reactive serine in human adenovirus hexon polypeptide
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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 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.
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).
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.
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 Eagles 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.
ADENOVIRUS HEXON REACTIVE SERINE 95
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.
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.
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 1Energie 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 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).
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 re- moved 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-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.
Analytical Sodium Dodecyl Sulfate (SDS- Polyacrylamide Gel Electrophoresis
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,
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 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).
Sucrose Density Gradient Analysis
(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.
(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.
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 injectio