 hr and centrifuged in the cold at 50,000 g for 30 min. This procedure is supposed to release the peripentonal hexons (Prage et al., 1970). The supernatant (B) was removed and analyzed by SDS-gels,
135
l:VI~:IWT,
SUNl)(JUIST,
PETTl:HSSO~,
AND PIIILIPSOI%
TABL15 4 SCIIEMF; _..-.
--.-
oli
S~:overwI~L
LIISINTKORATIOX -.... .-
---
Method
0~
\'II~I~Ns~ -..--.~
.-
1:taction -.--
Characterization -
Dialysis against: 0.005 M Ttis-maleate buffet pH 6.3, 1 m:M EI)TA at 4”, 18-20 ht Ulttacenttifugation 50 000 g/30 min r
-.
-
----
--
t
I
Supetnatant (A) contains mainly pentons and fibers
j SDS-gel
I Immunodiffueioq See Fig. 5
Sediment, redissolved in: 0.01 ‘II ‘Es-IIAc buffet pH 8.0 kept at 25”, for 2-3 ht Tntracenttifugation 50 000 g/30 min ..-..-, r .- .-. - --
Supctnatant. (B) contains mainly petipentonnl hexons
. Sediment., redissolved in 0.01 M glycine 0.001 M Ttis.IICl buffet pI1 7.2, O.Ol$; Ttiton X-100; frozen and thawed 16 t.imes at -20” and 37”, respectively Ulttacenttifugation 50 Wo g/30 min -
I-
_
-,
Supetnatant, (C) contains mainly the hexor~s at thr facets of the icosnhedton LSediment (1)) contains mainly the cots structure
I 1
SDS-gel Immunodiffusion SW Fig. 6
(‘SDS-gel 1 Immunodiffusion I UC-gel” lLSeeFig. 7 SDS-gel i) UC-gel (See Fig. 8 -.-. .-
a This scheme was described in a previous communication (Ptagc eL (11., 1970). * SDS-gel refers to analytical polyactylamide gels in sodium dodecyl sulfate. UCgel lytical polyactylamidc gels in acid urea. Both methods ate described in Materials md
refers to anaMethods.
immunodiffusion, and UC-gels after UIYYLoxt.raction. tidial immunodiffusion showed that about 20(& of the hctxo~~s wcrc rcleased, presumably thfl peripontonal hexons, which theoretically would amount to 25% of
(H). Small amounts of polypcptidcs V and VII wrc also prcwnt in this fraction. UCgels shuwctd the prcsencc of proteins VI and X and tract: amounts of proteins V :md VII
the total 1~x01~s in the virion (I’ragc et al., 1970). Immunodiffusion only rcvealcd the but protein VI could not prcsenct: of llcxo~ls be dtttwted probably bccausc of insuflicicnt amounts. SDS-g& rcwalcd l~~~on~ (II), traces of pcnton bases (III), and fibers (IV), and also polvpctptides VI, III+ and X (Fig. 6). Polypcptidr IIIa was difficult to quantitatc, since: it was poorly rcsolwd from
The pellet from the previous stcbp ww resuspcnd(~d in 0.01 :1f glycinc 0.001 ‘11 Tris. HCl buffer, pH 7.2, 0.01% Triton X-100 and frozen and thawed 16 times in order to break the rcmaindcr of the capsid and rclcasct tht> cow (Pragc ef al., 1970). After c:entrifugnt.ion 50 000 q for 30 min thtl supcrnutant (C) was analyzed by nnalvtiwl PDSgels, immunodiffusion, and Ii6gels :lfter acid urea ext,raction. Radial immunodiffusion of the super-
polypeptidt:
IV in l$jo
~~1s. Knowing
the
ratio b&wcen polypcptidcs III and IV in isolat.c:d pcntons, WCcould, however, wtimatc the approximate amount of IIIn in the mixed peak of IIIa and IV cve11 whl pcantons were prcscnt. Since thcrc was more polypcptide II1 than IV in pent,ons, polypcptidc IITa must, bc prcwnt in ~up(~rrl:~t:Lnt
(Ilot.
show11).
nat.ant, (C) rc>vcsled that
this fraction
c*on-
taincd the, bulk of hcxons (75-SO~j) presumably originating from the triangular facets of the virion (l’ragc et al.. 1970). SDSncrylnmidc gc.1 (,lclctrophorcsis sho\vc,d a prominent peak of polypt:pt.id(~ 1I :md smaller
STRUCTURAL
PROTEINS
139
OF ADENOVIRUSES
peaks of polypeptides IIIa, VI, VIII, IX, against sera toward protein VI and hexon and X (Fig. 7A). Acid urea extracted two (Fig. 7C). proteins bz (VI) and bl (VIII) as judged by The DNA containing sediment (D) Table electrophoresis on UC-gels (Fig. 7B). Im- 4) was extracted with acid urea or treated munodiffusion showed precipitation arcs with SDS before analyses on UC- or SDSgels.
