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VIROLOGY 102,94-106 (1980)

Reactive Serine in Human Adenovirus Hexon Polypeptide

CHRISTIANE DEVAUX AND PIERRE BOULANGER’

Laboratoire de Virologie MolBculaire, INSERM, Place de Verdun, 59045 Lille, France

Accepted December 18, 1979

Affinity labeling with diisopropylfluorophosphate (DFP) is used to study the enzymes associated with human adenovirus type 2 infection. A DFP-labeled 120K polypeptide is constantly found at a late stage of the virus cycle. Biochemical and immunological data indicate that this 120K polypeptide corresponds to the hexon subunit. No detectable enzymatic activity is found associated with native or urea-denatured adenovirus 2 hexon capsomer, and no DFP-labeled components appeared to be incorporated into virus or assembly intermediate particles. DFP reacts with urea-denatured hexon in vitro with an apparent dissociation constant of 5 x 1O-s M. The occurrence of at least one transient reactive serine residue on the hexon polypeptide subunit and its possible implication in folding and/or assembly of hexon subunits into a hexon trimer is discussed.

INTRODUCTION

Virus particles may contain enzymes, ei- ther cellular enzymes adventitiously incorporated during virus maturation, or virion enzymes not provided by the host cell and required for the viral growth cycle. Adeno- virus has been found to contain an endonuclease, associated with the penton base capsomer (Burlingham and Doerfler, 1972; Marusyk et al., 1975; Cajean-Feroldi et al., 1977), which has been shown to be of cellular origin (Reif et al., 1977). A protein kinase activity, likely of cellular origin, has also been identified (Blair and Russell, 1978).

The importance of proteolysis in the adenovirus multiplication cycle is demon- strated by the processing of certain virus proteins at late stages of infection (Ander- sen et al., 1973; Bhatti and Weber, 1978) and by the isolation of temperature-sensitive mutants defective in cleavage of virus protein precursors, and blocked in virion maturation at restrictive temperature (Weber, 1976). It seemed, therefore, of interest to investigate the proteolytic activity of adenovirus-infected cells, in comparison with that of uninfected cells.

However, since it was impossible to study most of the enzymes of the infected cell

1 To whom all correspondence and requests for re- prints should be addressed.

by affinity labeling with substrate analogs binding specifically to their catalytic site, this search was limited to a class of enzymes susceptible to labeling with a probe reacting uniquely and specifically with one amino acid residue of the active site. Diisopropylfluoro- phosphate (DFP), an organic fluorophos- phate, reacts with a unique serine residue located in the active site of serine enzymes, e.g., serine proteases, and more generally serine esterases (Balls and Jansen, 1952; Schaffer et al., 1953, 1954; Koshland, 1963). As DFP reacts stoichiometrically with the serine enzymes, 32P- or 3H-labeled DFP can be profitably used to label the catalytic site.

In the present study, labeled DFP was reacted in vitro with cytoplasmic extracts from cells infected with human adenovirus type 2, at early and late stages of infection.

MATERIALS AND METHODS

Cells and Virus

Wild-type (WT) human adenovirus type 2 (HAd2) was originally supplied by J. F. Wil- liams. HAd5 was a gift from W. C. Russell, and HAd3 from R. G. Marusyk. The viruses were propagated on KB cells maintained in suspension culture at 3 x lo5 cells/ml in Eagle’s spinner medium supplemented with 5% horse serum. HeLa cells were cultured

0042~6822/80/050094-18$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

94

ADENOVIRUS HEXON REACTIVE SERINE 95

as monolayers in Eagle’s minimai essential medium supplemented with 10% calf serum. Cells were infected at a multiplicity of infection of 25-50 FFU per cell.

Virus was titrated on HeLa cell monolayer, using the fluorescent focus assay technique (Philipson et ccl., 1968). Temperature- sensitive (ts) mutants of HAd2 have been selected in our laboratory after nitrous acid mutagenization of a WT stock (Martin et al., 1978). HAd2 ts 104, 106, 118, and 121 have been characterized serologically: ts 104 as fiber-penton-base negative, and ts 106, 118, and 121 as hexon defective. ts 106, 118, and 121 belong to three different complementation groups. ts 106 (complementation group K) produced reduced quantities of hexons, whereas ts 118 (group H) and ts 121 (group A) failed to synthesize detectable amounts of hexons at nonpermissive temperature (Martin et al., 1978). The permissive temperature was 33”, and the nonpermissive 39.5”.

