JOURNAL OF VIROLOGY, Apr. 1977, p. 113-117 Vol. 22, No. 1 Copyright ©D 1977 American Society for Microbiology Printed in U.S.A. Polyacrylamide Gel Electrophoretic Analysis of Herpes Simplex Virus Type 1 Immunoprecipitates Obtained by Quantitative Immunoelectrophoresis in -Containing Gel BODIL NORRILD* AND B. F. VESTERGAARD Institute of Medical Microbiology, University of Copenhagen, DK-2100 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 ofthe polypeptides are presented. Crossed immunoelectrophoresis in antibody- line was described previously (18). containing agarose gel has been used to charac- HSV-1 antigens. The cells were infected with a terize herpes simplex virus type 1 (HSV-1) anti- multiplicity of 10 infectious viral particles per cell, In as described previously (18). Three hours after infec- gens in infected cells (17, 18). other laborato- tion, the cells were labeled with "C-labeled ries, HSV-1 polypeptides have been extensively hydrolysate (>45 mCi/matom) at a concentration in analyzed by the use of sodium dodecyl sulfate- the maintenance medium of 3 uCi/ml, with L- polyacrylamide gel (SDS- P5S]methionine (275 Ci/mmol) at a concentration of PAGE) (4, 5, 11, 14). In these studies the HSV 10 uCi/ml, or with D-[1-'4C]glucosamine (59 mCi/ polypeptides were characterized by their molec- mmol) at a concentration of 0.5 ,uCi/ml. All isotopes ular weight, ranging from 30,000 to 260,000. were purchased at Amersham, England. The label- The number of polypeptides obtained varied, ing period was 24 h. The viral antigens were ex- depending on the polyacrylamide gel system tracted from HSV-infected cells by ultrasonic treat- ment in a 0.020 M glycine-0.0076 M tris(hydroxy- used (11, 14). The viral polypeptides present in methyl)aminomethane (Tris) buffer (pH 8.6, 25°C) infected cells were compared with the polypep- containing 5% (vol/vol) nonionic detergent Triton tides of extensively purified virus particles (7, X-100 (Serva, Heidelberg, Germany) (B. Norrild, 0. 8, 10, 13, 15), and it was found that infected cells Bjerrum, and B. F. Vestergaard, Anal. Biochem., contained both structural and nonstructural in press). The final protein concentration in the polypeptides. By using HSV-1 temperature- soluble fraction was 10 mg/ml. sensitive mutants, it has been possible during Crossed immunoelectrophoresis. This was per- infection to analyze the time of production and formed in a 1% (wt/vol) agarose gel (Litex A/S, the localization of some of the viral polypep- batch HSB no. 252, Glostrup, Denmark) in 0.18 M Tris-0.060 M barbital buffer (pH 8.6, 16°C) with 1% tides in order to clarify the function of these (vol/vol) Triton X-100, as previously described (18). substances during the virus maturation process The first-dimension electrophoresis was performed (2, 3). at 10 V/cm for 1.5 h, and the second-dimension elec- The relationship between the HSV-1 polypep- trophoresis in the antibody-containing gel was run tide pattern obtained by SDS-PAGE analysis at 2 V/cm for 18 h. The amounts of antigen used in and the pattern obtained the first-dimension electrophoresis are specified for by crossed immunoelectrophoresis has not yet the individual experiments. The second-dimen- been established. In the present paper we pres- sional gel contained 12 ,ul of a purified rabbit HSV-1 ent a method that makes it possible to obtain a antiserum per cm2 (17). Isolation of immunoprecipitates from the sec- correlation between the individual HSV-1 anti- ond-dimensional gel of the crossed immunoelectro- gens isolated as precipitates from crossed im- phoresis. Each precipitate, numbered according to munoelectrophoresis and the polypeptide struc- the anodic migration velocity of the corresponding ture of the antigenic components in the precipi- antigen (17), was cut out of the unstained gel. Con- tates. tamination from other precipitates was avoided, and identical precipitates from 15 parallel plates were MATERIALS AND METHODS pooled. The polypeptides from the precipitate-con- Cell culture. Human epidermoid carcinoma no. 2 taining agarose gel pieces were eluted by gentle (HEp-2) cells were used. The propagation ofthis cell shaking at 37°C in 0.06 M Tris-H3P04 buffer (pH 6.9) 113 114 NORRILD AND VESTERGAARD J. VIROL. with 2% (wt/vol) SDS and 2.5% (vol/vol) 2-mercapto- ethanol, as published elsewhere (Norrild et al., in 6 8 press). The eluate containing the solubilized radio- ,.. ;.v fx actively labeled polypeptides from individual HSV-1 . precipitates and the unlabeled polypeptides from ~# the were used for SDS-PAGE analysis. Ar Analytical SDS-PAGE. The discontinuous poly- 3 acrylamide