Structural proteins of adenoviruses

67,197-208 (1975)

VIROLOGY

Structural

Proteins

of Adenoviruses

XII. Location and Neighbor Relationship Among Proteins of Adenovirion

Type 2 as Revealed by Enzymatic

lodination,

lmmunoprecipitation

and Chemical Cross-Linking

EINAR Departments

EVERITT,’ ofMicrobiologyand

LEONARD Molecular

LUTTER,

AND

Biology, The Wallenberg Uppsala, Sweden Accepted

April

LENNART Laboratory,

PHILIPSON The University

of Uppsala,

30, 1975

A refined topographical model of adenovirus type 2 was constructed based on results with enzymatic iodination of intact and disrupted virions and immunoprecipitation of intact virions with specific antisera. Results with reversible chemical cross-linkage of the structural proteins support this model, which suggests that proteins VI, VII and VIII are located within the virion. The core protein V is also an internal protein probably located at the vertices in close connection with the penton bases, hexons and protein IIIa molecules. Proteins IIIa and IX apparently lack tyrosine residues exposed on the outside of the virion but have external antigenic determinants. INTRODUCTION

The localization of protein components in membranes of erythrocytes (Phillips and Morrison, 1970; Tsai et al., 1973), normal and transformed cells (Mastro et al., 1974; Hynes 1973; Marchalonis et al., 1971) and several enveloped viruses such as vaccinia (Sarov and Joklik, 1972; Katz and Margalith, 1973), influenza (Stanley and Haslam, 1971), avian myeloblastosis (Fritz, 1974), Sindbis (Sefton et al., 1973), and murine RNA tumor viruses (Witte et al., 1973) has been studied by enzymatic iodination. Other organelles including ribosomes (Litman and Cantor, 1974) and the nonenveloped viruses, FMDV (Talbot et al., 1973) and bovine enteroviruses (Carthew and Martin, 1974) have also been subjected to iodination by the lactoperoxidase technique originally described by Marchalonis (1969). The high molecular weight lactoperoxidase enzyme (MW, ’ Present address: National Cancer Institute, NIH, Bethesda, MD 20014. 197 Copyright@ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

80 000) (Barman, 1969) is believed for steric reasons to catalyze only the labeling of tyrosine residues of externally located proteins. Immunoprecipitation has been used as an alternative method to determine external protein components in bacterial ribosomes (Stiiffler et al. 1973). Bifunctional cross-linking chemical reagents have also been employed to determine the topography of proteins in organelles. The neighborhoods of protein components in ribosomes (Lutter et al., 1974) and plasma membranes (Ii and Ji, 1974) have been studied with this technique. This report compares the labeling pattern in adenovirus polypeptides after enzymatic iodination of intact and disrupted virions. The external location of antigenic determinants was also studied by immunoprecipitation of virions using specific antibodies prepared against highly purified structural proteins. The virions were also cross-linked with reversible chemical crosslinkers to reveal adjacent proteins. The results were used to construct a topograph-

198

EVERI’IT,

LUTTER

ical model of the virion, which is refined in its details compared with the previous model (Everitt et al., 1973). MATERIALS

AND

METHODS

Cells and Virus Infection

of Hyperimmune

PHILIPSON

cylindrical and the second on 13% slabgels under the same conditions. The gels were stained and processed for autoradiography as described previously (Everitt and Philipson, 1974). Enzymatic

HeLa cell strain S-3 was grown in spinner cultures in Eagle’s spinner medium (Eagle, 1959) with 7% calf serum. The prototype strain of adenovirus type 2 was propagated and purified as described previously (Pettersson et al., 1967; Everitt et al., 1971). Purified virus was contaminated to less than 0.01% with host cell proteins (Everitt et al., 1973). Preparation

AND

Sera

Specific antisera against purified hexon, fiber and the major core protein were prepared as described by Pettersson et al. (1967, 1968) and Prage and Pettersson (1971). Sera against proteins VI, VIII and IX were prepared as recently described (Everitt and Philipson, 1974). Purification and preparation of serum against protein IIIa will be described elsewhere (Everitt et al., in preparation). Sodium Dodecyl Sulphate (SDS)-Polyacrylamide-Gel Electrophoresis