Radiochemicals

32P-Labeled diisopropylfluorophosphate (DFP, 400-500 @Zi/mg, 75-90 mCi/mmol) without solvent and rH]DFP (3-4 Ci/mmol) in propylene glycol were both purchased from the Radiochemical Centre Ltd. (Amer- sham, U. K.). r2P]DFP was dissolved in propylene glycol before use, at lo-20 mCi/ml. [32P]DFP was used at the beginning of this study, but PH]DFP was then pref- erentially used for prolonged biochemical investigations.

[35S]Methionine (600-700 Ci/mmol) was purchased from the Radiochemical Centre, and t3H]valine (25-30 Cilmmol) and [3H]1eu- tine (50-60 Ci/mmol) from the Commissariat a 1’Energie atomique (Saclay, France).

In Vivo Labeling of Adenovirus Proteins

HAdB-Infected KB cells were labeled from 18 to 36 hr after infection with labeled amino acid (10 $X/ml) in culture medium containing 10% of the concentration of the corresponding amino acid of normal medium. Ex- traction and purification of HAd2 major capsid proteins have been described in detail elsewhere (Boulanger and Puvion, 1973; Boulanger et al., 1978).

In Vitro Affinity Labeling with Radioac- tive DFP

HeLa cells (15 x 106) grown in monolayer were mock-infected or infected with HAd2 at a multiplicity of infection of 50 FFU per cell. Cells harvested at 6 and 24 hr after infection, respectively, were washed in phosphate-buffered saline (PBS:150 m&f NaCl, 3 m&f KCl, 8 m&f Na, HP04, 15 m&f KHZPOI, 5 mM MgCl2 6H2O, 1 m2M CaCl,, pH 7.4), and resuspended in 0.5 ml of hypo- tonic buffer (TE:50 mM Tris-HCl, 2 n-&f Na-EDTA, pH 7.8). After three cycles of freezing and thawing, the cell lysate was briefly sonicated (50 W for 15 set with a B-12 Branson sonifier microtip). The nuclei, large organelles, and cell debris were removed by centrifugation at 10,000 g for 15 min and the supernatant, referred to as S-10, was subjected to DFP labeling.

32P- or 3H-Labeled DFP (50-100 ~1) in propylene glycol solution were added to 0.5 ml of S-10, to a final activity of 0.8-1.5 mCi per S-10 sample, and the sample was im- mediately homogenized on a Vortex mixer, then left at room temperature for 2 hr with magnetic stirring.

For analysis in dissociating SDS-polyacrylamide gel, DFP-labeled protein material was separated from soluble labeled compounds by precipitation with 10% tri- chloroacetic acid (TCA) at 0” for 30 min. The precipitate was washed twice with 0.1% TCA and cold ethanol and dissolved by heating at 100” for 2 min in SDS-gel sample buffer (62.5 mJ4 Tris-HCl buffer, pH 6.8, containing 4% SDS, 10% 2-mercaptoethanol, and 6 M urea). For immunological studies, the soluble label was eliminated by prolonged dialysis against TE buffer containing 10% glycerol.

Analytical Sodium Dodecyl Sulfate (SDS- Polyacrylamide Gel Electrophoresis

Analysis of labeled polypeptides was performed in SDS-containing 15% polyacrylamide slab gel (acrylamide:bisacrylamide ratio of 50:0.235) overlaid by a 5% spacer gel (acrylamide:bisacrylamide ratio of 50: 1.33) in the discontinuous buffer system described by Laemmli (1970). The gels were stained with Coomassie brilliant blue R-250,

96 DEVAUXANDBOULANGER

dried under vacuum, and autoradiographed on Kodak Kodirex film. 3H-Labeled protein samples were revealed by fluorography on Kodak Royal X-Omat film at -70” in PPO- impregnated gel (Bonner and Laskey, 1974).

Preparative SDS -Polyacrylamide Gel E lec- trophoresis

SDS-Denatured samples were electro- phoresed under the same conditions described above. Stained polypeptides were extracted by slicing the gel, mincing the gel slices, and eluting the polypeptides electrophoretically using a Gradipore elution device (Gradipore, Townson and Mercer Ltd., Lane Cove, Australia).

Sucrose Density Gradient Analysis

(a) Sucrose gradient centtifugation of proteins. Samples (0.2-0.3 ml) of DFP-labeled cell extract or of in vivo labeled virus proteins used as markers were layered on a 12-ml, 5 to 20% linear sucrose gradient in 20 mM Na-borate buffer, pH 8.0, containing 1 M NaCl and 1 mM Na-EDTA, and centrifuged at 30,000 rpm for 16 hr at 20 in a Spinco SW-41 rotor. Fractions (0.3 ml) were collected from the bottom of the tube and assayed for TCA-precipitable and im- munoprecipitable radioactivity.

