APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1992, p. 3517-3521

Vol. 58, No. 11

0099-2240/92/113517-05$02.00/0 Copyright X 1992, American Society for Microbiology

Activity of Glutaraldehyde at Low Concentrations against Capsid Proteins of Poliovirus Type 1 and Echovirus Type 25 MARTINE CHAMBON,* JEAN-LUC BAILLY, AND HELENE PEIGUE-LAFEUILLE Laboratoire de Bact6riologie-Virologie, Faculte6 de Medecine et Pharmacie, 28 Place Henri-Dunant, 63001 Clermont-Ferrand Cedex, France Received 8 June 1992/Accepted 1 September 1992

The activity of glutaraldehyde (GTA) against capsid proteins of poliovirus type 1 and echovirus type 25 was studied to understand the mode of action of this reagent against enteroviruses. The viruses were treated with GTA concentrations ranging from 0.005 to 0.10%Y. In the poliovirus particles, high-molecular-weight products were formed by 0.05% GTA, whereas in the echovirus particles, they were formed at 0.005% GTA. These products consist of complexes composed essentially of VP1 and VP3. There seemed to be differences in the composition of the complexes in the two viruses. Cross-linkings between the two polypeptides of the poliovirus capsid may be due to the accessibility to GTA of lysine residues on the loops of VP1 and VP3, which twist out from the surface of the shell.

study. Both strains were obtained from the World Health Organization Collaborating Center, National Reference Center for Enterovirus, Lyon, France. Tissue cultures. Vero cells, a line of African green monkey kidney cells, were obtained from Flow Laboratories, Inc. (McLean, Va.). MRC5 cells were obtained from BioMerieux Laboratory (Marcy-L'Etoile, France) and Whittaker Bioproducts (Fontenay sous Bois, France). MRC5 cells were used for passages 25 to 38. Cell lines were propagated in Eagle minimum essential medium (J. BIO Laboratoire, Les Ulis, France) supplemented with 10% fetal calf serum (J. BIO), penicillin (200 IU/ml), streptomycin (50 ,ug/ml), and glutamine (1%). GTA preparation, storage, and control. Stock solutions of 10% GTA were prepared in phosphate-buffered saline (PBS) from a 25% GTA solution (reagent grade I; Sigma OSI, Paris, France). Storage and control were performed as described previously (1). Virus growth. High-titer stocks of echovirus type 25 JV4 strain were produced in MRC5 cells and used for the preparation of labeled viral proteins. The Vero cell line was used for the preparation of the stock of poliovirus type 1 Mahoney strain. Confluent monolayers were inoculated at a multiplicity of infection of 1 to 10 MPNCU (most probable number of cytopathic units) per cell. Virus titration. Virus titers were determined in microtitration plates. Echovirus titration assays were performed with MRC5 cells. Poliovirus titration assays were performed with Vero cells. The endpoint dilution method with identical parameters (fourfold dilution rate, 11 replicates per dilution) for all virus titrations was used. Details of this method and the evaluation of titers have been reported elsewhere (1). Labeling of viruses. Virus capsid proteins were labeled with 20 ,uCi of [35S]methionine (Amersham, Les Ulis, France) per ml of methionine-free Eagle minimal essential medium (Select-Amine Kit; Gibco BRL). Confluent monolayers of MRC5 cells (about 1.6 x 107 cells per 75 cm2) were used to label the echovirus, and confluent monolayers of Vero cells (about 1.2 x 107 cells per 75 cm2) were used to label the poliovirus. All the cell cultures were inoculated with the viruses at a multiplicity of infection of 50 for 3 h. Medium was replaced by methionine-free minimal essential medium with 20 ,uCi of [35S]methionine (Amersham) per ml