Exponential slab-gels (1.5 mm thick, 70 mm wide and 70 mm long) were made by mixing 5 ml of a 16% acrylamide solution with an excess (around 15 ml) of a 10% acrylamide solution in a gradient mixer. The general procedure has been described by McGuire et al. (1974). The buffer systems for discontinuous SDS gels described by Maize1 (1971) were used, and a ratio of acrylamide to bisacrylamide of 3O:O.S was maintained. To ensure a proper gradient the 16% acrylamide solution was made 50% (v/v) with respect to glycerol. Polymerization was induced by addition of 0.03 and 0.04% ammonium persulphate to the heavy and light acrylamide solutions, respectively. TEMED was present at 0.05% in both solutions. The gels were cast at a rate of 2.3 ml/min, and they polymerized within 30 min. When the two-dimensional SDSpolyacrylamide-gel system was used the first dimension was electrophoresed on 10%

Iodination

a) Disrupted uirions. Highly purified adenovirions were dialyzed against 5 mM Tris-HCl, pH 8.1, dissociated with 10% pyridine (Prage et al., 1970) and incubated at room temperature for 30 min. The material was dialyzed against 5 mM Tris-HCl, pH 8.1, and then iodinated by adding lactoperoxidase (Sigma Chemicals Co., St. Louis, MO) to give a ratio of enzyme protein to virion proteins of 1:250. Carrierfree Na[Y] (Amersham Radiochemical Centre, U.K.) was added to a concentration between 10 and 100 pCi/ml and the reaction initiated by furnishing hydrogen peroxide to a concentration of 4 PM. After incubation at room temperature (22”) for 30 min, the remaining hydrogen peroxide was reduced with 0.1% 2-mercaptoethanol, after which the proteins were precipitated with 10% trichloroacetic acid (TCA) in 10 mM KI. The precipitate was washed three times with 5% TCA in 10 mM KI, three times with ether:ethanol (1:l) and finally once with ether. The samples were dissolved in the disruption solution for SDS-polyacrylamide electrophoresis described earlier (Everitt and Philipson, 1974). b) Intact uirions. Three flasks of HeLa cells grown in monolayers were infected as previously described (Everitt et al., 1971). After 35 hr at 37” the cells were shaken off, washed in phosphate-buffered saline and incubated for 10 min at a cell density of 5 x lo6 cells/ml in hypotonic buffer (0.01 M NaCl, 1.5 mM MgCl, in 0.01 M Tris-HCl, pH 7.4) at 0” and the nuclei isolated as previously described (Everitt and Philipson, 1974). The nuclei were suspended in phosphate-buffered saline to give a final concentration of 4 x 107-5 x lo7 cell equiv/ml. The nuclei were sonicated for 2 x 15 set at 0”. After centrifugation at 3000 g for 5 min the virus-containing supernatant fluid was iodinated by adding 500 PCi of carrier-free Na [‘251], lactoperoxidase (5