(b) Sucrose gradient centrifugation of virus particles. Extracts of cells harvested at 24 hr after infection were layered on a 12-ml, 25-40% linear sucrose gradients in 20 r&l4 Na-borate buffer, pH 8.0, containing 200 mM NaCl and 1 mJ4 Na-EDTA, and centrifuged at 22,000 rpm for 95 min at 4” in a SW-41 rotor. Fractions (0.6 ml) were collected from the bottom of the tube and assayed for acid-precipitable radioactivity.

Antisera

Preparation of polyspecific antisera against HAd2 and HAd5 virions, of polyspecific antisera against virus-soluble eap- sid components, and of monospecific antisera has been described in detail elsewhere (Martin et al., 1975; Boudin et al., 1979). Antiserum against type-specific determinants of HAd2 hexon capsomers was prepared in rabbits by injection of hexon papain

cores (Boulanger, 1975; Boulanger et al., 1978). Antiserum against the hexon polypeptide unit in a denatured state was a generous gift from L. Philipson (Oberg et al., 1975).

Immunoprecipitation

Labeled antigenic components were identified by immunoselection with Staphylo- coccus aureus protein A (Kessler, 1975). Formalin-treated S. aureus cell (Cowan 1 strain) were purchased from Eivai Bios Lab (Horsham, Sussex, U. K.) and used in the immune complex assay as previously described (Boulanger et al., 1979).

Enzymatic and Acid Hydrolyses

(a) Peptide jingerpkting. Comparative peptide fingerprinting was performed in SDS-containing, highly crosslinked polyacrylamide slab gel (15% acrylamide, acrylamide:bisacrylamide ratio of 50:1.33). Polypeptide samples eluted electrophoreti- tally from SDS-gel slices were hydrolyzed within a 5% spacer gel with increasing amounts of S. aureus V, protease (Drapeau et al., 1972) for 30 min at room temperature (Cleveland et al., 1977).

(6) Acid hydrolyses. Hydrolysis of DFP- labeled protein samples was carried out with 5.6 N HCI at 110” in sealed tubes for 2, 6, and 20 hr. After freeze-drying, the hydrolyzates were analyzed by electrophoresis or electrochromatography on cellulose thin-layer plates. Electrophoresis (first dimension) was conducted at 900 V for 90 min in 8% acetic acid-2% formic acid, pH 1.9. Chromatography (second dimension) was carried out in pyridine: acetic acid:n-butanokwater (100:30:150: 120, v/v).

r2P]DFP-serine was analyzed by electrochromatography and autoradiography. rH]DFP-serine was analyzed by mono- dimensional high-voltage eleetrophoresis and scanning with a thin-layer Scanner RTLS-IA (Panax Equipment Ltd., Red- hill, Surrey, U. K.). Labeled serines were identified by comparison with control DFP-serine obtained from DFP-labeled trypsin and chymotrypsin.


a b c d e f

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FIG. 1. SDS-polyacrylamide gel autoradiogram of r*P]DFP-labeled KB cells, mock-infected (a), or infected with human adenovirus 2 (HAd2), and harvested at 6 hr (b) and 24 hr (c) after infection. (d, e) control r5S]methionine-labeled HAd2 virion. The 120K polypeptide, labeled with r*P]DFP, as in (c), was eluted electrically and reelectrophoresed in SDS-gel (f).

Purijkation of HAd2 Virus Particles and Capsid Proteins

The purification of virions and of the soluble capsid components hexon, penton base, penton, and fiber has been described (Boulanger and Puvion, 19’73; D’Halluin et al., 1978; Boudin et al., 1979).

Assay for Protease Activity of HAd2 Hexon

rH]valine-labeled hexon protein was incubated at pH 6.0 to 9.0 with heat- and acid-denatured bovine hemoglobin as substrate (Barrett, 1967). Proteolysis was monitored by measurement of the A,,,., in the TCA-soluble supernatant of incubation mixtures. Labeled hexon was used in order to correct for possible self-proteolysis.

Assayfor E&erase Activity of HAd2 Hexon

Trypsin-like activity was assayed by the method of Erlanger et al. (1961) using &-benzoyl-DL-arginine p-nitroanilide-HCl (BAPNA, Sigma Chemical Co.) as substrate. Chymotrypsin-like activity was assayed by the method of Twumasi and Liener, (1977) using Z-L-tyrosine-4-nitro- phenyl ester (Z-Tyron, Fluka, Switzer- land) as substrate.

