WIESTi-E LlfiflA RY flfiehigan State University This is to certify that the thesis entitled MOLECULAR AND IMMUNOLOGIC CHARACTERIZATION OF MAREK'S DISEASE HERPESVIRUS ANTIGENS presented by Carol Glaubiger has been accepted towards fulfillment of the requirements for Magi-£12.5— degree in Microbiology WVWw Major professor Date 7/2 12/” / 0-7 639 OVERDUE FINES: "25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to remove charge from circulation records .. sf T"; “x (1 i’l-¥\\\\‘ L MOLECULAR AND IMMUNOLOGIC CHARACTERIZATION OF MAREK'S DISEASE HERPESVIRUS ANTIGENS BY Carol Glaubiger A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1981 ABSTRACT MOLECULAR AND IMMUNOLOGIC CHARACTERIZATION OF MAREK'S DISEASE HERPESVIRUS ANTIGENS BY Carol Glaubiger The identification of Marek's disease herpesvirus (MDHV) A and B antigens was attempted by S.aureus immuno- precipitation followed by polyacrylamide gel electr0phore- sis in the presence of sodium dodecyl sulfate (SDS-PAGE). Using MDHV-A antigen reactive sera and a highly purified MDHV—A antigen preparation (49), MDHV-A antigen was conclusively identified as a glyc0protein with an approximate molecular weight of 61-65,000. An antigen of apparently identical molecular weight was also demonstrated in the culture medium of herpesvirus of turkeys (HVT)-infected cells. Identification of MDHV-B antigen from infected cell lysates by immunOprecipation with MDHV-B antigen reactive sera (106) was inconclusive. 35S-methionine labeling resulted in the immunOprecipita- tion of virus-specific polypeptides of 83,000 and 150,000 14 daltons while C-glucosamine labeling resulted in the immun0precipitation of only a 230,000 dalton molecule. ACKNOWLEDGMENTS My sincere appreciation and thanks goes to Dr. Leland F. Velicer for his guidance and encouragement. I would also like to thank the members of my guidance committee, Dr. Keyvan Nazerian and Dr. Maria Patterson, for their helpful suggestions. A special appreciation goes to the members of my laboratory, fellow graduate students, and numerous others, who provided me with advice, assistance, and support during the course of this study. I would also like to express my gratitude and love to my parents, for their patience and encouragement. I would like to acknowledge the financial support of the Department of Microbiology and Public Health and NIH grant CA-23408, which made it possible for me to ‘ pursue this degree. ii TABLE LIST OF TABLES . . . . LIST OF FIGURES . . . INTRODUCTION . . . . LITERATURE REVIEW . . . Pathogensis of MD . Properties of MDHV . MDHV-Induced Antigens Resistance to MD . . Host Immune Response to MDHV Vaccinal Immunity . MATERIALS AND METHODS . Cells and Virus . . Radioactive Labeling Preparation of Labeled Culture Cell Lysates for Immunoprecipitation Radioactivity Assays Protein Determinations OF CONTENTS I Media and Antisera for ImmunOprecipitations ImmunOprecipitation SDS-PAGE and Fluorography . RESULTS Analysis of 35 Media . . . . . ImmunOprecipitation Analysis of Culture 35S-methionine Labeled Cells Medium of Using RoA . . . ImmunOprecipitation Analysis of Culture 358-methionine Labeled Cells Medium of Using ICS . . . iii S-methionine and 14C mine Labeled Polypeptides Found in Culture nfection -glucosa- Page 37 40 43 Page ImmunOprecipitation Analysis of Culture Medium of 14C-glucosamine Labeled Cells. . 46 ImmunOprecipitation Analysis of Culture Medium of HVT-Infected Cells . . . . . 46 Analysis of 358-methionine and 14C- glucosamine Labeled Polypeptides of Cell Lysates . . . . . . . 51 ImmunOprecipitation Analysis of 35S- methionine Labeled Cell Lysates with Sera Reactive with B Antigen . . . . 56 ImmunOprecipitation Analysis of 14C-glucosa- mine Labeled Cell Lysates with Sera Reactive with B Antigen . . . . 60 ImmunoPrecipitation Analysis of Cell Lysates of MDHV and HVT-infected Cells with Sera Reactive with B Antigen . . . . . . . 60 DISCUSSION . . . . . . . . . . . . . 66 APPENDIX . . . . . ‘. . . . . . . . . 82 REFERENCES . . . . . . . . . . . . . 90 iv LIST OF TABLES Table Page 1. Published Comparison of MDHV-A and MDHV-B Antigens. . . . . . . . . . 