Agarose Gel. as described previously (18). Three hours after infection, 10 uci/ml, or with D-[1-'4C]glucosamine (59 mci/

Transcription

1 JOURNAL OF VIROLOGY, Apr. 1977, p Copyright D 1977 American Society for Microbiology Vol. 22, No. 1 Printed in U.S.A. Polyacrylamide Gel Electrophoretic Analysis of Herpes Simplex Virus Type 1 Immunoprecipitates Obtained by Quantitative Immunoelectrophoresis in Antibody-Containing Agarose Gel BODIL NORRILD* AND B. F. VESTERGAARD Institute of Medical Microbiology, University of Copenhagen, DK-21 Copenhagen, Denmark Received for publication 24 August 1976 Crossed immunoelectrophoresis was used to characterize herpes simplex virus type 1 (HSV-1) antigens produced by infected HEp-2 cells. We report on a method for analyzing the polypeptide content in individual antigen-antibody precipitates eluted from the second-dimensional agarose gel. Four glycoprotein antigens of HSV-1, Ag-8, Ag-11, Ag-6, and Ag-3, were isolated and analyzed for polypeptide content. The molecular weights of the polypeptides are presented. Crossed immunoelectrophoresis in antibodycontaining agarose gel has been used to characterize herpes simplex virus type 1 (HSV-1) antigens in infected cells (17, 18). In other laboratories, HSV-1 polypeptides have been extensively analyzed by the use of sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS- PAGE) (4, 5, 11, 14). In these studies the HSV polypeptides were characterized by their molecular weight, ranging from 3, to 26,. The number of polypeptides obtained varied, depending on the polyacrylamide gel system used (11, 14). The viral polypeptides present in infected cells were compared with the polypeptides of extensively purified virus particles (7, 8, 1, 13, 15), and it was found that infected cells contained both structural and nonstructural polypeptides. By using HSV-1 temperaturesensitive mutants, it has been possible during infection to analyze the time of production and the localization of some of the viral polypeptides in order to clarify the function of these substances during the virus maturation process (2, 3). The relationship between the HSV-1 polypeptide pattern obtained by SDS-PAGE analysis and the immunoprecipitation pattern obtained by crossed immunoelectrophoresis has not yet been established. In the present paper we present a method that makes it possible to obtain a correlation between the individual HSV-1 antigens isolated as precipitates from crossed immunoelectrophoresis and the polypeptide structure of the antigenic components in the precipitates. MATERIALS AND METHODS Cell culture. Human epidermoid carcinoma no. 2 (HEp-2) cells were used. The propagation of this cell line was described previously (18). HSV-1 antigens. The cells were infected with a multiplicity of 1 infectious viral particles per cell, as described previously (18). Three hours after infection, the cells were labeled with "C-labeled protein hydrolysate (>45 mci/matom) at a concentration in the maintenance medium of 3 uci/ml, with L- P5S]methionine (275 Ci/mmol) at a concentration of 1 uci/ml, or with D-[1-'4C]glucosamine (59 mci/ mmol) at a concentration of.5,uci/ml. All isotopes were purchased at Amersham, England. The labeling period was 24 h. The viral antigens were extracted from HSV-infected cells by ultrasonic treatment in a.2 M glycine-.76 M tris(hydroxymethyl)aminomethane (Tris) buffer (ph 8.6, 25 C) 113 containing 5% (vol/vol) nonionic detergent Triton X-1 (Serva, Heidelberg, Germany) (B. Norrild,. Bjerrum, and B. F. Vestergaard, Anal. Biochem., in press). The final protein concentration in the soluble fraction was 1 mg/ml. Crossed immunoelectrophoresis. This was performed in a 1% (wt/vol) agarose gel (Litex A/S, batch HSB no. 252, Glostrup, Denmark) in.18 M Tris-.6 M barbital buffer (ph 8.6, 16 C) with 1% (vol/vol) Triton X-1, as previously described (18). The first-dimension electrophoresis was performed at 1 V/cm for 1.5 h, and the second-dimension electrophoresis in the antibody-containing gel was run at 2 V/cm for 18 h. The amounts of antigen used in the first-dimension electrophoresis are specified for the individual experiments. The second-dimensional gel contained 12,ul of a purified rabbit HSV-1 antiserum per cm2 (17). Isolation of immunoprecipitates from the second-dimensional gel of the crossed immunoelectrophoresis. Each precipitate, numbered according to the anodic migration velocity of the corresponding antigen (17), was cut out of the unstained gel. Contamination from other precipitates was avoided, and identical precipitates from 15 parallel plates were pooled. The polypeptides from the precipitate-containing agarose gel pieces were eluted by gentle shaking at 37 C in.6 M Tris-H3P4 buffer (ph 6.9)