Enzymes

Trypsin (TPCK-treated) and chymotrypsin (crystallized) were purchased from Worthington Biochemical Corporation (Freehold, N. J.). Papain, twice crystallized, as a sodium acetate suspension, was purchased from Sigma Chemical Com- pany (Saint Louis, MO.), and StaphyZococ- cus V, protease, crystallized, from Miles Laboratories Ltd. (Slough, U. K.).

Protein

Protein concentrations were estimated by the method of Lowry et al. (1951) using bovine serum albumin as the standard.

Nomenclature

The nomenclature proposed by Ginsberg et al. (1966) for the major capsid components (hexon, penton base, penton, and fiber) is used. The structural polypeptides are referred to according to the terminology proposed by Maize1 et al. (1968) and Anderson et al. (1973).

RESULTS

Evidence for DFP-Labeled Proteins Spe- ci;fic for Cells Infected with Human Adenovirus 2 (HAd2)

HAdS-Infected cell extract proteins labeled in vitro with P”P]DFP were analyzed in SDS-polyacrylamide gel at early and late stages after infection and compared with mock-infected cell extracts. As shown in Fig. 1, the three polypeptide patterns are very similar, with only minor differ- ences. Two protein bands, of 35,000 (35K) and 22,000 (22K) molecular weight, which were absent from the mock-infected pat- tern, were visible in the early extract. In the late extract, a major polypeptide of 120K was found, as well as a 35K polypeptide. The DFP-labeled 120K polypeptide was eluted electrophoretically from the gel, and reelectrophoresed in SDS- polyacrylamide gel with adenovirus polypeptide markers. The 120K species comi- grated with HAd2 hexon polypeptide (Figs. le, f).


Inasmuch as the 35K and 22K species were not constantly observed in both early and late extracts, further studies focused on the late 120K protein species.

Absence of DFP-Labeled Proteins within Adenovirus Particles

The HAd2-infected late cell extract labeled in vitro with r2P]DFP was analyzed in 25-40% linear sucrose gradients. No 32P label appeared at the sedimentation position of mature adenovirion (‘750 S), nor of assembly intermediate particles (550- 600 S; D’Halluin et al., 1978). Most of the label remained at the top of the gradient, corresponding to “soluble” components, not incorporated in virus particles (not shown). The fractions from the top of the gradient were pooled and sedimented in a 5-20% linear sucrose gradient, in order to separate the viral and cellular structural components. No [32P]DFP label was observed at the position of hexon capsomers, which have an apparent sedimentation coefficient of 12-13 S (Wilhelm and Ginsberg, 1972). The 32P radioactivity sedimented at 3-4 S, at the position of the hexon polypeptide subunit marker (Velicer and Ginsberg, 1970), obtained by SDS denaturation of the hexon capsomer (not shown).

SDS-Polyacrylamide gel analysis of the gradient fractions showed that the 3-4 S region contained the DFP-labeled 120K polypeptide of the late extract, and the DFP-labeled 35K and 22K polypeptides of the early extract (Fig. 2). As the DFP-labeled 120K protein specific for HAdZ-infected cell extracts was not found incorporated into virus particles, the question arose whether this 120K polypeptide was of viral or cellular origin, i.e., virus-coded or cellular coded, and stimulated during the virus infection.

Identi$cation of the Late Virus-Coded DFP-Labeled 120K Polypeptide

If the DFP-labeled 120K polypeptide was virus coded and corresponded to hexon polypeptide, its apparent molecular weight would follow the variation in the molecular weight of the hexon polypeptide subunit observed among the different human adeno-

m e I

,120K

-35K

-22K

FIG. 2. SDS-polyacrylamide gel autoradiogram of rH]DFP-labeled material sedimenting in sucrose gradient at 3-4 S. (m) Mock-infected KB cell extract; (e) “early” HAdZ-infected cell extract; (1) “late” HAd& infected cell extract. In contrast to the late 120K species, the 35K and 22K polypeptides of the early extract were inconstantly observed.

virus serotypes. Since HAd5 hexon polypeptide unit is known to have a molecular weight (99-100K) lower than that of HAd2 hexon (120K; Marusyk and Cummings, 1978), similar in vitro labeling experiments with r2P]DFP were carried out with HAd5-infected cell extract to determine whether the major late DFP-labeled species migrated in SDS-gel in a position corresponding to a 100K viral polypeptide or to a 120K cellular polypeptide.