11 Figure 1. LIST OF FIGURES Analysis of culture media from 35$- methionine and 14C-glucosamine labeled, MDHV-infected (INF) and uninfected (CON) cells 0 O O O O O I I O O O O O Immun0preci§itation analysis of culture media from 55-methionine labeled, MDHV- infected (INF) and uninfected (CON) cells with rabbit anti-A serum (RaA). . . . . ImmunOprecipitation analysis of culture media from 58—methionine labeled, MDHV- infected (INF) and uninfected (CON) cells with immune serum from naturally infected chickens (ICS) . . . . . . . . . . Immun0precipitation analysis of culture media from 4C-glucosamine labeled, MDHV— infected (INF) and uninfected (CON) cells with rabbit anti-A serum (RaA). . . . . ImmunOprecipitation analysis of culture media from 4C-glucosamine labeled, MDHV— infected (INF) and uninfected (CON) cells with immune serum from naturally infected chickens (ICS) . . . . . . . . . . ImmunOprecipitation analysis of culture medium from 355-methionine labeled, HVT- infected cell . . . . . . . . . . Analysis of detergent lysates from 35$- methionine and l4C-glucosamine labeled, MDHV-infected (INF) and uninfected (CON) cells . . . . . . . . . . . . ImmunOprecipitation analysis of detergent lysates from 35S-methionine labeled, MDHV- infected (INF) and uninfected (CON) cells with sera reactive with B antigen. . . . vi Page 38 41 44 47 49 52 54 57 Figure 9. lo. 11. 12. Page Immun0precipitation analysis of detergent lysates from 4C-glucosamine labeled, MDHV-infected (INF) and uninfected (CON) cells with sera reactive with B antigen. . 61 Immun0precipitation analysis of 35$- methionine labeled lysates from HVT- infected cells with sera reactive with B antigen. . . . . . . . . . . . 63 Con A affinity chromatography of 35$— methionine labeled cell sonicates. . . . 86 Isoelectric focusing analysis . . . . . 88 vii INTRODUCTION Marek's disease (MD) is a common neOplastic disease of chickens. Until the early 1960's, MD was classified with other neOplastic diseases of chickens as part of the avian leukosis complex. Careful clinical studies by Campbell (3) and Biggs (4) separated the complex into two diseases: Marek's disease which was later shown to be caused by Marek's disease herpesvirus (MDHV) (17,18,62); and lymphoid leukosis which has an RNA tumor virus as the etiologic agent (61). Until it was brought under control by vaccination in the early 1970's, MD was a major cause of economic loss to the poultry industry. In the United States alone, annual losses due to MD by the late 1960's were estimated at about 200 million dollars (75). The antigens of the MD system have been defined by various immunological methods. The immunofluorescent technique has identified the membrane antigens of the productive infection (2,15,31,65,?8,105), and the tumor associated antigen of the transformed state (54,56,59, 66,92,111). Immunodiffusion analysis has identified the predominantly extracellular A antigen, and the predominantly cell-associated B antigen, both character- istic of the productive infection. Although the nature of the host immune response to MD has been extensively studied, the relatedness of the antigens defined by different immunological methods remains unknown. Also, the relationship of these antigens to the mature virion, their location in the infected cell, and their role in the disease process still remains to be elucidated. The objective of this thesis was to further the immunologic and molecular characterization of the A and B antigens on polyacryamide gel electrOphoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) following S.aureus immunOprecipitation. The physical and chemical prOperties of these antigens have been extensively studied (49,50,106) and the ability to further study these antigens was dependent upon the availability of previously prepared, highly purified antigens and mono- specific antisera (49,106). The identification of the A and B antigens on SDS-PAGE will aid in the understanding of the relation- ship of these antigens to the MD syndrome, and will additionally serve as a foundation for further molecular studies of the carbohydrate and polypeptide portions of these antigens and future analysis of the MDHV genome. LITERATURE REVIEW Pathogenesis of MD ~MD is characterized by the appearance of lymphoid tumors in viscera, muscle, and