I
A
n nIa/IP:
m xl
!
1-
II
X IL/
i;
!
00
FIG. 5A. Analytical
13y0 SDS-polyacrylamide gel pattern of material released in supernatant (A) (Table 4) of adenovirus type 2. The gels were traced densitometrically as described in Materials and Methods.
FIG. 5B. Immunodiffusion of supernatant (A) from adenovirus type 2 (Table 4) in the center well and specific antisera toward hexon (2), penton (3), fiber (4), protein V (5), protein VI (6), and frozen and thawed virions (1) in the peripheral wells.
L d 5
Enl
10
FIG. 6A. Analytical 13’% SDS-polyacrylamide gel pattern of material released into supernatant (B) (Table 4) of adenovirus type 2, shown by densitometric tracing as described in Materials and Methods.
FIG. 6B. Immunodiffusion of supernatant (B) from adenovirus type 2 (Table 4) in the center well against specific antisera toward hexon (2), penton (3)) fiber (4)) protein V (5), protein VI (6), and frozen and thawed virions (1) in the peripheral wells.
140
EVERITT,
SUNDQUIST,
PETTERSSON,
Analytical UC-gels showed the presence of protein a (V) and protein c (VII) (Fig. SB). SDS-gels showed prominent peaks of polypeptides II, V and VII and trace amounts of polypeptide VI (Fig. SA). The presence df hexons in the core fraction was probably due to an unspecific adsorption of hexons to the cores. Since polypeptide VI was detect.ed in all fractions which contained hexons, we wanted to establish if these proteins were attached to each other. Therefore supernatants (B) and (C) were analyzed by centrifugation in sucrose gradients with and without 0.15 M NaCl as described in Materials and Rlethods. The fractions corresponding to the hexon peak and the top fract’ions were precipit’ated with 10% trichloroacetic acid
0
5
cm
10
FIG. 7A. Densitometric tracing of analytical 13y0 SDS-polyacrylamide gels showing separation of material released into supernatant (C) (Table 4) of adenovirus type 2.
b2
AA bl
,-0
5
cm
FIG. 7B. Analytical UC-polyacrylamide gel separation of high-salt, acid urea-extracted material from supernatant (C) of adenovirus type 2 (Table 4).
AND
PHILIPSON
FIG. 7C. Immunodiffusion of supernatant (C) from adenovirus type 2 (Table 4) in the center well and specific antisera toward hexon (2), penton (3), fiber (4), protein V (5)) protein VI (6), and frozen and thawed virions (1) in the peripheral wells.
and analyzed on SDS-gels. The stained gels were traced with a densitometer and the location of the peak of polypeptide VI was determined (Fig. 9). The results showed that polypeptide VI was attached to the hexon unit after fractionation of supernatant (B) in gradients containing no salt, while polypeptide VI was recovered at the top of the gradient in the presence of 0.15 M of NaCl. The same result was obtained for supernatant (C) (not shown). Polypeptides VIII and IX were not detectable in individual fractions of the hexon peak after sucrose gradient centrifugation, probably due to insufficient concentrations. When all fractions of the hexon peak after salt free sucrose gradient centrifugation of supernatant (C) were pooled and analyzed on SDS-gel, polypeptides II, VI, and VIII were detected suggesting that both polypeptide VI and VIII may be associated with the hexons of the triangular facets, since polypeptide VIII was not observed in supernatant (B) (Fig. 6A). On the other hand, polypeptides IX and X were observed in supernatant (C) free from the hexons at sucrose gradient centrifugation without salt, which may suggest speci,-
STRUCTURAL
PROTEINS
ii&y to the association of polypeptides VIII and II. Polypeptides VI, VIII and IX could not be detected in hexons purified from the
141
OF ADENOVIRUSES
zoo-
A
A
1
III A P
looL 25
bottom
cm
.-
B C
b
frscrmn
number
30 top
FIG. 9. Sucrose gradient centrifugation of the material released in supernatant (B) from adenovirus type 2 (Table 4). Gradients without NaCl (panel A) and with 0.15 M NaCI (panel B) were made as described in Materials and Methods and centrifuged in the SW 40 rotor at 198,000 g for 14 hr at 4’. --, Continuously recorded optical density at 280 nm. O-O, The peak area of the hexon polypeptide (II) in analytical 13% SDS-polyacrylamide gels densitometrically traced at 620 nm scaled from zero to 3 OD due to the large amounts of protein applied to the gels. O-----O, The peak of the polypeptide VI in analytical 13% SDS-polyacrylamide gels densitometrically traced at 550 nm and scaled from zero to 1 OD.