Figure 3 shows that the difference in electrophoretic migration between the [32P]DFP-labeled polypeptides of HAd5- and HAdS-infected cells was identical to that shown between ~5S]methionine-labeled HAd2 and HAd5 hexon polypeptides. Thus, the occurrence of a DFP-labeled 1OOK polypeptide in HAd5-infected cell extracts


FIG. 3. SDS-Polyacrylamide gel autoradiogram of HAdZ- and HAd5-infected KB cells, labeled in tiwo with [Wlmethionine (a-c), or in vitro with r*P]DFP (d-f). (a, d) Mock-infected cells; (b, e) HAdB- infected cells; (c, f) HAd5-infected cells; (v) control ~5SJmethionine-labeled HAd2 virion. HAd5 hexon subunit migrates as a 1OOK polypeptide, HAd2 hexon subunit as a 120K polypeptide (Marusyk and Cum- mings, 1978).

strongly suggested a viral origin for this reactive serine-containing protein.

Further evidence for the viral origin of the DFP-labeled 120K polypeptide was given in experiments performed with HAd2 temperature-sensitive (ts) mutants. ts 118 and ts 121 have been shown to be hexon- negative mutants and fail to synthesize detectable amounts of hexon at 39.5”. ts 106 is also altered in hexon production, but synthesizes appreciable amounts, though reduced, of hexon at nonpermissive temperature. ts 104 is a fiber-penton- base-defective mutant (Martin et al., 1978). As shown in Fig. 4, a DFP-labeled 120K polypeptide was not found in ts 11% and ts 121-infected cells at 39.5, whereas a 120K polypeptide was labeled with DFP in ts lobinfected cell extracts. The 120K label species of ts 106 was reduced compared with WT or ts 104.

The DFP-labeled late 120K protein of HAd2 which corn&rated with hexon polypeptide in SDS-polyacrylamide gel did, then, follow the variations in molecular weight of the hexon polypeptide unit among the adenovirus serotypes, and also

followed the variations in production of hexons of the ts mutants. It might be concluded that the DFP-labeled 120K polypeptide was virus-coded and corresponded to HAd2 hexon polypeptide.

Immunological Characterization of the HAd.2 DFP-Labeled 12OK Polypeptide

Antisera against whole HAd2 virions, purified HAd2 hexon, HAd5 hexon group- specific determinants, and type-specific determinants of HAd2 hexon (viz., anti- hexon papain core; Boulanger, 1975), failed to precipitate the DFP-labeled 120K polypeptide, even when using the sensitive S. aureus protein A technique. None of these antisera were capable of reacting with isolated or nascent hexon polypeptide subunits, as suggested by previous results obtained at supraoptimal temperatures (Leibo- witz and Horwitz, 1974; Warocquier and Boulanger, 1976). The hexon capsomer has been shown to be a trimer (Boulanger and Puvion, 1974; Grtitter and Franklin,

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IL

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FIG. 4. SDS-polyacrylamide gel autoradiogram of rH]DFP-labeled extracts from KB ceils infected with wild-type (WT) or temperature-sensitive (ts) mutants of HAd2. (a) Mock-infected cells; (b) WT-infected; (c) ts 118infected; (d) ts l21-infected; (e) ts 104-infected; (f) ts W-infected; (v) control WT HAd2 adenovirion. ts 118, 121, and 106 are hexon-defective mutants; ts 104 is a fiber-penton-base-defective mutant (Martin et al., 19’78).


19’74; JGmvall et al., 1974) comprised of three 120K polypeptide units (Horwitz et al., 1970). Only the hexon trimeric edifice has been found to react with antisera against assembled hexon capsomers and virus particles (Oberg et al., 1975).

Biochemical Characterization of the HAd2 DFP-Labeled 120K Polypeptide

As shown above, the DFP-labeled 120K polypeptide sedimented in sucrose gradient at 3-4 S, i.e., the position of the hexon polypeptide unit (Velicer and Ginsberg, 1970), whereas hexon capsomer (the trimer) sediments at 12-13 S (Wilhelm and Gins- berg, 1972). This suggested that the DFP- labeled 120K polypeptide visible in SDS- polyacrylamide gel was in monomeric form.

This hypothesis was supported by the sensitivity of the DFP-labeled 120K polypeptide to papain digestion. Native hexon capsomers have been found to be partially resistant to papain, leaving a papain- resistant core migrating in SDS-polyacrylamide gel as a polypeptide of 85K molecular weight (Boulanger, 1975). When [32P]DFP-labeled infected cell late extract was subjected to papain digestion, under the same conditions as native hexon cap- Somers, no 85K or 120K polypeptide was observed. The DFP label migrated with the buffer front, as low-molecular-weight peptides (not shown). The papain sensitivity of the 120K polypeptide suggested a con- formational state different from that present in hexon capsomer, such as absence of the quaternary structure of the hexon polypeptide.