TOPOGRAPHY

OF PROTEINS

pg) and H,Oz at 10 min intervals, each time at a final concentration of 5 PM. After 30 min the reaction was stopped with mercaptoethanol, and 300-~1 portions of the labeled nuclear extract were layered onto preformed gradients of Angiografin (Schering AG, Berlin, West Germany). The linear gradients from 5080% Angiografin in 0.01 M Tris-HCl, pH 8.1, were centrifuged in an SW56 rotor at 190,000 g for 15 hr at 4’. After collection, the virus band was diluted with two volumes of 0.02 M Tris-HCl, pH 8.1, and centrifuged in an SW40 rotor through 30% sucrose in the same buffer onto a cushion of CsCl (p = 1.40 g/cm3). The virus material was precipitated with TCA, washed and dissolved as described above. Immunoprecipitation Adenovirions labeled with [3H]thymidine and purified as previously described (Everitt et al., 1971) were used. The virus was rapidly frozen in 10% glycerol, 1 mM MgCl, in 0.02 M Tris-HCl, pH 7.5, at -70” and thawed at 37” before use. Such virus preparations will retain the specific infectivity for at least 6 months and after at least five cycles of freezing and thawing (unpublished). Twenty microliters (25,000 cpm and 4.5 x 10” particles) of virus material was mixed with 20 ~1 of the appropriate serum, diluted in phosphate-buffered saline and incubated at 37” for 30 min in the direct test. When the double antibody technique was used, antiIgG was added at equivalence, determined by immunodiffusion, and the mixture incubated for another 30 min. At the end of the incubation 100 ~1 of 1% Nonidet P-40 (NP40) in phosphate-buffered saline was added, and, after thorough mixing, the material was layered onto sucrose gradients on top of a cushion of CsCl (p = 1.40 g/cm3). Linear gradients from lo-25% (w/v) sucrose in 5 mM Tris-HCl buffer, pH 7.5, and 0.2 mM EDTA were centrifuged in an SW56 rotor at 50,000 g for 10 min. The gradients were fractionated from the bottom and aliquots of the 200-~1 fractions were assayed for radioactivity on Whatman 3 MM filters after TCA precipitation.

IN THE

Reversible

199

ADENOVIRION

Chemical

Cross-Linking

Cross-linking experiments were performed using the cleavable protein crosslinker tartryl diazide (TDA). Its synthesis and use have been described previously (Lutter et al., 1974). TDA is capable of crosslinking protein amino groups which are at most 0.6 nm from each other. A typical cross-linking reaction involves incubating adenovirus (2.2 mg of protein/ml) in 50 mM triethanolamine-HCl, pH 8.5, with 20 mM TDA for 30 min at 20”. The reaction is stopped by the addition of methylamine-HCl, pH 7.7, to a final concentration of 0.1 M. The protein is then extracted as described above for SDS-gel electrophoresis. A protein-protein complex cross-linked together by TDA can be cleaved by mild periodate treatment, which breaks the carbon-carbon bond between the vicinal hydroxyl groups of the tartrate moiety and releases the constituents of the complex. A two-dimensional polyacrylamide-gel electrophoretic system which takes advantage of this cleavability can be used to analyze the constituents of the various cross-linked complexes formed (Lutter et al., 1974). The cross-linked adenovirus protein is electrophoresed in a cylindrical SDS-containing 10% polyacrylamide gel. This gel is then soaked for two 5-hr changes in a buffer containing 50 mM NaH,P04, 0.1% SDS, 10 mM NaIO,, pH 6.0, followed by electrophoresis in a second-dimension gel-slab with 13% polyacrylamide. All proteins participating in cross-linked complexes will electrophorese as complexes in the first dimension and as monomers in the second dimension, so they will be found below the diagonal formed by the uncross-linked monomer proteins. RESULTS

Enzymatic

Iodination

The presence of tyrosine residues accessible to lactoperoxidase in intact adenovirus was first investigated. Material from sonically treated isolated nuclei of productively infected HeLa cells was iodinated and the labeled virus was subsequently purified according to a procedure causing a’ minimum of damage to the virions; Freon

200

EVERITT,

LUTTER

extractions and CsCl gradient centrifugations as described in Materials and Methods were therefore excluded. This procedure was adopted to minimize labeling of partially disintegrated virions. The SDS-gel pattern of labeled polypeptides of intact virions isolated in this way was compared to the pattern obtained after labeling pyridine-disrupted virions as shown in Fig. 1. In the intact virions, label was predominantly confined to proteins corresponding to polypeptides II (hexon), III (penton base), and IV (fiber), whereas in disrupted virions label was also present in polypeptides IIIa, V-VIII and X-XII but not polypeptide IX. Adenovirus labeled after CsCl purification (Everitt et al., 1971) followed by reisolation of labeled virus showed iodination of polypeptide V and when purified virions were labeled after one cycle of freezing at -70” and thawing at 37”, polypeptides V, VII and, to a lesser extent, VI were also labeled in reisolated virions in addition to the polypeptides labeled in intact virions.