a excess pool of structural proteins in infected cells by the procedure of Pettersson et al. (1967). Disruption
/L 0
5
cm
FIG. 8. Analytical 137?eSDS- and UC-polycrylamide gel patterns (panels A and B, respecively) of material from the sediment (D) (Table ) of adenovirus type 2, shown by densitometric I racing as described in Materials and Methods. he sediment was taken up in SDS-gel sample uffer or extracted with high salt in acid urea nd subjected to electrophoresis as described in i aterials and Methods.
of Viriom
with Pyridine
Pyridine disrupts adenovirions into t#hree fract.ions, which may be separated on sucrose gradient.s (Fig. 10). The fast sedimenting peak (a) contains the adenovirus core, the second (p) contains aggregates of nine hexons, and the third and slowest sedimenting peak (y) contains hexons and pentons presumably from the peripentonal region (Prage et al., 1970). The three peaks were analyzed by immunodiffusion and SDS-gel and UC-gel electrophoresis after acid urea extraction. The core fractions ((Y) gave faint precipi-
142
EVERITT,
SUNDQUIST,
PETTERSSON,
tation arcs with sera against proteins V and VII (not shown). The second peak (p) gave precipitation lines with antiserum against protein VI and antiserum against virions, but smprisingly no precipitation lines with antihexon serum (Pig. 12C). The arc against antivirion serum shows identity with the anti-VI precipitation line (not shown). The
AND
PHILIPSON C
1
B
a
FIG. 11B. Analytical UC-gel electrophoresis after high salt, acid urea extraction of material in peak (u) of Fig. 10.
FRACTION
NUMBER
A
FIG. 10. Rate sonal salt free sucrose gradient centrifugation (at 90,000 g for 150 min in the SW 27 rotor at 4’) of W-amino acid and 3H-thymidine labeled adenovirus type 2 virions disrupted by 10% pyridine (Prage et al., 1970). The gradients were made as described in Materials and Methods. Three fractions were collected: peak (or) at the bottom, peak (0) in the intermediate position, peak (y) near the top of the gradient. The horizontal bars indicate fractions pooled. P denotes the radioactivity in the pellet of the tube. Sedimentation is from right to left.
Ix
A
A-
c 0
5
cm
lo
FIG. 12A. Analytical 1376 SDS-polyacrylamide gel pattern of material in peak (a) of Fig. 10. The gel was traced densitometrically as described in Materials and Methods.
m
B
a
/./ 5
, cm
10
FIG. 11A. Analytical 13y0 SDS-polyacrylamide gel pattern of material in peak (01) of Fig. 10. The gel was traced densitometrically as described in Materials and Methods.
P
0
5
cm
FIG. 12B. Analytical UC-gel electrophoresis after high-salt, acid urea extraction of material in peak (0) of Fig. 10.
STRUCTURAL
PROTEINS-OF
143
ADENOVIRUSES
B
b 0
Jc 5
cm
FIG. 13B. Analytical UC-gel electrophoresis after high salt, acid urea extraction of material in peak (7) of Fig. 10.
FIG. 12C. Immunodiffusion pattern of material in peak (0) (the center well) surrounded by specific antisera against hexon (2), penton (3), fiber (4), protein V (5), protein VI (6), and frozen and thawed virions (1) in the peripheral wells.
m
B 3
A
FIG. 13C. Immunodiffusion pattern of material in peak (7) (the center well) surrounded by specific antisera against hexon (2), penton (3), fiber (4), protein V (5), protein VI (6), and frozen and thawed virions (1) in the peripheral wells.