Comparative peptide fingerprinting by electrochromatography of the DFP-labeled 120K polypeptide and of control purified hexon polypeptide proved worthless as the DFP-labeled peptide of the 120K polypeptide would have a chromatographic be- havior in organic solvents different from that of the corresponding unlabeled peptide(s) of the control hexon. Peptide fingerprinting on a column of Chromobeads P (Boulanger et al., 1969) presented the same drawback, due to the different affinity of DFP-labeled peptides for hydrophobic re- gions of the resin.

FIG. 5. Comparative peptide fingerprinting of lT-I]DFP-labeled 120K polypeptide from HAdZ-infected KB cells (a-e), and rH]valine-leucine-labeled HAd2 hexon polypeptide subunit (f-j). Gel slices containing the labeled material were hydrolyzed in SDS-polyacrylamide spacer gel with increasing amounts of Staphylococcus V, protease (Cleveland et al., 197’7). (a, f) 0.25, (b,-g) 0.50, (c, h) 1.0, (d, i) 2.5, (e, j) 5.0 pg of protease per slot. (v) HAd2 low-molecular-weight polypeptide markers: VII (18,500); VIII (13,000), IX (12,000), .according to Anderson et al. (1973).

The possible relationship between the DFP-labeled 120K polypeptide and the hexon polypeptide unit was, therefore, studied by partial proteolytic cleavage in SDS-polyacrylamide gel (Cleveland et al., 1977). To prevent a possible loss of in- formation, the control HAd2 hexon was labeled with both PH]valine and rH]leu- tine. rH]valine-leucine-labeled hexon polypeptide unit and [3H]DFP-labeled 120K polypeptide were prepared in SDS-polyacrylamide gel and gel slices containing the labeled material were hydrolyzed with increasing amounts of Staphylococcus V8 protease in SDS-polyacrylamide slab gel. Figure 5 shows that one major DFP- labeled species of 11.5K molecular weight and a minor DFP-labeled species of 12.5K originating from cleavage of the 120K polypeptide corresponded to similar break- down products of rH]valine-leucine-labeled hexon polypeptide. Other discrete bands labeled with PH]DFP corn&rated


with [3H]valine-leucine-labeled peptides from control hexon polypeptide. These peptide patterns strongly suggested an amino acid sequence relationship between the DFP-labeled 120K polypeptide and the hexon polypeptide.

Similar peptide patterns were obtained with HAd5 and HAd3 DFP-labeled 100K polypeptides, suggesting that the reactive serine was located in a highly conserved amino acid sequence (not shown).

Occurrence of DFP Labeling of the HAd2 120K Polypeptide on a Serine Residue

Unspecific trapping of DFP label in the hexon polypeptide was unlikely since the DFP label bound to the 120K polypeptide resisted several cycles of heating at 100” in 4% SDS-6 M urea and electrophoresis in SDS-polyacrylamide gel. Never- theless, the nature of the amino acid residue labeled with DFP was determined by comparison with DFP-labeled trypsin and chymotrypsin used as controls.

As shown in Fig. 6, following 6 hr of hydrolysis at 100” in 5.6 N HCl, the rH]DFP-labeled 120K polypeptide re- leased a ninhydrin-positive and radioac- tive spot corresponding to [3H]DFP- serine. The same result was obtained in electrochromatography of P”P]DFP spots and autoradiography (not shown). A 6-hr hydrolysis was not sufficient to completely degrade the hexon polypeptide, but a 20-hr hydrolysis resulted in low recovery of DFP-serine, the ester bonds being cleaved by acid hydrolysis.

Absence of Detectable Enzymatic Activity Associated with HAd2 Hexon

Since reactive serine residues are usually involved in the catalytic sites of many enzymes (Koshland, 1963), and although no enzymatic activity has thus far been found associated with hexon (Philipson and Pet- tersson, 1973), this eventuality was ex- amined by testing for protease and esterase activities.

No protease activity was detected against denatured bovine hemoglobin as substrate. No trypsin- or chymotrypsin-like esterase activity was found associated with purified

hexon capsomers, in the presence or absence of urea (concentrations ranging from 0.4 to 8.0 M).

In Vitro Labeling of Purijkd Hexon Capsomer with PH]DFP

Incubation of purified adenovirus particles, assembly intermediate particles (D’Halluin et al., 1978), or isolated hexon capsomers in vitro with PH]DFP resulted in no covalent binding of labeled DFP with these structures.

