JOURNAL OF VIROLOGY, Apr. 1980, p. 119-129 Vol. 34, No. 1 0022-538X/80/04-0119/11$02.00/0 Separation of Neutralizing and Hemagglutination-Inhibiting Antibody Activities and Specificity of Antisera to Sodium Dodecyl Sulfate-Derived Polypeptides of Polyoma Virionst JOSEPH B. BOLEN AND RICHARD A. CONSIGLI* Section of Virology and Oncology, Division ofBiology, Kansas State University, Manhattan, Kansas 66506 Antisera to the sodium dodecyl sulfate (SDS)-polyacrylamide gel-derived pol- yoma virion polypeptides were used in immunoprecipitation experiments with ethylene glycol-bis-N,N'-tetraacetic acid (EGTA)-dissociated polyoma virions and capsids to determine the specificity of the antipolyoma polypeptide sera. Additionally, a technique for applying "2I-labeled immunoglobulins to SDS- polyacrylamide gels was used to explore the antigenic specificities of the antisera. The results demonstrated that antisera directed against the SDS-gel-derived VP1, VP2, and VP3 did not react with native polyoma proteins, but would react with the appropriate antigens on denatured polyoma proteins. Antisera against the histone region of such gels reacted with native and denatured polyoma VP1. Separation of neutralizing antibodies from hemagglutination inhibition (HAI) antibodies to polyoma in antisera directed against the histone region of polyacryl- amide gels was done by using a polyoma capsid affinity column. The antibodies eluted from this column which did not react with capsids possessed only neutral- izing activity, whereas antibodies which bound to capsids possessed only HAI activity. These isolated immunoglobulin G fractions were then used in immuno- precipitation experiments to demonstrate that the antigenic determinants respon- sible for the HAI activity of the serum were contained on a 16,000-dalton polypeptide, whereas those antigenic determinants responsible for neutralizing activity were contained on a 14,000-dalton polypeptide. Both ofthese polypeptides present in the histone region of the SDS-gels appeared to be derived from the major virion protein VP1. In a previous report from this laboratory (11), with polyoma virions for specific binding sites antisera prepared from sodium dodecyl sulfate on the surface of mouse kidney cells (MKC) and (SDS)-polyacrylamide gel electrophoresis were unable to inhibit virion infection of MKC. (PAGE) of polyoma virion polypeptides was Capsids could, however, block virion adsorption used to demonstrate the cytoplasmic synthesis to guinea pig erythrocytes (GPRBC). Virion ad- of polyoma VP1, VP2, and VP3 and subsequent sorption to and infection of MKC was found to transport of these proteins to the nucleus of be independent of the ability of virions to agglu- infected cells. Additionally, anti-VP1, anti-VP2, tinate GPRBC (1). Since capsids lack the recep- and anti-VP3 sera did not form precipitin bands tor(s) necessary for infection but possess the with virions during immunodiffusion assays, nor moieties necessary for hemagglutination (HA), did these antisera demonstrate the capacity to we have been able to use capsids as an affinity cause hemagglutination inhibition (HAI). These matrix to separate the HAI antibodies from the antisera possessed little virus-neutralizing activ- neutralizing antibodies present in the anti- ity. Antiserum produced against the histone re- histone sera. gion of the SDS-gels, however, demonstrated This investigation was therefore directed to cytoplasmic and nuclear immunofluorescence in provide a more detailed study of the antibody polyoma-infected cells, was capable of neutral- specificities to the SDS-derived polyoma virion izing virus infectivity and inhibiting hemagglu- polypeptides and to gain insight into the anti- tination, and formed precipitin bands with viri- genic relationship between the proteins of poly- ons in immunodiffusion assays. oma virions and capsids. The data presented in A recent report from our laboratory demon- this report indicate that little, if any, antibody is strated that polyoma capsids did not compete directed against the polyoma histones. Rather, the major antigen(s) in the histone region of the t Contribution no. 80-123-J from the Kansas Agricultural gels appears to be products derived from the Experiment Station, Kansas State University. major virion protein VP1. Furthermore, this re- 119 120 BOLEN AND CONSIGLI J. VIROL. port demonstrates that HAI antibodies can be CsCl gradients and run on SDS-polyacrylamide gels separated from neutralizing antibodies in the were used as the antigen source as described previ- antihistone serum. This ability to separate these ously (11). Antisera were produced in New Zealand activities allows, for the first time, the identifi- white rabbits, and inmmunoglobulin G (IgG) was puri- of the of fied from whole serum as described previously (11). cation polypeptides polyoma virions Preparation of .25I-labeled IgG. The IgG frac- responsible for hemagglutinin and receptor func- tions of the various antisera was labeled with 125I by tions. the Enzymobead method (product information bulle- tin 1060, Bio-Rad Laboratories). Unincorporated io- MATERIALS AND METHODS dine was removed on Sephadex G-25 column chro- Cell and virus propagation. Preparation of pri- matography in 0.01 M phosphate buffer, pH 7.2. The mary cultures of MKC has been described (12). Wild- specific activities of '25I-labeled IgG fractions were 1 type small-plaque polyoma virus was used to infect x 105 to 4 x 105 cpm per,g of protein. cells at a multiplicity of infection of 10. Infected cul- Preparation of EGTA-DTT- and iodoaceta- tures were maintained in serum-free Dulbecco-modi- mide-treated "WI-labeled virion and capsid poly- fied Eagle medium (10). peptides. "nI-labeled virions and capsids adjusted to Virus purification. Virus was purified from the the same protein concentration and HA titer were infected-cell lysate as previously described (10). CsCl dissociated as described by Brady et al. (2, 3), using gradients used to purify the virus were prepared as ethylene glycol-bis-N,N'-tetraacetic acid (EGTA) and described by Brunck and Leick (4) and described in dithiothreitol (DTT), into DNA-protein complex and greater detail previously (2, 3). Empty capsids were capsomere subunits. Iodoacetamide-treated 125I-la- purified from infected-cell lysates by isolating the light beled virions and capsids were prepared by modifying virus band from cesium chloride gradients (buoyant the procedure of Kasamatsu and Flory (9). Briefly, density, 1.28 g/cm3). The capsid band was collected, "26I-labeled virions and capsids were treated with 5% diluted with Tris buffer (0.01 M, pH 7.4) to a density SDS, 10 mM Tris (pH 7.4), 5 mM EGTA, and 5 mM of 1.20 g/cm3, layered onto a four-step CsCl velocity DTT for 45 min at room temperature. The dissociated gradient, and centrifuged at 35,000 rpm for 3 h as products were sedimented through a 10 to 30% linear described previously (2). The isolated capsids were sucrose gradient (10 mM Tris, pH 7.4-5 mM EGTA- dialyzed against Tris buffer (0.01 M, pH 7.4) for 18 to 0.15 M NaCl) for 40 min at 40,000 rpm (Beckman SW- 24 h at 4°C, and then the capsids were layered onto a 50.1). The polypeptides were pooled and dialyzed seven-step CsCl velocity gradient consisting of 0.6 ml against 0.5% SDS, 10 mM Tris (pH 8.0), and 1 mM each of 1.294, 1.283, 1.272, 1.266, 1.261, 1.256, and 1.252 EGTA. DTT was added to 10 mM, the proteins were of CsCl per cm3. The gradients were centrifuged at incubated at 450C for 30 min, and iodoacetamide 35,000 rpm for 3 h. The capsid band was collected, (Sigma Chemical Co.) was added to a final concentra- diluted with Tris buffer (0.01 M, pH 7.4) to a density tion of 14 mM. The reaction was allowed to proceed in of 1.20 g/cm3, and layered onto a five-step CsCl veloc- the dark for 45 min at room temperature. The treated ity gradient consisting of0.5 ml each of 1.294 and 1.266 polypeptides were dialyzed against 0.5% Triton X-100, g of CsCl per cm3 and 0.8 ml each of 1.261, 1.254, and 10 mM Tris (pH 7.8), and 5 mM EGTA overnight. 1.252 of CsCl per cm3. The gradients were centrifuged Immunoprecipitation. EGTA-DTT- or iodoacet- at 35,000 rpm for 2.5 h. The capsid band was collected amide-treated 125I-labeled virions and capsids (50 pi) and centrifuged on a second five-step CsCl gradient as were incubated with 10 pl of the various antipolyoma described above. polypeptide IgG for 90 min at room temperature. Goat The capsid band was isolated and dialyzed at 4°C (IgG fraction) anti-rabbit IgG (Miles Laboratories, for 18 to 24 h against Tris buffer (0.01 M, pH 7.4) to Inc.) was added (50 pl), and the preparation was al- remove residual CsCl. The above scheme for purifying lowed to react for 90 min at room temperature. The empty capsids was determined to be necessary to precipitate was pelleted by centrifugation, washed ensure the removal of residual pseudovirions. Capsid three times with buffer (10 mM Tris, pH 7.8-0.25% preparations which did not undergo the extensive pu- Triton X-100-5 mM EGTA-0.15 M NaCl), and elec- rification steps above were found to be free of histones trophoresed on 15% SDS-polyacrylamide tube gels as when SDS-polyacrylamide gels were stained with Coo- described previously (11). The gels were cut into 2- or massie brilliant blue. However, when such prepara- 4-mm slices, and radioactivity was determined by liq- tions were subsequently labeled with "I and rerun on uid scintillation spectroscopy. similar gels, the developed autoradiograms demon- 1251-labeled IgG staining of polyoma polypep- strated the presence of histones. All capsid prepara- tides in SDS-polyacrylamide slab gels.