YII
0
5
cm
10
FIG. 13A. Analytical
13% SDS-polyacrylamide gel pattern of material in peak (y) of Fig. 10. The gel was traced densitometrically as described in Materials and Methods.
antigenic determinants of the hexons therefore seemed to be masked by protein VI or protein IX and thus were unaccessible to antibodies specified by the hexon. The top fraction (y) gave specific precipitation lines with antisera against hexons, pentons, fibers and protein VI (Fig. 13C). SDS-gels and UC-gels after urea extrac-
tion clearly showed that the core contained polypeptides V and VII (Fig. 11). Fractions of the intermediate peak (the groups of nine hexons) were shown to contain a large amount of polypeptide II and polypeptides VI and IX on SDS-gels (Fig. 12A), and protein bz was recovered after yrea extraction (Fig. 12B). To rule out that polypeptides VI and IX contaminated peak 6 from the top of the gradient the fractions from this peak were pooled, dialyzed against 0.002 M TriseHCl buffer, pH 7.5, 0.2 mM EDTA, and recentrifuged in sucrose gradi-
144
EVERITT,
SUNDQUIST,
PETTERSSON,
ents containing no salt. SDS-gels showed that polypeptides II, VI, and IX were present in the same relative proportion after recentrifugation as in peak (p) (not shown). At recentrifugation in 0.15 M NaCl polypeptide VI but not IX disappeared from the groups of nine hexons. The top fraction (y) contained polypeptides II, III, IV, VI, VIII, and X (Fig. 13A). Polypeptide IIIa is probably present as well, since peak IIIa/IV
GROUPS OF NINE
AND
PHILIPSON
is higher than would be expected if only polypeptides from the penton were present. Trace amounts of polypeptides V and VII were also present on the top of the gradient. DISCUSSION
The polypeptide pattern of adenovirus type 2 has been studied by SDS-disc-gel electrophoresis according to Maize1 (1971). This system has better resolution than the
HEXONS II
MAGNIFIED IN
YT
lx
II
ma
FIG. 14. An hypothetical model of the location of isolated proteins in the virus particle of adenovirus type 2. The molar ratio between the internal basic proteins VII and V was 5 (Table 5), which is maintained in the schematic representation. Protein VII may cover about 50% of all phosphates of the DNA (Prage and Pettersson, 1971). The molar ratio of protein VI to hexons was 1.8. To account for the masked antigenic specificities of the hexon in the group of nine structures (Fig. 12C) protein VI may be located both at the inner and outer surface of the hexons. Peripentonal hexons also possess protein VI. The acidic, low molecular weight protein IX is probably the cementing substance between the hexons, since it is the only polypeptide, except for protein VI, associated with groups of nine hexons after pyridine treatment (Fig. 12A). Protein VIII associated with the facet hexons after sequential disintegration and showing basic properties (Fig. 7A) may reside at the inner surface of the triangular facets to neutralize the DNAphosphate groups. Polypeptide IIIa may be associated with material in the peripentonal region of the virion (Fig. 6A). The localization of protein X is unclear at present. (A) A schematic view of a vertical section of adenovirus type 2. The different proteins are indicated by their Roman numerals, according to Fig. 1. Magnified views of groups of nine hexons and the peripentonal region are given in B, C, and D. (B) A vertical section througha group oi nine hexons structure showing the location of proteins VI, VIII, and IX. (C) A horizontal view from the outside of the group of nine hexons showing the location of proteins VI and IX. (D) A magnification of the peripentonal region showing the proteins II, III, IIIa, IV, and VI.