Glutaraldehyde (GTA) is used as a fixative in electron microscopy and cytochemistry. It is an excellent crosslinking agent for proteins (6). These primary practical properties have been investigated in numerous studies on the chemical processes involved in the reaction between GTA and proteins. The reactions of model compounds such as amino acids and low-molecular-weight peptides have been studied (3, 11). Various workers have also investigated the effect of GTA on proteins and on enzymes in particular (5, 7, 11). Since the introduction of GTA as a chemosterilizer, there have been a number of reports on the effects of GTA on the protein constituents of different bacteria (for a review, see reference 2) and on spores (16). In comparison, little work has been done on viruses. Sangar et al., who worked with foot-and-mouth disease virus, showed that protein subunits were altered by GTA and that the overall structural integrity of the virion was not maintained (19). More recently, Passagot et al. examined the reduction in antigen titer of hepatitis A virus particles treated with GTA and showed that it was about 80% after 30 min with 0.10% GTA (13). The three-dimensional structure of the picornavirus capsid is now well known. The picornavirus particle consists of a plus single-stranded RNA genome enclosed within an icosahedral protein capsid. RNA and capsid proteins are the two potential targets for GTA in those viruses. In an earlier report, we studied the activity of GTA against the RNA of poliovirus type 1 (1). In the present study and as a follow-up to the previous work, we used sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis to investigate the effect of GTA on capsid proteins of poliovirus type 1 and echovirus type 25. We chose echovirus type 25 because it is a representative member of the echoviruses and because a comparative study of the biological and antigenic properties of wild and JV-4 prototype strains is currently being made in our laboratory (14, 15).

MATERIALS AND METHODS V'irus strains. Poliovirus type 1 Mahoney 1-397 strain and echovirus type 25 JV-4 reference strain were used in the *

Corresponding author. 3517

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CHAMBON ET AL.

for 24 h (14). When monolayers showed complete cytopathic effect, the supernatants were collected and centrifuged at 20,000 x g for 30 min at 4°C. The virus was pelleted at 125,000 x g for 4 h and then suspended in PBS. The virus suspension was finally purified by isopycnic centrifugation in CsCl at 165,000 x g for 40 h at 4°C. The virus-containing fractions were dialyzed against PBS for 24 h at 4°C (1). 35S-labeled virus preparations were then aliquoted (50,000 cpm per aliquot) and stored at -20°C until used in inactivation assays and electrophoresis. Inactivation assays. Purified and labeled virions were mixed with appropriate GTA dilutions to obtain final GTA concentrations of 0.005, 0.01, 0.02, 0.05, and 0.10% (wt/vol) and were incubated for 5, 30, 60, or 120 min. The procedure of inactivation has been described elsewhere (1). The preparations were centrifuged with an 180 A-100 fixed-angle rotor (Beckman) and an Airfuge (air-driven ultracentrifuge; Beckman) at an air pressure of 27 lb/in2 (186 kPa) for 15 min. Supernatants and virus pellets were then analyzed by electrophoresis in denaturing conditions. Electrophoresis of virus proteins. SDS-PAGE was performed as described by Laemmli (10). Samples were heated in boiling water for 5 min and layered onto a 12.5% poly-

acrylamide gel (0.75-mm thickness). Electrophoresis was run at a constant current (6 mA for 18 h). Gels were fixed, soaked in Amplify (Amersham), dried, and subjected to autoradiography (Hyperfilm, MP; Amersham). '4C-methylated molecular weight standards were obtained from Amersham. Immunoblot of echovirus type 25. Electrophoresis was performed as described above. Proteins were transferred to 0.45-,um-pore-size nitrocellulose sheets (Schleicher et Schuell, Inc., Dassel, Germany) as described by Towbin et al. (21) at 12 V for 10 min and then at 24 V for 50 min with a Transblot cell apparatus (Biolyon, Dardilly, France). The sheets were blocked with a 10% solution of dried milk in Tris-buffered saline (TBS) plus 0.05% Tween 20 (TBS-T) for 1 h at room temperature. Membrane-bound proteins were detected with a polyclonal rabbit immune serum raised against the echovirus type 25 JV-4 reference strain. The immunization procedure for rabbits and the reactivity of antiserum are described elsewhere (14). The sheets were soaked in TBS plus 5% dried milk and rabbit antiserum at a 100-fold dilution. The sheets were then washed three times (once each in TBS-T plus 5% dried milk, TBS-T, and TBS to reduce background staining) and incubated for 2 h with peroxidase-labeled goat immunoglobulin G at a 1:1,500 dilution (Nordic Immunological Laboratories, Tilburg, The Netherlands). After three washings, peroxidase-labeled antibodies were visualized with 4-chloro-1-naphthol (3 mg/ml in methanol; Sigma) and H202 in TBS. RESULTS Effect of GTA on poliovirus. (i) Investigation of nonspecific adsorption phenomena. GTA is known to adsorb on the surfaces of various materials (12, 17). To determine whether this phenomenon occurred during the reactions described in this study, we designed the following assay. Samples of [35S]methionine-labeled poliovirus were incubated at 25°C with 0.05% GTA for 30 and 120 min and with 0.10% GTA for 120 min. The control assay sample was incubated for 120 min without GTA. At the end of incubation, viral particles were collected by ultracentrifugation. Tubes of reaction mixture were thoroughly rinsed with electrophoresis denaturing buffer to desorb the viral material. The pellets and the