Since the results of DFP labeling in HAdB-infected cell extracts suggested a binding of DFP on hexon polypeptide units, purified hexon capsomers were denatured with 8 M urea at room temperature, rH]DFP was added, and the sample was further incubated at room temperature for 4 hr. Excess unbound DFP and urea were eliminated by extensive dialysis and the sample was analyzed in SDS-polyacrylamide gel. As controls, native and urea-denatured bovine serum albumin, ovalbumin, trypsin, and chymotrypsin were reacted with rH]DFP and analyzed in the same manner in SDS-gel.

Figure 7 shows that traces of label were present in a 80K species in the serum albumin, and in a 45K species in the ovalbumin sample. The 45K polypeptide might correspond- to nonspecific trapping of DFP label in the ovalbumin molecule. The labeled 80K polypeptide present in the serum albumin might correspond to a contaminating enzyme. In DFP-labeled chymotrypsin, a labeled polypeptide of 11K was found, which was expected as this enzyme has a pluripeptidic structure and had been heated in SDS-urea-2- mercaptoethanol buffer: the 11K polypeptide corresponded to the reactive serine- containing peptide. The DFP-labeled trypsin showed two major polypeptide bands, a band at 23K, as expected for the molecular weight of the enzyme polypeptide, and a band at llK, which might be a self-proteolytic cleavage product of the enzyme or chymotrypsin contaminants of the trypsin preparation. This latter hypothesis was troubling since the trypsin used was treated with TPCK.

DEVAUX AND BOULANGER

FIG. 6. High-voltage electrophoresis on cellulose thin-layer plate of HAd2 12OK polypeptide (a) and of commercial chymotrypsin (b), labeled in vitro with rH]DFP and hydrolyzed in 5.6 N HCl at 110” for 6 hr. (0) Origin; (H) unhydrolysed 120K polypeptide; (CT) unhydrolysed chymotrypsin. Arrow indicates the position of rH]DFP-labeled serine. Electrophoresis was conducted at 900 V at pH 1.9. Anode at the right.

Figure 7i shows that urea-denatured hexon capsomer bound a significant amount of rH]DFP, a binding which resisted several cycles of heating in SDS-urea-2- mercaptoethanol sample buffer and electrophoresis in SDS-gel. This labeling might be considered to be the result of covalent binding of DFP to the protein molecule. The DFP labeling was higher when a urea-denatured sample was heated for 5 min

at temperatures of 80-loo”, with or without 2-mercaptoethanol (1.5-2.0 n-N), before reacting with DFP at room temperature for 4 hr. In contrast, no DFP labeling was obtained when hexon was denatured by heating at 100” in the presence of l-5% SDS, prior to reaction with DFP (not shown).

Since urea was removed by dialysis, along with unbound DFP, the following


FIG. 7. In vitro labeling of proteins with PH]DFP. (a) Native bovine serum albumin (BSA); (b) native ovalbumin (OVA); (c) urea-denatured BSA; (d) urea- denatured OVA; (e) urea-denatured chymotrypsin (CT); (f) urea-denatured trypsin (T); (g) native CT; (h) native T; (i) urea-denatured HAd2 hexon cap- Somers; (v) control lW]methionine-labeled HAd2 virion. The molecular weights of HAd2 polypeptide markers are estimated to be: VI (24,000); VII (18,500); and IX (12,000) according to Anderson et al. (1973).

experiment was designed to determine whether the DFP labeling occurred during unfolding of the hexon polypeptide chain in the presence of urea, or during refolding of the chain as the urea concentration decreased in the dialysis step. Urea-denatured hexon capsomer was labeled with PH]DFP for 4 hr at room temperature. Urea and unbound DFP were removed by dialysis against TE buffer, or against TE buffer containing a large excess (20 m&f) of cold DFP. Hexon- bound DFP label was determined by TCA precipitation of the samples and subse- quent SDS-gel analysis. It was found that the labeling of the 120K polypeptide was similar in the presence or absence of cold DFP, suggesting that DFP labeling occurred during the unfolding and/or dis- assembly of hexon polypeptide subunits.

The urea-denatured hexon, labeled in vitro with DFP, was no longer precipitable by antisera against hexon capsomer, but did react with an antiserum against the hexon polypeptide unit (not shown). These results indicated that, after removal of urea, the

DFP-labeled hexon did not recover its immunological reactivity, which implied a change in tertiary and quaternary structure.