gel system of Dimmock and Watson (6) was used with the modification that urea was omit- ted (Norrild et al., in press). Vertical slab gels (20 by 20 by 0.2 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 bisacryl- amide was 0.2% (wt/vol) in both gels. Marker pro- FIG. 1. Crossed immunoelectrophoretic analysis. teins for molecular weight estimations were: f- First dimension: 30 Pl of 14C-amino acid-labeled galactosidase (Boehringer), molecular weight HSV-1 antigen preparation. Migration against the 130,000; phosphorylase a (Boehringer), molecular anode to the left of the picture. Staining with weight 94,000; bovine albumin (Sigma), molecular Coomassie brilliant blue. The arrows indicate the weight 67,000; catalase (bovine liver) (Boehringer), part ofthe precipitates used in SDS-PAGE analysis. molecular weight 60,000; ovalbumin (Fluka), mo- lecular weight 45,000; and erythrocyte membrane weights in the range of 60,000 to 69,000, all , kindly supplied by 0. Bjerrum, the Pro- labeled with the same intensity, and two tein Laboratory, Copenhagen. Marker proteins (5 weakly labeled polypeptides of molecular Ag) and antigen polypeptide extracts (40 AD) were weights 115,500 and 124,000. used in each gel run. All samples were solubilized The main polypeptides of Ag-11 had molecu- in 2% (wt/vol) SDS and 2.5% (vol/vol) 2-mercapto- ethanol and heated for 3 min at 100'C. The samples lar weights of 111,000, 115,500, and 124,000 and were loaded onto the gel after mixing with sucrose a less intensively labeled polypeptide at a mo- and phenol red. The peptides obtained from a total lecular weight of 60,100. precipitate of the HSV-1 radioactively labeled anti- The main polypeptide present in Ag-6 had a gen preparation precipitated in solution with puri- molecular weight of 131,000. The more weakly fied rabbit HSV-1 antiserum were used as reference labeled polypeptide had a molecular weight of pattern. An appropriate volume of antiserum was 95,000. added to the antigen solution, and after 1 h at 37°C Ag-3 was isolated from the crossed immunoe- the precipitation was allowed to proceed for 18 h at lectrophoretic plates together with other anti- 4°C. The precipitate was collected by centrifugation at 3,000 x g for 30 min, washed twice in phosphate- gens precipitating in the same area of the gel buffered saline, and finally resuspended in 0.06 M (Fig. 1). The polypeptides found had molecular Tris-H3PO4 buffer, pH 6.9. For each gel run, 20 ,ul of weights of49,700, 60,100, 85,900, and 131,000. the reference precipitate was solubilized by the SDS-PAGE analysis of a total reference pre- addition of 2% (wt/vol) SDS and 2.5% (vol/vol) 2- cipitate with D-[1-14C]glucosamine-labeled anti- mercaptoethanol. The sample was heated and gen identified two broad bands with molecular loaded as described above. The electrophoresis was weight ranges of 60,100 to 68,900 and 111,000 to performed at a constant current of 50 mA for 9 h at 131,000, respectively (Table 1). 4°C. The gels were washed and dried. Autoradio- graphy was done as described previously (Norrild et DISCUSSION al., in press). Quantitative immunoelectrophoresis of HSV-1 antigens from infected HEp-2 cells re- RESULTS veals both HSV type-common and type-specific The HSV-1 antigens characterized by crossed immunoprecipitates (Fig. 1) (17). Ag-8, Ag-11, immunoelectrophoresis are shown in Fig. 1. and Ag-3 are type common, and Ag-6 is type 1 The following antigen-antibody precipitates specific. All four antigens are glycoproteins were isolated and solubilized as described in (18). The amount of viral antigen in immuno- Materials and Methods: Ag-8, Ag-11, Ag-6, and precipitates detectable in the wet, unstained Ag-3. Each precipitate was cut out as indicated gel is probably not less than 1 ,tg. This is calcu- by the arrows in the figure. SDS-PAGE analy- lated from crossed immunoelectrophoretic data ses of the polypeptides obtained from the indi- obtained with an antigen preparation extracted vidual precipitates are presented in Fig. 2, by nonionic detergent in a similar way as the showing the X-ray data. Nineteen different HSV antigens (1, 16). These authors electro- polypeptides were present in the total reference phorese 20 pmg of purified protein in the first precipitate, with molecular weights ranging dimension. The precipitates of the three major from 30,000 to 260,000 (Table 1). proteins, spectrin, major "intrinsic" protein, The polypeptides of Ag-8 had molecular and MN-glycoprotein, constitute 30, 24, and 7%, VOL. 22, 1977 SDS-PAGE ANALYSIS OF HSV-1 IMMUNOPRECIPITATES 115 MW x 10-3 IMMUNE PPT 8 11 6 3 IN SOLUTION 258 242