2 114 NORRILD AND VESTERGAARD with 2% (wt/vol) SDS and 2.5% (vol/vol) 2-mercaptoethanol, as published elsewhere (Norrild et al., in press). The eluate containing the solubilized radioactively labeled polypeptides from individual HSV-1 precipitates and the unlabeled polypeptides from the antibodies were used for SDS-PAGE analysis. Analytical SDS-PAGE. The discontinuous polyacrylamide gel system of Dimmock and Watson (6) was used with the modification that urea was omitted (Norrild et al., in press). Vertical slab gels (2 by 2 by.2 cm) were run with a 1-cm stacking gel of 3.5% (wt/vol) acrylamide and a separation gel of 7.5% (wt/vol) acrylamide. The content of bisacrylamide was.2% (wt/vol) in both gels. Marker proteins for molecular weight estimations were: f- galactosidase (Boehringer), molecular weight 13,; phosphorylase a (Boehringer), molecular weight 94,; bovine albumin (Sigma), molecular weight 67,; catalase (bovine liver) (Boehringer), molecular weight 6,; ovalbumin (Fluka), molecular weight 45,; and erythrocyte membrane proteins, kindly supplied by. Bjerrum, the Protein Laboratory, Copenhagen. Marker proteins (5 Ag) and antigen polypeptide extracts (4 AD) were used in each gel run. All samples were solubilized in 2% (wt/vol) SDS and 2.5% (vol/vol) 2-mercaptoethanol and heated for 3 min at 1'C. The samples were loaded onto the gel after mixing with sucrose and phenol red. The peptides obtained from a total precipitate of the HSV-1 radioactively labeled antigen preparation precipitated in solution with purified rabbit HSV-1 antiserum were used as reference pattern. An appropriate volume of antiserum was added to the antigen solution, and after 1 h at 37 C the precipitation was allowed to proceed for 18 h at 4 C. The precipitate was collected by centrifugation at 3, x g for 3 min, washed twice in phosphatebuffered saline, and finally resuspended in.6 M Tris-H3PO4 buffer, ph 6.9. For each gel run, 2,ul of the reference precipitate was solubilized by the addition of 2% (wt/vol) SDS and 2.5% (vol/vol) 2- mercaptoethanol. The sample was heated and loaded as described above. The electrophoresis was performed at a constant current of 5 ma for 9 h at 4 C. The gels were washed and dried. Autoradiography was done as described previously (Norrild et al., in press). RESULTS The HSV-1 antigens characterized by crossed immunoelectrophoresis are shown in Fig. 1. The following antigen-antibody precipitates were isolated and solubilized as described in Materials and Methods: Ag-8, Ag-11, Ag-6, and Ag-3. Each precipitate was cut out as indicated by the arrows in the figure. SDS-PAGE analyses of the polypeptides obtained from the individual precipitates are presented in Fig. 2, showing the X-ray data. Nineteen different polypeptides were present in the total reference precipitate, with molecular weights ranging from 3, to 26, (Table 1). The polypeptides of Ag-8 had molecular ,.. v ;. fx ~# J. VIROL. FIG. 1. Crossed immunoelectrophoretic analysis. First dimension: 3 Pl of 14C-amino acid-labeled HSV-1 antigen preparation. Migration against the anode to the left of the picture. Staining with Coomassie brilliant blue. The arrows indicate the part of the precipitates used in SDS-PAGE analysis. weights in the range of 6, to 69,, all labeled with the same intensity, and two weakly labeled polypeptides of molecular weights 115,5 and 124,. The main polypeptides of Ag-11 had molecular weights of 111,, 115,5, and 124, and a less intensively labeled polypeptide at a molecular weight of 6,1. The main polypeptide present in Ag-6 had a molecular weight of 131,. The more weakly labeled polypeptide had a molecular weight of 95,. Ag-3 was isolated from the crossed immunoelectrophoretic plates together with other antigens precipitating in the same area of the gel (Fig. 1). The polypeptides found had molecular weights of49,7, 6,1, 85,9, and 131,. SDS-PAGE analysis of a total reference precipitate with D-[1-14C]glucosamine-labeled antigen identified two broad bands with molecular weight ranges of 6,1 to 68,9 and 111, to 131,, respectively (Table 1). DISCUSSION