AND

PHILIPSON

(I)

(2)

-E-n-

-ma-lE-‘9:-

Immunoprecipitation

Enzymatic iodination of intact virions will reveal only external tyrosine residues of the proteins accessible to lactoperoxidase. Polypeptides that lack external tyrosines may therefore be overlooked although they are located on the external surface of the virion. Immunoprecipitation of intact virions with specific antibodies (Oberg et al., 1975) against highly purified structural proteins of adenovirions (Everitt and Philipson, 1974) was therefore used as an additional tool to reveal external proteins. The virus material was labeled with [3H]thymidine in the DNA to minimize the contribution in the assay of virus proteins released from degraded virus. Highly purified preparations which retain their infectivity were used to prevent precipitation of partly degraded virus. Immunoprecipitation was carried out as described in Materials and Methods with a direct and a double antibody technique. A normal guinea pig serum was included as a negative control and an anti-fiber serum was used as a positive control (Prage et al., 1970). Table 1 shows that antisera against

FIG. 1. SDS-polyacrylamide slab-gel autoradiograms of in vitro iodinated intact (1) and pyridine-disrupted (2) adenovirions. Slot (3) is mounted and shows the protein-stained pattern of the sample in slot (1). 50,000 cpm of each material was electrophoresed for 6 hr at 100 V on a 10-169’~ exponential and discontinous SDS slab-gel as described in Materials and Methods.

proteins II, IIIa and IX were able to precipitate between 17 and 74% of the [3H]thymidine radioactivity when analyzed by the double antibody technique but did not precipitate the virions with the direct method. Antisera against proteins VI-VIII did not aggregate the virions by either method. Chemical

Cross-Linking

Protein neighborhoods 0.6 nm were subsequently fied virus material was reversible cross-linking scribed in Materials and

at a distance of examined. Purireacted with a reagent, as deMethods. Thirty

TOPOGRAPHY

OF PROTEINS

IN THE

TABLE IMMUNOPRECIPITATION Serum

Anti-hexon

Reciprocal dilution

(II)

serum used

201

ADENOVIRION

1

OF TYPE 2 ADENOVIRIONS Anti-IgG

Reciprocal serum dilution effective in immunodiffusion with corresponding protein?

Percent radioactivity precipitated’

10 10

+ -

23-37’ 3

100 10 10

+ + -

25 68-74d 2

10 10

+

NT’ 90

128

Anti-VI

10 10

+ -

2 NT

32

Ant i-VII

50 10 10

+ + -

3 6 1

16

Anti-VIII

10 10

+ -

3 NT

4

Anti-IX

8 10 10

+ + -

19 17 4

4

10 10

+

3 3

-

Anti-IIIa

Anti-fiber

Normal

(IV)

guinea

pig

“Percent of applied [3H]thymidine radioactivity described in Materials and Methods. b Immunodiffusion with around 2-4 pg of purified described (Everitt and Philipson, 1974). c Range of two experiments. d Range of three experiments. ‘NT, not tested.

micrograms of cross-linked and control adenovirion proteins was analyzed in adjacent wells on an SDS slab-gel as described in Materials and Methods. As shown in Fig. 2, two new electrophoretic components reproducibly appeared; these were denoted TDA-1 and TDA-2, and they migrate ahead of polypeptides III and V, respectively. The molecular weights in SDS gels of the TDA-1 and TDA-2 complexes are 70,000 and 36,000 respectively (Fig. 3). An additional band appeared in some preparations which migrated at an estimated molecular weight of around 26,000. High molecular weight complexes were also formed,

sedimenting

to the

CsCl

antigen

was determined

cushion

128

4

under

in agarose

the conditions

gels as previously

not penetrating the separation gel and thus with molecular weights two to three times larger than the hexon polypeptide (120,000). The slightly faster migration of polypeptide VII in the cross-linked preparation as compared with the control material, may be due to intraprotein cross-linking. In order to identify the complexes a twodimensional SDS slab-gel system was used (Lutter et al., 1974; and Materials and Methods). Figure 4 as well as the molecular weight determinations of the TDA-1 and TDA-2 complexes in Fig. 3 reveal that the TDA-1 complex probably contains one