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FIG. 1. Investigation of nonspecific adsorption phenomena. Samples of [3nS]methionine-labeled poliovirus (50,000 cpm) were treated with GTA at 25(Cin PBS and collected by ultracentrifugation in an Airfuge (Beckman). The supernatants (S), pellets (P), and rinsing buffers (R) were subjected to electrophoresis at 6 mA for 18 h. Lanes: 1, molecular mass markers (in kilodaltons); 2, 3, and 4, control (untreated poliovirus particles); GTA for e.t5pr 5, 6, and 7, 30bfin; 8, 9, and 10, a.05% GTA for 120 min; 11, 12, and 13, 0.10so

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supernatants were collected after ultracentrifugation, and the rinsing buffers were then analyzed by SDS-PAGE (Fig. 1). In the control assay, most of the viral particles were recovered in the pellet (lane 3), and a small quantity of the virus remained in the supeuatant (lane 2). Viral material was also recovered in the rinsing bupfer (lane 4), tis.ceng that virion uptake occupfed on the surfaces of the tubes without GTA. Viruses were collected mainly in the pellets or the rinsing buffers. The products of the reaction of GTA with poliovirus particles after electrophoresis were the same whether they were recovered in the pellets or the rinsing buffers (lanes 6 and 7, 9 and 10, and 12 and 13). It was not possible with the method used in this study to determine whether the action of GTA against the immobilized virions was the same as that against the virions in suspension. However, the products of the reaction between GTA and these virions had similar molecular weights. (ii) Effect of GTA on poliovigus capsid proteins. Electrophoresis of virion polypeptides after GTA treatment resulted in a complex pattem of proteins (Fig. 1; lanes 6, 7, 9, 10, 12, and 13). The autoradiographic signal of V'Pl was much weaker than those of VP2 and VP3 when the incubation time was prolonged (lanes 6 and 9) or when the GTA concentration increased (lane 12). There was also a decrease in the 'VT3 signal, but to a lesser extent and mainly in the virus particles recovered in the pellets. V`P2 signal, however, remained unchanged. Sigh-molecular-weight products were generated in the poliovirus particles after reaction with GTA. Two of them, with molecular weights of about 64 and 92 kDa, behaved like homogeneous proteins. Two other products with similar molecular weights (about 160 kDa) were also formed. Several higher-molecular-weight reaction products formed a complex pattern. (ii1i) Kinetics of the effect of GTA on poliovirus capsid polypeptides. The effect of 0.02% GTA on poliovirus was tested for 5, 30, 60, and 120 min (Fig. 2B). The analysis of virion proteins by SDS-PAGE showed that the new product of about 64 kDa was detected early, after S min of exposure

GLUTARALDEHYDE AND ENTEROVIRUS CAPSID PROTEINS

VOL. 58, 1992

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FIG. 2. Electrophoretic pattern of poliovirus type 1 proteins after treatment with GTA for various times. (A) GTA at 0.05%. Lanes 1 and 2, control (untreated poliovirus particles); lanes 3 and 4, S min. (B) Lane 1, 0.02% GTA for 5 min; lanes 2, 3, and 4, 0.02% GTA for 30, 60, and 120 min, respectively; lane 5, 0.01% GTA for 120 min; lane 6, molecular mass markers. S, supernatant; P, pellet.

(Fig. 2B, lane 1). This product was formed in poliovirus particles incubated with GTA at 0.01% for 120 min without any other detectable reaction product (Fig. 2B, lane 5). However, when polioviruses were incubated with 0.05% GTA for 5 min, the 64-kDa product was detected with other, higher-molecular-weight products (Fig. 2A, lane 4). Furthermore, the formation of this product was clearly correlated with the decrease in the VP1 signal and, to a lesser degree, with that of the VP3 signal. Effect of GTA on echovirus type 25 capsid proteins. The effect of 0.005% GTA on echovirus type 25 was tested for 30 and 120 min (Fig. 3). After 30 min of incubation, four high-molecular-weight products were detected in the electrophoresis pattern (lane 4). The molecular weights of two of the products were estimated to be about 96 and 178 kDa, while the other two had higher molecular weights (>200 kDa). When the echovirus particles were treated for 120 min, 1 2 3 4 5 6 7 8 P S

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