skin, as well as infiltration of lymphoid cells in peripheral nerves (63). Marek (52) first characterized the classical form of MD in 1907, which presents itself as a paralysis of the wings and legs with gross enlargements of the periph- eral nerves. The acute form of MD, described by Biggs, et al. (5), has a high inCidence of visceral lymphoid tumors and a high incidence of mortality. Lymphomas are composed of a heterogeneous combination of cells including small and medium lymphocytes, mature blast- type cells with abundant basoPhilic cytoplasm and large nuclei, and plasma cells (72). The target cell for trans- formation is the T-lymphocyte, since most cells present in lymphomas are T-cells (28,89,99) and cell lines deve10ped from MD lymphomas are T-cell lines (66,68,75). PrOperties of MDHV The causative agent of MD was first isolated in cell cultures from tumor cells and identified as a group B herpesvirus (17,18,60). MDHV displays a typical herpesvirus morphology. The nucleocapsid is about 100 nm and has an icosahedral symmetry with 162 hollow centered capsomeres. A herpesvirus isolated from normal turkeys, named herpesvirus of turkeys (HVT), was found to be morphologically and antigenically related to MDHV but apathogenic for both chickens and turkeys (34,87,112). Vaccination of chickens with HVT was highly protective against the deveIOpment of MDHV-induced lymphomas (68) and HVT and MDHV appeared to share immunologically related antigens (21,87,106,109). The DNA of both MDHV and HVT is 3 daltons. It has a a double stranded molecule of l x 10 bouyant density in CsCl of 1.706 g/cm3 which corre5ponds to a guanine/cytosine content of 46% (44). Recent DNA homology studies indicate that there is apparently only a 2-5% homology between the two viral DNAs (26,35,47). A 2% DNA homology could allow for the production of only 2-3 HVT and MDHV common antigens with an average molecular weight of 5 x 104 daltons (26). Analysis by Chen, et a1. (14) of partially puri— fied MDHV virions by SDS-PAGE revealed at least 8 polypep— tides ranging in molecular weight from 110 x 103 to 155 x 103. Three polypeptides comprised about 50% of the total protein and two polypeptides could be labeled with glucosamine and were apparently associated with the viral enveloPe since pre-treatment of virions with Nonidet P-40 eliminated these proteins. The relation- ship of these proteins to the MDHV antigens defined by various serological tests was not established. Two types of virus-cell interactions can occur after infection with MDHV. Productive interaction results in the production of virus nucleic acid, virus proteins, sometimes virus particles, and cell death (59). Infection of feather follicles can result in fully enveloped and infectious virus (10), while infection of other cells of the chicken as well as tissue culture cells may result in the production of only virus specific antigens and noninfectious particles (1,21,60). Nonproductive interaction is encountered in lymphoid cells and can lead to cell proliferation and tumor formation. Virus DNA may be present in multiple cepies in these cells, but the expression of viral DNA is rarely observed (62,64). Evidence exists for the integration of viral DNA into host DNA (36). MDHV - transformed lymphoblastoid cell lines have been established (3,12,67,75) and all lymphoblastoid cell lines are of the T—cell type and carry a membrane associated tumor Specific antigen (MATSA) (54,56,59,66, 92,111). MDHV—Induced Antigens Several antigens have been detected in MDHV- infected cells using various serological tests. The agar-gel immunodiffusion test has revealed the presence of a number of precipitating antigens. Chubb,et a1. (16) initially detected only one precipitin line, although in later work (19) three major antigens were detected. The major antigen present in culture fluid of infected cells was designated A antigen and the two antigens detected in infected cell extracts were designated B and C antigens. It was also noted that the capacity of the virus to produce Aantigen was diminished during attenuation by passage in tissue culture and this suggested a correlation between the presence of A antigen and pathogenicity. This conclusion