STRUCTURAL
PROTEINS
method of Maize1 et al. (1968a) and 10 distinct polypeptides designated II, III, IIIa, IV-X according to the nomenclature proposed by Maize1 et al. (1968a), and Maize1 (1971) have been resolved. When excessive amounts of protein were analyzed, four additional bands were seen: IVa, Va, Via, and IXa (Fig. 2). Polypeptides IVal, IVa2, Va, Via, and IXa constitute each less than 0.1% of the total mass of protein in the virion based on radioactivity, which means that only a few copies of each of these peptides would be present per virus particle. They may therefore not represent unique polypeptides from the virion. Pereira and Skehel (1971) have suggested that the small polypeptides VIII, IX, and X of adenovirus type 5 are derived from the hexons and pentons because of proteolytic degradation during storage of the virus. We have compared gel patterns from virus prepared in the presence of a protease inhibitor and virus that was stored for 72 hr at 37’. Our results do not lend support to their hypothesis (Fig. 1). High-salt acid urea extraction of freshly prepared virions preferentially released polypeptides V, VI, VII, VIII, and X as revealed both by SDS- and analytical urea gels (Fig. 2). These proteins could be separated and purified by preparative polyacrylamide electrophoresis, and the relative amount of each of these in the adenovirions was determined (Table 2). Our estimated amounts agree with the figures reported by Maize1 et al. (196813) assuming that the gels described by Maize1 et al. (1968a) did not resolve polypeptides VI from VII and VIII from IX. Antisera produced against the proteins V, VI, and VII showed that all three antigens were dist’inct and different from the hexons, fibers, and penton bases (Fig. 4). The location of the adenovirus polypeptides was studied by analysis of fractions which were obtained after different stages of capsid degradation. The two methods described by Prage et al. (1970) were used, and they are outlined in Table 4. Both methods revealed that the adenovirus core contains two proteins corresponding to polypeptides V and VII (Fig. S and 11). Polypeptide IX seems to be the cementing polypeptide between the hexons, which
145
OF ADENOVIRUSES
originate from the facets of the adenovirus capsid since it is present in the aggregates of nine hexons which are released by pyridine (Fig. 12A). This polypeptide was released from the hexons when the capsid was disintegrated into single capsomers by freezing and thawing. Polypeptide VIII was recovered in association with hexons after freezing and thawing, but was separate from the groups of nine hexons after degradation with pyridine. Possibly this polypeptide is linked to the hexons in the capsid, but is detached by pyridine. Polypeptide VI appears to be associated with all hexons present in the capsid since (i) all fractions from disintegrated virus which contained hexons also contained polypeptide VI ; (ii) polypeptide VI has an affinity to hexons since it cosediments with hexons in sucrose gradients without salt irrespective of whether the hexons were obtained by freezing and thawing or by pyridine treatment. The location of polypeptide IIIa is difficult to determine, since this polypeptide is incompletely resolved from polypeptide IV on 13% SDS-gels. It appears, however, that polypeptide IIIa is preferentially released after pyridine treatment and together with the peripentonal hexons after dialysis against Tris-maleate buffer, pH 6.3. We therefore TABLE MOLAR
PROPORTION OF MINOR IN THE VIRION~
Protein6
V VI VII X
5
Molecules/virion
180 420 1070 50
POLYPEPTIDES
Molecules/hexon capsomerc 1.8 -
a The values are obtained from the radioactivity data given in Tables l-3 together with the fact that 10’2 physical particles of adenovirus type 2 correspond to 0.28 mg of protein (Prage et az., 1970). b Corresponding figures could not be obtained from protein VIII and IX, since these proteins have not been purified. c The number of morphological hexon units per virion is 240, and there are 3 molecules of polypeptide II per hexon (Valentine and Pereira, 1965; Maize1 et al., 1968,).