Quantitative immunoelectrophoresis of HSV-1 antigens from infected HEp-2 cells reveals both HSV type-common and type-specific immunoprecipitates (Fig. 1) (17). Ag-8, Ag-11, and Ag-3 are type common, and Ag-6 is type 1 specific. All four antigens are glycoproteins (18). The amount of viral antigen in immunoprecipitates detectable in the wet, unstained gel is probably not less than 1,tg. This is calculated from crossed immunoelectrophoretic data obtained with an antigen preparation extracted by nonionic detergent in a similar way as the HSV antigens (1, 16). These authors electrophorese 2 pmg of purified protein in the first dimension. The precipitates of the three major proteins, spectrin, major "intrinsic" protein, and MN-glycoprotein, constitute 3, 24, and 7%, Ar

3 VOL. 22, 1977 MW x 1-3 IMMUNE PPT IN SOLUTION SDS-PAGE ANALYSIS OF HSV-1 IMMUNOPRECIPITATES " _ _m _ SW FIG. 2. Autoradiogram ofan SDS-PAGE slab showing the polypeptides present in the individual antigens Ag-8, Ag-li, Ag-6, and Ag-3. The individual polypeptides were identified by comparison to the total precipitated HSV-1 polypeptide pattern used as a reference. All samples were analyzed by electrophoresis on the same gel slab, but only the relevant tracks ofthe autoradiogram were cut out for presentation in this figure. MW, Molecular weight; ppt, precipitate. S S * S * 4U respectively, of the total (16). The calculated amounts of antigen in these immunoprecipitates are 6, 5, and 1 ug, respectively. One microgram of the MN-glycoprotein gives rise to an immunoprecipitate comparable in quantity to the major precipitates obtained with the HSV-1 antigen preparation. If we assume that 1 pg of viral protein is present in each ofthe four major precipitates, at least 4 ug of viral protein is present in the antigen preparation applied in the first dimension. The total protein content in the solubilized antigen preparation was 1 mg/ ml, which equals 3 pig of protein in each firstdimensional run. In our calculation, which is an underestimation, 1.3% of the protein in the antigen preparation is virus specific and precipitable with HSV-1 antibodies. The polypeptide pattern obtained by SDS-PAGE analysis of the

4 116 NORRILD AND VESTERGAARD TABLE 1. Polypeptide pattern of individual antigens induced by HSV-1 infection of HEp-2 cells (molecular weight x 1-3) Precipitates from quantitative Data on purified HSV-1 Precipitation in solution of total immunoelectrophoresis virionsa HSV-1 antigen Ag-8 Ag-11 Ag-6 Ag C c (glycoprotein) 131(glycoprotein) (glycoprotein) (glycoprotein) (glycoprotein) (glycoprotein) (glycoprotein) (glycoprotein) 6.1 (glycoprotein) a From Spear and Roizman (15). bmean values of four estimations. c Molecular weight estimations unsure above 2,. individual precipitates shows that each antigen contains different polypeptide chains (Fig. 2). The molecular weights of the polypeptides are summarized in Table 1 for each of the isolated antigens. We do not yet know whether all or only some of the polypeptides isolated from individual antigens are glycosylated. The D-[1-14C]glucosamine-labeled HSV-1 total antigen precipitated in solution with HSV-1 antiserum contained glycosylated polypeptides of molecular weights that agreed with the data of Honess and Roizman (9) on HSV-1-infected HEp-2 cells (Table 1). These authors reported viral specific glycosylated polypeptides with molecular weights of 45,, 6,, and 13,. In purified HSV-1 virions, glycosylated polypeptides with molecular weights in the range of 57, to 62,, 69, to 7,, and 115, to 126, have been reported (7, 15). All the polypeptides of Ag-8 and Ag-11 are probably glycosylated (Table 1). The major polypeptide of the HSV-1-specific antigen Ag-6 (molecular weight 131,) has its counterpart J. VIROL. in the glucosamine-labeled antigen preparation (Table 1). We expect that at least this polypeptide of Ag-6 is glycosylated. It might be identical with the glycosylated polypeptide (molecular weight 13,) reported by Honess and Roizman (9) in HSV-1-infected cells and identical with the major virion envelope glycoprotein of molecular weight 123, and 126,, as published by Courtney and Powell (5) and by Spear and Roizman (15). Monospecific antiserum to Ag-6 (B. F. Vestergaard, manuscript in preparation) neutralizes HSV-1 but not HSV-2. These results are in good agreement with the data published on the type-specific neutralization obtained with serum prepared against the major glycoprotein 123, (5). The Ag-3 is known to be contaminated with other antigens (Fig. 1). The polypeptide pattern obtained showed four compounds, of which only the 6,1 and 131, polypeptides could be glycosylated (Table 1). At least one of these must therefore belong to the Ag-3 precipitate. In conclusion, it must be noted that the polypeptide structure of the viral antigens de-

