Reactive serine in human adenovirus hexon polypeptide

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<ul><li><p>VIROLOGY 102,94-106 (1980) </p><p>Reactive Serine in Human Adenovirus Hexon Polypeptide </p><p>CHRISTIANE DEVAUX AND PIERRE BOULANGER </p><p>Laboratoire de Virologie MolBculaire, INSERM, Place de Verdun, 59045 Lille, France </p><p>Accepted December 18, 1979 </p><p>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 enzy- matic 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 inter- mediate particles. DFP reacts with urea-denatured hexon in vitro with an apparent dissocia- tion 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. </p><p>INTRODUCTION </p><p>Virus particles may contain enzymes, ei- ther cellular enzymes adventitiously incor- porated 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 endonu- clease, associated with the penton base cap- somer (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). </p><p>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 pro- tein precursors, and blocked in virion mat- uration at restrictive temperature (Weber, 1976). It seemed, therefore, of interest to investigate the proteolytic activity of adeno- virus-infected cells, in comparison with that of uninfected cells. </p><p>However, since it was impossible to study most of the enzymes of the infected cell </p><p>1 To whom all correspondence and requests for re- prints should be addressed. </p><p>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. </p><p>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. </p><p>MATERIALS AND METHODS </p><p>Cells and Virus </p><p>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 Eagles spinner medium supplemented with 5% horse serum. HeLa cells were cultured </p><p>0042~6822/80/050094-18$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. </p><p>94 </p></li><li><p>ADENOVIRUS HEXON REACTIVE SERINE 95 </p><p>as monolayers in Eagles minimai essential medium supplemented with 10% calf serum. Cells were infected at a multiplicity of in- fection of 25-50 FFU per cell. </p><p>Virus was titrated on HeLa cell mono- layer, using the fluorescent focus assay tech- nique (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 complementa- tion 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 temper- ature was 33, and the nonpermissive 39.5. </p><p>Radiochemicals </p><p>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. </p><p>[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 1Energie atomique (Saclay, France). </p><p>In Vivo Labeling of Adenovirus Proteins </p><p>HAdB-Infected KB cells were labeled from 18 to 36 hr after infection with labeled amino acid (10 $X/ml) in culture medium contain- ing 10% of the concentration of the corre- sponding amino acid of normal medium. Ex- traction and purification of HAd2 major cap- sid proteins have been described in detail elsewhere (Boulanger and Puvion, 1973; Boulanger et al., 1978). </p><p>In Vitro Affinity Labeling with Radioac- tive DFP </p><p>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&amp;f NaCl, 3 m&amp;f KCl, 8 m&amp;f Na, HP04, 15 m&amp;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-&amp;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 re- moved by centrifugation at 10,000 g for 15 min and the supernatant, referred to as S-10, was subjected to DFP labeling. </p><p>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. </p><p>For analysis in dissociating SDS-poly- acrylamide gel, DFP-labeled protein ma- terial 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 heat- ing 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. </p><p>Analytical Sodium Dodecyl Sulfate (SDS- Polyacrylamide Gel Electrophoresis </p><p>Analysis of labeled polypeptides was per- formed in SDS-containing 15% polyacryl- amide 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, </p></li><li><p>96 DEVAUXANDBOULANGER </p><p>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). </p><p>Preparative SDS -Polyacrylamide Gel E lec- trophoresis </p><p>SDS-Denatured samples were electro- phoresed under the same conditions de- scribed above. Stained polypeptides were extracted by slicing the gel, mincing the gel slices, and eluting the polypeptides elec- trophoretically using a Gradipore elution device (Gradipore, Townson and Mercer Ltd., Lane Cove, Australia). </p><p>Sucrose Density Gradient Analysis </p><p>(a) Sucrose gradient centtifugation of proteins. Samples (0.2-0.3 ml) of DFP-la- beled 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, contain- ing 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. </p><p>(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&amp;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. </p><p>Antisera </p><p>Preparation of polyspecific antisera against HAd2 and HAd5 virions, of poly- specific 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 determi- nants of HAd2 hexon capsomers was pre- pared in rabbits by injection of hexon papain </p><p>cores (Boulanger, 1975; Boulanger et al., 1978). Antiserum against the hexon poly- peptide unit in a denatured state was a generous gift from L. Philipson (Oberg et al., 1975). </p><p>Immunoprecipitation </p><p>Labeled antigenic components were iden- tified 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). </p><p>Enzymatic and Acid Hydrolyses </p><p>(a) Peptide jingerpkting. Comparative peptide fingerprinting was performed in SDS-containing, highly crosslinked poly- acrylamide 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 tempera- ture (Cleveland et al., 1977). </p><p>(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 electro- phoresis or electrochromatography on cel- lulose 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). </p><p>r2P]DFP-serine was analyzed by elec- trochromatography 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. </p></li><li><p>ADENOVIRUS HEXON REACTIVE SERINE 97 </p><p>a b c d e f 1101(- </p><p>FIG. 1. SDS-polyacrylamide gel autoradiogram of r*P]DFP-labeled KB cells, mock-infected (a), or infected with human adenovirus 2 (HAd2), and har- vested 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). </p><p>Purijkation of HAd2 Virus Particles and Capsid Proteins </p><p>The purification of virions and of the soluble capsid components hexon, penton base, penton, and fiber has been described (Boulanger and Puvion, 1973; DHalluin et al., 1978; Boudin et al., 1979). </p><p>Assay for Protease Activity of HAd2 Hexon </p><p>rH]valine-labeled hexon protein was in- cubated 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 incuba- tion mixtures. Labeled hexon was used in order to correct for possible self-proteolysis. </p><p>Assayfor E&amp;erase Activity of HAd2 Hexon </p><p>Trypsin-like activity was assayed by the method of Erlanger et al. (1961) using &amp;-benzoyl-DL-arginine p-nitroanilide-HCl (BAPNA, Sigma Chemical Co.) as sub- strate. Chymotrypsin-like activity was as- sayed by the method of Twumasi and Liener, (1977) using Z-L-tyrosine-4-nitro- phenyl ester (Z-Tyron, Fluka, Switzer- land) as substrate. </p><p>Enzymes </p><p>Trypsin (TPCK-treated) and chymotryp- sin (crystallized) were purchased from Worthington Biochemical Corporation (Freehold, N. J.). Papain, twice crystal- lized, 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.). </p><p>Protein </p><p>Protein concentrations were estimated by the method of Lowry et al. (1951) using bovine serum albumin as the standard. </p><p>Nomenclature </p><p>The nomenclature proposed by Ginsberg et al. (1966) for the major capsid com- ponents (hexon, penton base, penton, and fiber) is used. The structural polypeptides are referred to according to the terminol- ogy proposed by Maize1 et al. (1968) and Anderson et al. (1973). </p><p>RESULTS </p><p>Evidence for DFP-Labeled Proteins Spe- ci;fic for Cells Infected with Human Adenovirus 2 (HAd2) </p><p>HAdS-Infected cell extract proteins la- beled in vitro with PP]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 poly- peptide was eluted electrophoretically from the gel, and reelectrophoresed in SDS- polyacrylamide gel with adenovirus poly- peptide markers. The 120K species comi- grated with HAd2 hexon polypeptide (Figs. le, f). </p></li><li><p>98 DEVAUXANDBOULANGER </p><p>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. </p><p>Absence of DFP-Labeled Proteins within Adenovirus Particles </p><p>The HAd2-infected late cell extract labeled in vitro with r2P]DFP was analyzed i...</p></li></ul>