202

EVERITT,

LUTTER

TDA-l--) T DA-2e

-91 -m A!? -x - XL-XII FIG. 2. The polypeptide pattern of cross-linked (1) and control (2) adenovirus proteins after SDS-polyacrylamide slab-gel electrophoresis. Equal amounts of protein (30 pg) were electrophoresed, as described in the legend to Fig. 1, and the polypeptides were stained with Coomassie blue as described in Materials and Methods and by Everitt and Philipson (1974).

mole each of polypeptides V and VII, whereas the TDA-2 complex appears to be a dimer of polypeptide VII. The low molecular weight complex with a molecular weight of around 26,000 was not detected after cleavage of the cross-linker, possibly due to its elution during the periodate soaking of the gels. This complex may consist of some of the fast-migrating constituents in the region of the gel where polypeptides X-XII are located. Figure 4 also reveals the cleavage products of other cross-linked complexes which were not apparent as separate electrophoretie components in the first-dimension electrophoresis. A dimer of polypeptide VI is apparent, which comigrated with polypeptide V in the first dimension. A complex of polypeptides IIIa and VII is also visible, and its first-dimension mobility is consistent with the molecular weights of its

AND

PHILIPSON

constituents. Also visible is what appears to be a complex of polypeptides V and VI, but the sum of molecular weights of its putative constituents implies that it should have a higher electrophoretic mobility in the first-dimension than is observed. It may be possible that this complex is composed of two moles of polypeptide VI (since dimers have been shown to be formed) and one mole of polypeptide V. The cleavage products of some relatively high molecular weight cross-linked complexes can be seen in the left portion of the slab gel in Fig. 4. It is difficult to draw neighborhood conclusions from these cleavage products since it is almost certain that some of the cleavage products that lie on the same vertical line come from different cross-linked complexes and are therefore not necessarily neighbors in the intact virion. Thus, although the evidence is not as clear as for the dimer pairs mentioned above, the cleavage products of the high molecular weight complexes give some support to the following neighborhoods: II-IIIa, II-V-VI-VII, II-III-IIIa-IV, and possibly a linkage between these complexes and polypeptide V. By determining the horizontal distribution of radioactivity in a two-dimensional gel like that shown in Fig. 4, it was found that approximately 70% of protein VII exists as monomer following cross-linking, whereas as much as 70% of protein V could be found in cross-linked complexes. Disruption

of Cross-Linked

Virions

To assess the size of the cross-linked complexes, CsCl-purified virions stored at -7O”, were iodinated and cross-linked after thawing at 37”. After thorough dialysis the virions were disrupted with 10% aqueous pyridine (Prage et al., 1970) and subsequently separated on sucrose gradients as previously described (Prage et al., 1970; Everitt et al., 1973). The control preparation revealed the sedimentation pattern described before (Everitt et al., 1973) with the core material sedimenting the fastest, the groups of nine-hexon structures (ninemers) at an intermediate position and the vertex capsomers together with low molecular weight material near

TOPOGRAPHY

OF PROTEINS

20

30 Distance

IN THE

10 migrated

203

ADENOVIRION

50

60

70

(mm)

FIG. 3. Determination of the molecular weights of the TDA-1 and TDA-2 cross-linked complexes. As molecular weight markers the virion SDS polypeptides III (7O,OfM-85,006), IIIa (63,009-68,600), V (46,500-50,006), and VI (22,000-24,600) were used. Molecular weight values are taken from Anderson et al. (1973) and Ishibashi and Maize1 (1974).