was later proven false since it appeared that loss of A antigen can occur without loss of pathogencity (84), and a serologically identical A antigen was still detected in HVT as well as other apathogenic strains of MDHV— infected cell fluids (80,87,108). A antigen is the antigen most commonly detected by sera from naturally infeéted birds (50,88). It can be produced in reasonably large amounts in tissue culture and is easily monitored by immunodiffusion (16,50). For these reasons, A antigen has been extensively studied by many labs. An early attempt in the purification of A antigen was made by Ross,et a1. (88) who reported a 20-fold purification with either a 45% recovery by electrOphoresis in 5% acrylamide or a 20% recovery by chromatography on either DEAF—Sephadex A50 or Sephadex G—200. The antigen was produced in duck cells and was detected by convalescent chicken sera. It was also identified as a glyc0protein by autoradiog- raphy and was found to have a molecular weight range of 70,000 to 90,000 daltons by gel filtration and a heterogeneous charge (pI 4.5 to 5.5). In a study by Settnes (93), crude A antigen was found to be trypsin sensitive and resistant to pH 1.7. Onuma,et a1. (69) described a common antigen, presumed to be A antigen, that was associated with both MDHV and HVT infections. It appeared as a single band in immunodiffusion tests from extracts obtained from feather-tips of MDHV infected chickens and from tissue culture fluids of cultures infected with MDHV and HVT. The molecular weight of the feather-tip antigen, estimated by gel filtration, was 33,000 daltons and this antigen also had a pI of 6.35. The antigens from culture fluids of MDHV and HVT infected cells had molecular weights of 46,000 and 43,000 respectively, and both had a pI of 4.5. The common antigen was unaffected by acetone or ether and was concluded to be a glyc0protein due to its sensitivity to pronase and sodium periodate. The antigen was soluble and separable from MDHV and HVT virions but it reacted in common with a virion antigen in immunodiffusion, leading to the conclusion that the antigen may be a virion envelOpe glyc0protein or an altered cell membrane glycoprotein. The authors indicated that inoculation with common antigen stimulated virus neutralizing antibody and that it might be part of the virus envelope. In later work, antisera against a partially purified common antigen, presumed to be A antigen, neutralized cell-free MDHV and HVT and also appeared to react with a late appearing membrane antigen defined by immunofluorescence (55,70, 71). Ross,et a1. (87) analyzed the B and C antigens present in extracts of chicken embryo cells infected with an attenuated strain of MDHV after partial purifi- cation of the antigens by gel filtration. The B antigen was relatively stable and of lower molecular weight on polyacrylamide gel electrOphoresis than C antigen. B and C antigens were also found in the culture medium of infected cells at low levels but were distinguishable from.A antigen. The results of immunodiffusion studies suggested that B antigen was common to MDHV and HVT and that C antigen was MDHV-specific. A rigorous purification scheme for A antigen was developed by Long,et al. (49,50), and for B antigen by Velicer,et al. (106), so that A and B antigens could be purified apart from each other and produced in quantities sufficient to achieve sharp antigen peaks for analysis. Long,et al. (49) purfied A antigen 200- fold with a 25% recovery by a combination of ion- exchange column chromatography, isoelectric focusing, and preparative polyacrylamide gel electroPhoresis. The antigen had a pI of 6.68 when l M urea and Brij 35 was used to maintain solubility, and a molecular weight of 44,800 by gel filtration on Sephadex c-zoo and 53,160 from calculation from sedimentation coefficients. The antigen was apparently not purified to homogeneity since four polypeptides were visible on SDS-polyacrylamide gel electrophoresis analysis of the antigen. Antibody to A antigen was prepared in a rabbit, and antibody to two contaminating antigens was removed by adsorption to yield monospecific antisera. Extensive characterization and purification of B antigen free of A antigen was