181

161 -" ___ 131 SW 124 _ 0 115.5 0 111 95 S 85.9 _m 0 81.3 78

68.99_ S 66.4 - 60.1 1 * S * U4

49.7 - 47.2 -

40.9 38.7

32.7

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 of the autoradiogram were cut out for presentation in this figure. MW, Molecular weight; ppt, precipitate.

respectively, of the total (16). The calculated present in the antigen preparation applied in amounts of antigen in these immunoprecipi- the first dimension. The total protein content in tates are 6, 5, and 1 ug, respectively. One mi- the solubilized antigen preparation was 10 mg/ crogram ofthe MN-glycoprotein gives rise to an ml, which equals 300 pig ofprotein in each first- immunoprecipitate comparable in quantity to dimensional run. In our calculation, which is the major precipitates obtained with the HSV-1 an underestimation, 1.3% of the protein in the antigen preparation. If we assume that 1 pg of antigen preparation is virus specific and precip- viral protein is present in each ofthe four major itable with HSV-1 antibodies. The polypeptide precipitates, at least 4 ug of viral protein is pattern obtained by SDS-PAGE analysis of the 116 NORRILD AND VESTERGAARD J. VIROL. TABLE 1. Polypeptide pattern of individual antigens induced by HSV-1 infection ofHEp-2 cells (molecular weight x 10-3) 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-3 275 258C 260 242c 184 181 155 161 146 126 (glycoprotein) 131(glycoprotein) 131 131 124 (glycoprotein) 124 124 112 115.5 (glycoprotein) 115.5 115.5 111 (glycoprotein) 111 98 93 95 95 87 85.9 85.9 81.3 78 78 71 68.9 (glycoprotein) 68.9 65 66.4 (glycoprotein) 66.4 59 (glycoprotein) 60.1 (glycoprotein) 60.1 60.1 60.1 57 53 50 49.7 49.7 47.2 44 40.9 37 38.7 33 32.7 25 a From Spear and Roizman (15). bMean values of four estimations. c Molecular weight estimations unsure above 200,000. individual precipitates shows that each antigen in the glucosamine-labeled antigen preparation contains different polypeptide chains (Fig. 2). (Table 1). We expect that at least this polypep- The molecular weights of the polypeptides are tide of Ag-6 is glycosylated. It might be identi- summarized in Table 1 for each of the isolated cal with the glycosylated polypeptide (molecu- antigens. lar weight 130,000) reported by Honess and We do not yet know whether all or only some Roizman (9) in HSV-1-infected cells and identi- of the polypeptides isolated from individual an- cal with the major virion envelope glycoprotein tigens are glycosylated. The D-[1-14C]glucosa- of molecular weight 123,000 and 126,000, as mine-labeled HSV-1 total antigen precipitated published by Courtney and Powell (5) and by in solution with HSV-1 antiserum contained Spear and Roizman (15). Monospecific anti- glycosylated polypeptides of molecular weights serum to Ag-6 (B. F. Vestergaard, manuscript that agreed with the data of Honess and Roiz- in preparation) neutralizes HSV-1 but not man (9) on HSV-1-infected HEp-2 cells (Table HSV-2. These results are in good agreement 1). These authors reported viral specific glyco- with the data published on the type-specific sylated polypeptides with molecular weights of neutralization obtained with serum prepared 45,000, 60,000, and 130,000. In purified HSV-1 against the major glycoprotein 123,000 (5). The virions, glycosylated polypeptides with molecu- Ag-3 is known to be contaminated with other lar weights in the range of 57,000 to 62,000, antigens (Fig. 1). The polypeptide pattern ob- 69,000 to 70,000, and 115,000 to 126,000 have tained showed four compounds, of which only been reported (7, 15). the 60,100 and 131,000 polypeptides could be All the polypeptides of Ag-8 and Ag-11 are glycosylated (Table 1). At least one of these probably glycosylated (Table 1). The major poly- must therefore belong to the Ag-3 precipitate. peptide of the HSV-1-specific antigen Ag-6 In conclusion, it must be noted that the poly- (molecular weight 131,000) has its counterpart peptide structure of the viral antigens de- VOL. 22, 1977 SDS-PAGE ANALYSIS OF HSV-1 IMMUNOPRECIPITATES 117 scribed above might be a minimal composition. 6. Dimmock, N. J., and D. H. Watson. 