the top (Fig. 5A). The cross-linked material exhibits a peak sedimenting in the core position, no pronounced peak of the ninemers and a peak near the top (Fig. 5B). The gradient fractions were pooled, TCAprecipitated and electrophoresed on SDS slab-gels. The gels were autoradiographed to detect the iodine label. As shown in Fig. 6 the three fractions of the control gradient contained the expected polypeptides (Everitt et al., 1973); the core fraction consisted mainly of polypeptides V and VII and the ninemers of polypeptides II and VI, but polypeptide IX, also confined to this fraction (Everitt et al., 1973), is unlabeled by enzymatic iodination as shown in Fig. 1. The top fraction contains the peripentonal hexons (polypeptide II) and the penton (comprising polypeptides III and IV). Polypeptide IIIa, which also accumulates in this fraction, is not labeled with iodine in intact virions (Fig. 1). The core peak from the cross-linked material also contained polypeptides V and VII. The intermediate region consisted of hexons together with trace amounts of the two core polypeptides, but the hexon-associated polypeptide VI was only faintly discerni-

ble. The top fraction consisted of the vertex capsomers. Evidently hexons were crosslinked to form aggregates of varying size and shape, all sedimenting in the intermediate position but not as a discrete peak. The TDA-1 and TDA-2 complexes were not detected as iodine-labeled entities, which might indicate that different copies of the internal proteins V-VII are labeled as compared to those that are cross-linked. The results suggest that no aggregates larger than the core are formed after cross-linkage. DISCUSSION

An attempt was made to investigate the topography of an adenovirus with the aid of enzymatic iodination, immunoprecipitation and chemical cross-linking. Results obtained with enzymatic iodination should be interpreted with care since only tyrosine and possibly some histidine residues of proteins which are accessible to lactoperoxidase are labeled. Failure to label does therefore not necessarily imply that the protein is internal in the virus particle. Results from experiments where proteins are cross-linked with bifunctional reagents

204

EVERITT,

LUTTER

FIG. 4. A stained two-dimensional SDS-polyacrylamide slab-gel electropherogram of cross-linked and cleaved adenovirus polypeptides. All spots appearing to the left of the diagonal are polypeptides derived from cross-linked complexes. The first dimension was electrophoresed at 100 V for 4 hr in a cylindrical (diameter, 3 mm) 10% SDS-polyacrylamide slab-gel and the second for around 6 hr in a 13% SDS-polyacrylamide slab-gel, as described in Materials and Methods.

using either chemical or antibody ligands may also be difficult to interpret for two reasons: i) Failure to react does not rule out closeness or availability for the ligand and ii) the first step of the two-step reaction of the reagent may change the conformation of the particle and thus given an incorrect second step. The three methods used in this investigation may, however, if interpreted with caution, together help to relate the proteins in the virion topographically. In the present investigation intact and pyridine-disrupted virions were iodinated and the labeled polypeptides were compared by SDS-gel electrophoresis (Fig. 1). In intact virions most of the label was confined to hexons, fibers and to some extent to penton bases. When virions were isolated and purified through the normal procedure including Freon extraction and

AND

PHILIPSON

CsCl centrifugation (Everitt et al., 1971) before iodination, label was also detected in the core polypeptide V (not shown). After the virions had been frozen and thawed once, the iodine label was, after reisolation of the virus, also present in polypeptides V and VII and to a lesser extent in polypeptide VI (Fig. 6). These observations strongly suggest that adenovirions are not rigid entities. Virions disrupted with pyridine (Prage et al., 1970) were labeled in all polypeptides except in polypeptide IX (Fig. 1). No attempts were made to quantitate the enzymatic label since the proteins may have different configurations after pyridine disruption. A qualitative comparison was therefore made by analyzing the same amount of total radioactivity in each gel. Electrophoretic analysis of the polypeptides after in vitro labeling of SDS-disrupted virions and in uiuo labeling with [3H]tyrosine showed comparable gel patterns (not shown). The possibility that antigenic determinants of some proteins were available on the external surface in spite of deficient iodine labeling was studied by immunoCPM

I 1400010000 6000

B

1

1

-

2000 10

20 FRACTION

30

I

1

40

50

NUMBER

FIG. 5. Sucrose gradient centrifugation of iodinated control and cross-linked adenovirions after pyridine disruption. The gradients, 10-256 sucrose in 2 mM Tris-HCl buffer, pH 7.5, and 0.2 mM EDTA, were centrifuged in an SW40 rotor at 90,000 g for 150 min. The sedimentation pattern of disrupted control (A) and cross-linked virus preparations (B) are shown.