Affinity of Hexon for DFP

Considering the hexon as an enzyme and DFP as a substrate, which was cleaved during the reaction, it was possible to determine the dissociation constant of the DFP-hexon complex by plotting the reciprocal of the rate of reaction versus the reciprocal of the substrate concentration. Urea-denatured hexon was reacted for 90 min at room temperature with increasing amounts of rH]DFP and then urea and unbound DFP were removed by dialysis. The rate of reaction was the number of 3H counts per minute covalently bound to TCA-precipitated hexon. The Line- weaver-Burk plot gave a value of 5 x 10mg M for the dissociation constant of the DFP-hexon complex.

Similar experiments gave a value of 5.5 x lo-” M for the dissociation constant of DFP-chymotrypsin complex, viz., an affinity for DFP loo-fold higher in chymotrypsin than in hexon.

DISCUSSION

The finding of a DFP-labeled 120K “late” protein in HAdB-infected cell raises several questions: (i) Does the labeled 120K species correspond to nonspecific trapping of label? (ii) Is the DFP-labeled protein virus- or host cell-coded and virus-induced? (iii) Does this affinity labeling by DFP correspond to an enzymatic activity, and which type of activity, protease, esterase, or both? (iv) Is the DFP-labeled protein vu-ion-incorporated or soluble in the cell pool?

The biochemical and immunological data presented here suggest that the DFP- labeled 120K polypeptide in HAdZ-infected cells corresponds to the hexon polypeptide unit. Nonspecific trapping of label can be ruled out since the DFP binding re- sists heating in the presence of denaturing agents (SDS and urea), and since labeled DFP was removed by acid hydrolysis as DFP-serine. That the DFP-labeled 120K

polypeptide is virus coded is suggested by peptide unit. (ii) One (or more) reactive the variations of migration of the DFP- serine is required for assembly of hexon labeled band in SDS-gel in relationship to monomers to form the immunologically the variations of apparent molecular weight active hexon trimeric edifice. A DFP block of hexon polypeptide units of different would prevent the hexon polypeptide units adenovirus serotypes, by DFP-labeled ex- assembly. (iii) A reactive serine residue is periments performed with hexon-defective located within a catalytic site, and cor- ts mutants at restrictive temperatures, responds to an enzymatic activity present and by comparative fingerprinting of DFP- in the hexon polypeptide before its as- labeled 120K and hexon polypeptide units. sembly into the hexon capsomers. (iv) These

The absence of a similar DFP-labeled three hypotheses are not mutually exclusive 120K entity in virus particles, in assembly since a limited self-proteolysis of hexon intermediate particles, and in assembled polypeptide may be required for normal hexon capsomers, as well as the absence of assembly. This catalytic activity would, antigenic reactivity of the DFP-labeled therefore, disappear from the assembled 120K polypeptide with antisera toward hexon capsomer molecules, explaining the hexon capsomers suggest that this DFP negative results in enzymatic assays per- 120K polypeptide corresponds to hexon formed with hexon capsomers. polypeptides remaining in the cell pool in This hypothesis is supported by the ob- the form of unassembled hexon subunits. servation of self-aggregation of hexon cap- HAd2 hexon has been shown to be a Somers following limited proteolysis of the trimeric association of three subunits of protein (Pettersson, 1971; Boulanger, 1975). 120K (Horwitz et al., 1970; Boulanger and Further experiments are required to deter- Puvion, 1974). When blocked by DFP on a mine the position in the amino acid se- reactive serine-containing site, the hexon quence and the exact function of the reac- subunit fails to gain its normal antigenic tive serine residue of the hexon polypeptide three-dimensional structure and possibly to assemble into hexon capsomer. This hy- ACKNOWLEDGMENTS pothesis is confirmed by the sensitivity of the DFP-labeled 120K polypeptide to This work was supported by the CNRS (ERA-

papain digestion, whereas treatment of 225), the INSERM (FRA-43), and the Universite

hexon capsomers results in papain-resistant du Droit et de la Sante (UER III). We thank Ray

cores of 85K (Boulanger, 1975). Also, the Marusyk for his constructive criticism, Guy Martin and Chantal Cousin for providing temperature-

sedimentation coefficient of the DFP 120K sensitive mutants of human adenovirus 2, and Len- polypeptide in sucrose gradients (3-4 S) is nart Philipson for the antiserum against hexon more compatible with that of the hexon polypeptide unit. The help of Pierre Lemay in elec-

subunit (Velicer and Ginsberg, 1970), than trochromatographic analysis is gratefully acknowl-

the hexon capsomer (12- 13 S; Wilhelm and edged. We are indebted to Mr. J. Croquette and

Ginsberg, 1972). This hypothesis is also S. Wojeik for expert secretarial aid.

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