achieved by Velicer, et al. (106) with Conconavalin A affinity chromatography, sucrose gradient sedimentation, isoelectric focusing, and gel filtration on Sephadex G-200. In the presence of 1-2 M urea and 0.05% Brij 35, purified B antigen had 10 a pI of 4.54 and a molecular weight of 58,250 by gel filtration. A greater than ZOO-fold purification of B antigen was achieved. Rabbits were immunized to prepare antisera that appeared monospecific for B antigen by immunodiffusion (106). A comparison of the published pr0perties of highly purified A and B antigens is summarized in Table l (106). In the previously cited purification work of Ross,et a1. (87) and Onuma (69,70,71) antigens were purified only by gel filtration and data were based on antigen positive regions rather than sharp peaks of antigen activity. In light of this, Velicer,et al. (106) points out that the discrepancies in the molecular weight values for A antigen and common antigen that have been reported could have been a result of the overlap on gel filtration of the A and B antigen peaks; A and B antigens both being common between MDHV and HVT infected cells; A and B antigen precipitin lines appearing as one ,under certain immunodiffusion conditions;_ and untreated A antigen tending to aggre- gate. The more extensive purfication of A and B antigens, free of each other and in quantities large enough to produce distinct antigen peaks, provides a basis for an accurate physio-chemical analysis of the antigens. ll ooo.am oedemm ucoAOAMMooo coflumuzofifloom Scum cofiuoasoamo om~.mm oom.ee ooNIu xoooeaom no coauouuaam How an oocwauouoo unmfloz Hmasooaoe ucoummmd mv.v mh.m unmanammooo coflumucmEHoom om.w mo.m ucflom owuuomaoomH mo» mm» mm nflum amo.o .oouo 2 H o» uooumaoom oz mo» cflmmauu ou o>fluflmcom no» no» ooflmocozlonaanuozlo an 4 caam>ocmocoo Eoum ousam mm» mm» o.~ mm on ucmumflmmm now no» :wmuoumoomaw no» no» a>m one >maz coosuon coeaoo maaoo Edflooz ca sowumuucoocoo amouooum ca coach no» no» woman oouoomcfl adamusuoc Scum whom an oouoouma ml>maz ¢r>mnz .mcomfiucm ml>=az pom ¢I>maz mo GOmfiHmmEoo oonmflandmil.a Manda 12 In a preliminary study by van Zaane and Gielkins (113), polypeptides from cells infected with different strains of MDHV and HVT were analyzed by immunOprecipita- tion with homologous and heterologous sera from infected birds followed by SDS-polyacrylamide gel electr0phoresis. The pattern of virus-specific polypeptides observed in immunOprecipitations with homologous sera from cells infected with pathogenic MDHV, apathogenic MDHV, and HVT were similar. Four major virus specific protein bands were immunoprecipitated and were apparently late gene products because their amounts increased late in infec- tion. Immun0precipitation with heterologous sera showed that the four virus Specific proteins from these viral strains were antigenically related. Two-dimensional gel electroPhoresis of these polypeptides revealed differ- ences in the polypeptide patterns between HVT and the MDHV strains. In light of the DNA homology studies between HVT and MDHV, it is possible that these four polypeptides might be coded for by the homologous regions of the HVT and MDHV genomes. Immun0precipita— tions of culture media from cells infected with the above strains of virus detected a protein, presumed to be A antigen, with a molecular weight of 60 - 65,000 that also appeared to be a glyc0protein. Immun0precipi- tations with heterologous sera showed antigenic 13 similarities between the presumptive A antigen from all strains. Four different antigens have been observed in infected cells by immunofluorescence: a diffuse nuclear antigen, a diffuse cyt0plasmic antigen, a granular cytOplasmic antigen and a membrane antigen (MA) on the surface of infected cells (2,15,31,65,78,105). Cyt0plasmic and nuclear antigens are present only in cells containing virus particles (23,65). The relation- ship between these antigens and viral structural proteins are still undefined. Nazerian and Chen (61) found that some cells positive for MA were not producing virus and suggested that MA could be similar to MA detected on cells infected by other herpesviruses such as Epstein- Barr virus. It was also reported that rabbits immunized with purified virus developed antibody to MA. Although it was recognized that the virus might not have been completely purified, it appeared that MA might be related to structural proteins of the virus. Nazerian (58) also reported the loss of MA in cells infected with attenuated MDHV that had also lost A antigen and con- cluded that A antigen and MA might be related, although it was recognized that these observations could also be coincidental. 