TOPOGRAPHY

CONTROL

OF PROTEINS

CROSS -LINK.

1231234

FIG. 6. SDS-polyacrylamide slab-gel autoradiograms of the fractions from the sucrose gradient separation of control (Fig. 5A) and cross-linked iodinated virus (Fig. 5B) preparations disrupted by pyridine. The fractions indicated by bars in Figs. 5A and B were precipitated with trichloroacetic acid and analyzed by SDS-polyacrylamide gels as described in Materials and Methods. The Roman numerals of some polypeptides in the virion marker are shown to the left.

of virions with specific antibodies against purified structural proteins. Antisera against proteins IIIa and IX precipitate virions with the double antibody technique (Table 1) although these proteins fail to become labeled in intact virions. The efficient precipitation of virions with anti-IIIa suggests an external location and is in accordance with the proposed site at the vertex region of the virion (Everitt et al., 1973). Antisera against proteins VI-VIII did not precipitate virions even in the presence of anti-IgG antibodies, and the corresponding polypeptides were not iodinated in intact virions. The antibodies have previously been used to identify the in vitro translation products obtained in two cell-free systems programmed with adenoprecipitation

IN THE ADENOVIRION

205

virus-specific messenger RNA (aberg et al., 1975), and all sera were thus shown to be highly specific. The neighbor relationship of the polypeptides in the intact virion were investigated using the chemical cross-linking reagent tartryl diazide. TDA is capable of cross-linking protein amino groups which are at most 0.6 nm from each other. Subsequently this cross-link can be cleaved, which makes it possible to use a twodimensional gel-electrophoresis system to identify the constituents of the complexes formed (Lutter et al., 1974). Two new electrophoretic components appeared following cross-linking; one was composed of polypeptides V and VII, whereas the other was found to be a dimer of polypeptide VII. Also detected were a complex of IIIa and VII, a dimer of polypeptide VI, and a complex consisting of polypeptides V and VI. Based on these results we propose a refined topographical model of an adenovirus as presented in Fig. 7. This model differs from our previous tentative model (Everitt et al., 1973) in that all protein VI residues are located internally. The iodination and immunoprecipitation studies support this position as does the finding that protein VI is entirely confined to the core structure (Prage, unpublished) when adenovirus type 3 virions are disrupted with 10% pyridine. Furthermore, since native protein VI has a molecular weight of 50,000 which is a dimer of the VI polypeptide and since there are only 2 moles of polypeptide VI per hexon capsomer (Everitt et al., 1973; Everitt and Philipson, 1974), the present results suggest an internal position of all protein VI molecules. Protein VI is obviously derived from a precursor polypeptide with a molecular weight of 27,000 (Anderson et al., 1973; Oberg et al., 1975) which is acidic and also present in precursor particles to complete virions (Sundquist et al., 1973; Ishibashi and Maizel, 1974). Since protein VI appears to be internal in the virion it is conceivable that the precursor to protein VI is cleaved into the basic protein VI (molecular weight of polypeptide: 24,000 (Everitt et al. (1973))) and a smaller acidic peptide at assembly.

206

EVERITT,

LUTTER

AND

PHILIPSON

Polypeptide

SDS-gel

PIPII

FIG.

7. A refined

topographical

model

of adenovirus

Similarly, the present results suggest that all the proteins so far identified which are derived by cleavage at assembly (i.e., VI-VIII) (Anderson et al., 1973; Sundquist et al., 1973; Ishibashi and Maizel, 1974; ijberg et al., 1975) are internal constituents; this correlation may have bearing on the mechanism of DNA folding and packing. Most moieties of the core protein V are placed immediately internal to the pentons in the vertex regions of the virion (Fig. 7). The results from cross-linkage of protein V (Fig. 4) and its accessibility to iodination when CsCl-purified virions are labeled with the lactoperoxidase method support this localization. Protein V is also partially dissociated together with the pentons at low pH (Everitt et al., 1973), again favoring a position close to the vertex region. Part of protein IIIa is presumably located at the exterior of the vertex regions of the virion. It was already suggested that protein IIIa might be a vertex protein (Everitt et al., 1973), and the results from chemical crosslinkage support this idea (Fig. 4). Immunoprecipitation of intact virions with antiIIIa antibodies using a double antibody