1969. Proteins specified by influenza virus in infected cells: analysis A higher specific activity of the antigen prepa- by polyacrylamide of antigens not ration could possibly resolve additional poly- present in the virus particle. J. Gen. Virol. 5:499-509. peptides. However, SDS-PAGE analysis of L- 7. Heine, J. W., R. W. Honess, E. Cassai, and B. Roiz- P5S]methionine-labeled gave results man. 1974. Proteins specified by herpes simplex vi- antigens rus. XII. The virion polypeptides of type 1 strains. J. identical to that of 'IC-amino acid-labeled anti- Virol. 14:640-651. gens. The data of Spear and Roizman (15) are 8. Heine, J:, W., P. G. Spear, and B. Roizman. 1972. included in Table 1 in order to compare our Proteins specified by herpes simplex virus. VI. Viral reference peptide pattern to their data on puri- proteins in the plasma membrane. J. Virol. 9:431- 439. fied virions. The polypeptides are listed only on 9. Honess, R. W., and B. Roizman. 1975. Proteins speci- the basis of molecular weight. Experiments are fied by herpes simplex virus. XIII. Glycosylation of in progress for the analysis of the glycosylation viral polypeptides. J. Virol. 16:1308-1326. of the polypeptides isolated from Ag-8, Ag-11, 10. Honess, R. W., and D. H. Watson. 1974. Herpes sim- plex virus-specific polypeptides studied by polyacryl- Ag-6, and Ag-3. amide gel electrophoresis of immune precipitates. J. Gen. Virol. 22:171-185. ACKNOWLEDGMENTS 11. McCombs, R. M. 1974. Antigens specified by herpesvi- We would like to thank Merete L6ff and Helle Arpe for ruses. III. Viral-induced nuclear polypeptides. Virol- excellent technical assistance, and J. Forchhammer and 0. ogy 57:448-458. Bjerrum for technical advice and stimulating discussions. 12. Powell, K. L., A. Buchan, C. Sim, and D. H. Watson. This work was supported by the Danish Cancer Society. 1974. Type-specific protein in herpes simplex virus envelope reacts with neutralizing antibody. Nature (London) 249:360-361. LITERATURE CITED 13. Powell, K. L., and D. H. Watson. 1975. Some structural 1. Bjerrum, 0. J., and T. C. Beg-Hansen. 1976: The im- antigens ofherpes simplex virus type 1. J. Gen. Virol. munonological approach to the characterization of 29:167-178. membrane proteins. Human erythrocyte membrane 14. Roizman, B., G. Hayward, R. Jacob, S. Wadsworth, N. proteins analyzed as a model system. Biochim. Bio- Frenkel, R. W. Honess, and M. Kozak. 1975. Human phys. Acta 455:66-89. herpesviruses I: a model for molecular organization 2. Bone, D. R., and R. J. Courtney. 1974. A temperature- and regulation ofherpesviruses-a review, p. 3-38. In sensitive mutant ofherpes simplex virus type 1 defec- G. de-The, M. A. Epstein, and H. zur Hausen (ed.), tive in the synthesis of the major capsid polypeptide. Oncogenesis and herpesviruses II. Iarc Noll, Lyon. J. Gen. Virol. 24:17-27. 15. Spear, P. G., and B. Roizman. 1972. Proteins specified 3. Courtney, R. J., and M. Benyesh-Melnick. 1974. Isola- by herpes simplex virus. V. Purification and struc- tion and characterization of a large molecular-weight tural proteins ofthe herpesvirion. J. Virol. 9:143-159. polypeptide of herpes simplex virus type 1. Virology 16. Steck, T. L. 1974. The molecular organization of the 62:539-551. proteins in the human red blood cell membrane. J. 4. Courtney, R. J., R. M. McCombs, and M. Benyesh- Cell Biol. 62:1-19. Melnick. 1971. Antigens specified by herpesviruses. 17. Vestergaard, B. F. 1973. Crossed immunoelectropho- II. Effect of arginine deprivation on the synthesis of retic characterization of herpesvirus homonis type 1 cytoplasmic and nuclear proteins. Virology 43:356- and 2 antigens. Acta Pathol. Microbiol. Scand. Sect. 365. B 81:808-810. 5. Courtney, R. J., and K. L. Powell. 1975. Immunological 18. Vestergaard, B. F., and T. C. Bog-Hansen. 1975. Detec- and biochemical characterization of polypeptides in- tion of -binding herpes simplex virus duced by herpes simplex virus types 1 and 2, p. 63-73. type 1 and type 2 antigens by crossed immuno-affi- In G. de-The, M. A. Epstein, and H. zur Hausen (ed.), noelectrophoresis. Scand. J. Immunol. 4(Suppl. 2): Oncogenesis and herpesviruses II. Iarc Noll, Lyon. 211-215.