14 Another MD related antigen, designated the MATSA antigen, was detected by immunofluorescence on the sur- face of MD tumor cells and on almost all cells in MD lymphoblastoid cell lines (54,56,67,75,lll). MATSA was not related to viral structural proteins because it was not present on cells productively infected with MDHV (56,111). It appeared that MATSA was distinct from histocompatibility antigen and embryonic antigen. MATSA from different tumor cell lines appeared to be immunologically related but not identical (111). The exact role of MATSA in the host immune response to MD is not known, but it appears that chickens infected with MDHV and HVT do elicit a transient cell mediated response to MATSA (96,100). Resistance to MD Resistance to MD can be natural or acquired. Natural resistance to the disease is genetically inherited and falls under two classifications: early resistance, exPerienced at hatching, and late resistance, which is expressed with increasing age (95). Both early and late forms of natural resistance are expressed through lesion regression (95). After infection with MDHV, resistant chickens deve10p lesions of MD that eventually disappear while simultaneously infected, susceptible chickens will deve10p lesions that are 15 accompanied by high levels of mortality. Natural resistance does not appear to be antibody dependent (94,100) but cell-mediated immunity appears to be important (102). A recent study by Lam and Linna (42) indicated that late natural resistance could be transferred to newly hatched, susceptible chickens with spleen cells from resistant chickens. These cells were apparently neither T-cell, B-cell, nor macrOphage in origin, but possibly part of the "third lymphocyte" papulation which shares many characteristics with natural killer cells in ability to kill a wide range of tumor targets. Acquired resistance is by vaccination, either by natural exposure to apathogenic strains of virus, or by artificial vaccination with attenuated or apathogenic strains of MDHV or with antigenically related HVT (68,83,95). The Marek's disease system is the only example of a malignant disease that can be completely protected against by vaccination. The first important vaccine was made by Churchill (19) who found that chickens could be protected by inoculation with a virus that was attenuated by repeated passage in tissue culture. Since then, a number of live vaccines have been deve10ped but HVT is the strain most widely used in vaccination (63,68). 16 Host Immune Response to MDHV Infection Infection with MDHV results in the initial suppression of both the humoral and cell—mediated responses of the host (72). This initial impairment might be a factor leading to lymphoma formation, or might be a consequence of the degenerative and prolifera— tive changes that occur in the lymphoid tissues of infected birds. There is a distinct humoral response to infec- tion with MDHV, but the significance of the humoral response in the overall protection of the host is not well understood. Anti-viral antibody has been detected by immunodiffusion (16), immunofluorescence (78,79), virus neutralization (9,24), indirect hemaglutination tests (22) and complement fixation test (53). Antibodies appear 3-5 weeks post—infection and are present for a long time, possibly due to the persistent infection caused by MDHV (63). Passively acquired maternal anti- body has been shown to have a significant protective effect against morbidity and mortality caused by MDHV (7). In chickens with maternal or passively administered antibody, the initial productive infection and acute inflammation was greatly reduced (72). The number of tissues with viral antigen and the amount of antigen in positive tissues was lower and it appeared that the 