-

Structural

Unit

-

Hexon

-

Pentonbase Neutralizing Fiber

0

-

Core

-

-

Hexon-associated

-

-

Core

, -

Hexon associated Protein specific

protein(?) for

groups

hexons

type

2. See text

antigen(?)

protein

protein

protein (AAP)

of nine

for explanations.

technique also strongly favors an external location (Table 1). The fact shown in Fig. 4 that protein IIIa, an external protein, can be cross-linked to protein VII, an internal protein, may indicate that part of protein IIIa extends into the interior of the virion. Preliminary evidence suggests that this protein may be important for virus neutralization (unpublished). Since virions during the early phase of uncoating presumably release proteins in the vertex regions (Sussenbach 1967; Lonberg-Holm and Philipson, 1969; Everitt, unpublished) an antiIIIa-protein IIIa complex may prevent subsequent uncoating after penetration of the antibody-virus complex. The protein IX specific for groups of nine hexons (Everitt et al., 1973) did not contain accessible tyrosines (Fig. l), but antigenic determinants were exposed at the surface of the virion (Table 1). The relative low efficiency with which anti-IX antibodies were able to precipitate intact virions may suggest that the antigenic determinants are buried between the surrounding hexons. The polypeptides X-XII finally may represent the cleavage products after processing of the precursors to polypeptides VI-

TOPOGRAPHY

OF PROTEINS

-VIII, since the former small polypeptides are absent in particles which probably are intermediates in assembly (Sundquist et al., 1973; Ishibashi and Maizel, 1974; Edvardsson et al., unpublished). Like the mature products, polypeptides VI-VIII, they are probably internal, which may suggest that the virion carries the processing enzyme since young intact virions may contain unprocessed precursors (Ishibashi and Maizel, 1974). This refined topographical map of the adenovirus proteins may be of importance in understanding the early events in virus infection and the mechanism of assembly for this group of viruses. ACKNOWLEDGMENTS We thank Mrs. Solveig Andersson and Mrs. Berit Nordensved for excellent technical and secretarial assistance, respectively. We also thank Mr. Hannu Ukkonnen for drawing the adenovirus model and Dr. Ulf Pettersson for a critical review of the manuscript. This investigation was supported by grants from the Swedish Cancer Society and the Wallenberg Foundation, Sweden, to the Department of Microbiology and by grants from the Swedish Cancer Society, Swedish Medical Research and Natural Science Research Councils to the Department of Molecular Biology. REFERENCES ANDERSON,C. W., BAUM, P. R., and GESTELAND,R. F. (1973). Processing of adenovirus P-induced proteins. J. Viral. 12, 241-252. BARMAN, T. E. (1969). “Enzyme Handbook,” Vol. 1, pp. 234-235. Springer Verlag, Vienna. CARTHEW,P. and MARTIN, S. J. (1974). The iodination of bovine enterovirus particles. J. Gen. Viral. 24, 525-534.

EAGLE, H. (1959). Amino acid metabolism in mammalian cell cultures. Science 130, 432-437. EVERITT, E., and PHILIPSON, L. (1974). Structural proteins of adenoviruses. XI. Purification of three low molecular weight virion proteins of adenovirus type 2 and their synthesis during productive infection. Virology 62, 253-269. EVERITT, E., SUNDQUIST,B., and PHILIPSON,L. (1971). Mechanism of the arginine requirement for adenovirus synthesis. I. Synthesis of structural proteins. J. Virol. 5, 742-753. EVERI~~, E., SUNDQUIST, B., PETTER.SSONU., and PHILIPSON, L. (1973). Structural proteins of adenoviruses. X. Isolation and topography of low molecular weight antigens from the virion of adenovirus type 2. Virology 52, 130-147.

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