146
EVERITT,
SUNDQUIST,
PETTERSSON,
propose that polypeptide IIIa is located in the peripentonal region. Polypeptide X was recovered in several fractions after sequential degradation and its location in the capsid is unknown. The topography of minor polypeptides within the virion was also studied by Maize1 et al. (1968b), and they reported a similar, but less detailed pattern than ours. The higher resolving power of the SDS-disc-gels (Maizel, 1971) and the urea extraction procedure allowed a better separation and enabled us to purify some of the minor polypeptides of the adenovirion. Our results are summarized in a hypothetical model of the adenovirus particle (Fig. 14). ACKNOWLEDGMENTS The superb technical assistance of Miss Solveig Rosen and Miss Ingela Hiibinette is gratefully acknowledged. We also want to thank Mr. Hannu Ukkonnen for drawing the adenovirus model. REFERENCES CONANT, R. M., and BARRON, A. L. (1967). Enhanced diffusion of enterovirus antigens in agar in the presence of protamine. virology 33, 547-549. DAYHOFF, M. 0. (Ed.) (1969). “Atlas of Protein Sequence and Structure,” Vol. 4, p. D-164. National Biochemical Research Foundation, Silver Spring, Maryland. EAGLE, H. (1959). Amino acid metabolism in mammalian cell cultures. Science 130, 432437. EVERITT, E., SUNDQUIST, B., and PHILIPSON, L. (1971). Mechanism of the arginine requirement for adenovirus synthesis. I. Synthesis of structural proteins. J. Viral. 5, 742-753. GREEN, M., and PIR.I, M. (1963). Biochemical studies on the adenovirus multiplication. IV. Isolation, purification and chemical analysis of adenovirus. Virology 20, 199-207. Hauss, F. N. (1963). Solutes and solvents for liquid scintillation counting. Packard Tech. Bull. No. I, revised. Packard Instrument Company, Downers Grove, Illinois. HJ$:RTI~N, S., JERSTEDT, S., and TISELIUS, A. (1965). Electrophoretic “particle sieving” in polyacrylamide gels as applied to ribosomes. Anal. Biochem. 11, 211-218. Lavr:~, W. G. (1970). Isolation of an argininerich protein from particles of adenovirus type 2. Virology 41, 488-500. LOENING, U. E. (1967). The fractionation of high molecular weight ribonucleic acid by poly-
AND
PHILIPSON
acrylamide gel electrophoresis. Biochem. J. 192, 25-257. LONBERG-HOLM, K., and PHILIPSON, L. (1969). Early events of virus-cell interaction in adenovirus system. J. Viral. 4, 323338. LOWRY, 0. H., ROSEBROUGH, N. J., F~RR, A. F., and RANDALL, R. J. (1951). Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MAIZEL, J. V., JR. (1971). Polyacrylamide gel electrophoresis of viral proteins. In “Methods in Virology” (K. Maramorosch and H. Koprowski, eds.), Vol. V, pp. 179-246. Academic Press, New York. MAIZEL, J. V., JR., WHITE, D. O., and SCHARFF, M.D. (1968a). The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12. Virology 36, 1X-125. MAIZEL, J. V., JR., WHITE, D. O., and SCHARFF, M.D. (1968b). The polypeptides of adenovirus. II. Soluble proteins, cores, top components, and the structure of the virion. 1Ti’irology 36, 126-136. MANCINI, G., CARBONAHB, A. O., and HEREMAKS, J. F. (1965). Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2, 23&254. NEVILLE, D. N., JR. (1971). Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J. Biol. Chem. 246, 63284334. PEREIRB, H. G., and SKEHEL, J. J. (1971). Spontaneous and tryptic degradation of virus particles and structural components of adenoviruses. J. Gen. Viral. 12, 13-24. PETTERSSON, U. (1971). Structural proteins of adenoviruses. VI. On the antigenic determinants of the hexon. Virology 43, 123-136. PETTERSSON, U., and H~GLUND, 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~GLUNU, S. (1967). Structural proteins of adenoviruses. I. Purification and characterization of adenovirus type 2 hexon antigen. Virology 33, 575590. PETTERSSON, U., PHILIPSON, L., and H~GLUND, S. (1968). Structural proteins of adenoviruses. II. Purification and characterization of adenovirus type 2 fiber antigen. Virology 35, 204-215. PRAGE, L., and PETTERSSON, U. (1971). Structural proteins of adenoviruses. VII. Purification and properties of an arginine-rich core protein from adenovirus type 2 and type 3. Virology 45, 364-373.
STRUCTURAL
PROTEINS
PRAQE, L., PETTERSSON, U., H~GLUND, S., LONBERG-HOLM, K., and PHILIPSON, L. (1970).
Structural proteins of adenoviruses. IV. Sequential degradation of the adenovirus type 2 virion. Virology 42, 341358. RUSSELL, W. C., MCINTOSH, K., and SKEHEL, J. J. (1971). The preparation and properties of adenovirus cores. J. Gen. Vi’irol. 11, 3546. SCHULZE, I. T., and COLOWICK, S. P. (1969). The modification of yeast hexokinases by pro-
OF ADENOVIRUSES
147
teases and its relationship to the dissociation of hexokinase into subunits. J. Biol. Chem. 244, 2396-2316. VALENTINE, R. C., and PEREIRA, H. G. (1965). Antigens and structure of the adenovirus. J. Mol. Biol. 13, 13-20. WEBER, K., and OSBORN, M. (1969). The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 44064412.