17 antibody exerted its effect by reducing the extent of the initial virus infection. Chickens with a high titer of antibody during infection appeared to survive the disease or live longer than those with a low antibody titer (59). However, since MDHV infection will cause a severe degeneration of the bursa and thus affect antibody titer, it is not clear whether mortality in low antibody producing chickens is a cause or effect of antibody production. Sharma,et al. (100) showed that bursectomy of age-resistant birds and the resulting agammaglobulinemia did not affect the susceptibility of these birds to MD, implying that the mechanism of tumor regression that is characteristic of age-resistant birds is not dependent on humoral functions. In addition, bursectomized chickens immunized with an apathogenic strain of MDHV were still protected against a trans- plantable MD-induced tumor (90), however the protective effect of HVT vaccination was impaired though not abolished after bursectomy and x-irradiation (82). Comparative studies on active antibody synthesis in _genetically resistant and susceptible chickens did not reveal any quantitative or qualitative differences (25). It appears that although humoral immunity has an amelioratory effect on the severity of Marek's disease, it is not a crucial part of the host's immune response to the disease. 18 A considerable amount of evidence exists demon- strating the importance of the cell-mediated immune response in the host's defense against MD. Fauser, et a1. (24) first provided evidence for cell-mediated immunity to MD based upon delayed hypersensitivity and MIF tests using a semi-purified A antigen preparation. Byerly and Dawe (8) reported a stronger delayed hypersensitivity reaction with a crude MDHV-associated antigen preparation derived from infected cells, than with antigens from tissue culture medium or from feather follicles. The T-cell system, in addition to being the target cell for transformation by MDHV (28,89,99), has been implicated in immune surveillance against the development of lymphomas. Sharma,et al. (102) showed thatgenetically resistant chickens made deficient in T-cells by neonatal thymectomy and gamma-irradiation became highly suscep- tible to MD while their untreated hatchmates remained resistant to tumor formation. The availability of MD lymphoblastoid cell lines (3,12,67,75) has allowed for the in vitro study of immune responses to the antigens present on these cells. Sharma,et a1. (96) used a 51Cr-release microcytotoxicity test to demonstrate a specific cell-mediated response in Spleen cells from 51 MDHV infected chickens directed against Cr-labeled lymphoblastoid target cells. The response was exPressed 19 in the absence of antibody and complement and occurred briefly after virus infection and early in the disease process, before gross lymphomas appeared. The cytotoxic response, presumably directed against MATSA, indicated that cell—mediated immunity was also important in mediat— ing the host response against MDHV-inducted tumors. Lee,et a1. (43,45,46) has reported on the existence of suppressor macrophages in the spleens of MDHV-infected chickens, that suppressed the PHA proliferation re5ponse of T-cells and also inhibited the DNA synthesis of lymphoblastoid cells. However, Sharma (97) demonstrated that spleens of normal chickens contain suppressor macr0phages that are inhibitory to cells of a rapidly dividinghfl)tumor cell line and also inhibited mitogen-induced blastogenesis of lymphocytes. It is not known if the mechanism of inhibition of MD infected chicken derived macr0phages is the same as that of macrOphages from normal chickens but it appears that suppressor macrOphages may play a nonSpecific role in anti-tumor immunity in the chicken. Vaccinal Immunity Vaccination with nononcogenic virus results in the persistent viremia of the vaccinating virus. These nononcogenic viruses have been shown to be lymphotrOphic with isolation patterns very similar to oncogenic 20 viruses (11,77,110). However, unlike oncogenic viruses, virus replication and antigen eXpression is minimal and tissue necrosis is absent (77). Purchase and Sharma (81) first demonstrated the involvement of the immune