5 VOL. 22, 1977 scribed above might be a minimal composition. A higher specific activity of the antigen preparation could possibly resolve additional polypeptides. However, SDS-PAGE analysis of L- P5S]methionine-labeled antigens gave results identical to that of 'IC-amino acid-labeled antigens. The data of Spear and Roizman (15) are included in Table 1 in order to compare our reference peptide pattern to their data on purified virions. The polypeptides are listed only on the basis of molecular weight. Experiments are in progress for the analysis of the glycosylation of the polypeptides isolated from Ag-8, Ag-11, Ag-6, and Ag-3. ACKNOWLEDGMENTS We would like to thank Merete L6ff and Helle Arpe for excellent technical assistance, and J. Forchhammer and. Bjerrum for technical advice and stimulating discussions. This work was supported by the Danish Cancer Society. LITERATURE CITED 1. Bjerrum,. J., and T. C. Beg-Hansen. 1976: The immunonological approach to the characterization of membrane proteins. Human erythrocyte membrane proteins analyzed as a model system. Biochim. Biophys. Acta 455: Bone, D. R., and R. J. Courtney A temperaturesensitive mutant ofherpes simplex virus type 1 defective in the synthesis of the major capsid polypeptide. J. Gen. Virol. 24: Courtney, R. J., and M. Benyesh-Melnick Isolation and characterization of a large molecular-weight polypeptide of herpes simplex virus type 1. Virology 62: Courtney, R. J., R. M. McCombs, and M. Benyesh- Melnick Antigens specified by herpesviruses. II. Effect of arginine deprivation on the synthesis of cytoplasmic and nuclear proteins. Virology 43: Courtney, R. J., and K. L. Powell Immunological and biochemical characterization of polypeptides induced by herpes simplex virus types 1 and 2, p In G. de-the, M. A. Epstein, and H. zur Hausen (ed.), Oncogenesis and herpesviruses II. Iarc Noll, Lyon. SDS-PAGE ANALYSIS OF HSV-1 IMMUNOPRECIPITATES Dimmock, N. J., and D. H. Watson Proteins specified by influenza virus in infected cells: analysis by polyacrylamide gel electrophoresis of antigens not present in the virus particle. J. Gen. Virol. 5: Heine, J. W., R. W. Honess, E. Cassai, and B. Roizman Proteins specified by herpes simplex virus. XII. The virion polypeptides of type 1 strains. J. Virol. 14: Heine, J:, W., P. G. Spear, and B. Roizman Proteins specified by herpes simplex virus. VI. Viral proteins in the plasma membrane. J. Virol. 9: Honess, R. W., and B. Roizman Proteins specified by herpes simplex virus. XIII. Glycosylation of viral polypeptides. J. Virol. 16: Honess, R. W., and D. H. Watson Herpes simplex virus-specific polypeptides studied by polyacrylamide gel electrophoresis of immune precipitates. J. Gen. Virol. 22: McCombs, R. M Antigens specified by herpesviruses. III. Viral-induced nuclear polypeptides. Virology 57: Powell, K. L., A. Buchan, C. Sim, and D. H. Watson Type-specific protein in herpes simplex virus envelope reacts with neutralizing antibody. Nature (London) 249: Powell, K. L., and D. H. Watson Some structural antigens ofherpes simplex virus type 1. J. Gen. Virol. 29: Roizman, B., G. Hayward, R. Jacob, S. Wadsworth, N. Frenkel, R. W. Honess, and M. Kozak Human herpesviruses I: a model for molecular organization and regulation ofherpesviruses-a review, p In G. de-the, M. A. Epstein, and H. zur Hausen (ed.), Oncogenesis and herpesviruses II. Iarc Noll, Lyon. 15. Spear, P. G., and B. Roizman Proteins specified by herpes simplex virus. V. Purification and structural proteins of the herpesvirion. J. Virol. 9: Steck, T. L The molecular organization of the proteins in the human red blood cell membrane. J. Cell Biol. 62: Vestergaard, B. F Crossed immunoelectrophoretic characterization of herpesvirus homonis type 1 and 2 antigens. Acta Pathol. Microbiol. Scand. Sect. B 81: Vestergaard, B. F., and T. C. Bog-Hansen Detection of concanavalin A-binding herpes simplex virus type 1 and type 2 antigens by crossed immuno-affinoelectrophoresis. Scand. J. Immunol. 4(Suppl. 2):