system in HVT vaccination. They found that HVT did not protect chickens that had previously been treated with high doses of cyclOphos- phamide, a drug that causes a permanent impairment of the humoral reSponse and a temporary impairment of the cellu- lar immune reSponse. Although they could not discern which of these two host responses was important in vaccinal immunity, subsequent work done by Else (23) and Rennie, et al. (79) showed that bursectomy and the resulting agammaglobulinemia did not substantially impair vaccine protection. Ross (85), using a plaque inhibition test as a measure of cell-mediated immunity, showed that plagues formed by MDHV-infected cells could be inhibited by T-lymphocytes present in the peripheral blood of chickens vaccinated with attenuated MDHV. When the same procedure was attempted using sensitized lympho- cytes from HVT vaccinated birds, only a low level of plaque inhibition was observed (86). However, when antisera from both HVT or attenuated MDHV vaccinated birds was supplemented with normal spleen cells, equally 21 Vgood plaque reduction was observed. The author suggests that two mechanisms might exist for inhibiting growth and spread of MDHV in tissue culture; one mediated by sensitized lymphocytes and the other by an antibody— dependent cell-mediated cytotoxicity. The nature of the important antigens involved in vaccinal immunity to MD was first examined by Kaaden, et al. (33). The protective effects of purified HVT virions and HVT-infected cellular membranes was studied. Purification of HVT from infected cells yielded viral preparations of low infectivity that contained many unenveIOped nucleocapsids. This viral preparation provided an effective immunity to vaccinated chickens, reducing specific mortality by 74%. When cellular membrane fractions from HVT infected cells, purified on sucrose gradients, were used to vaccinate chickens, specific mortality was reduced by 94%. After solubili- zation, the membranes from HVT-infected cells formed two specific precipitin lines in immunodiffusion tests with MDHV-Specific chicken sera. In subsequent work, (32) plasma membranes obtained from MDHV and HVT infected cells were highly purified by isopynic centrifu- gation in dextran and determined to be free of virus particles by electron microsc0pic examination. Two specific precipitin bands were formed in the 22 immunodiffusion assays with solubilized plasma membranes from infected cells, and polyacrylamide gel electro- phoresis analysis of this material also showed two virus induced protein bands. Antisera prepared against plasma membranes from MDHV or HVT infected cells neutralized extracellular infectious HVT. When chickens were inoculated twice with plasma membranes from HVT infected cells, MD mortality was reduced by 94%. This work indicated that virus infection with MDHV or HVT induced the formation of unique proteins on the infected cell surface that were also immunogenic. However, this work still leaves Open the question of the relationship of these membrane components to viral structural proteins as well as to the soluble antigens defined by immunodiffusion. Work concerning the antigenic nature of vaccinal immunity was done by Lesnik and Ross (48). Chickens were immunized with noninfectious materials extracted with nonionic detergents from cells infected with attenuated MDHV. Protection was obtained with both soluble and insoluble antigens of Nonidet P-40 extracts, but only with the insoluble fractions of deoxycholate extracts. The growth and spread of MDHV was reduced in immunized chickens based on the observed pr0portion of circulating blood leukocytes that contained virus 23 and the proportion of birds having A antigen in their feather follicles. In addition, the authors noted that when soluble and insoluble fractions were examined for the presence of precipitating antigens, it appeared that the amount of antigen in the preparation did not correlate with the capacity to protect, suggesting that immunity could be conferred by antigens that did not take part in the precipitation tests. Solubilized glyc0proteins from membrane-rich fractions of HVT-infected cells were isolated by Wyn- Jones and Kaaden (112) with Concanavalin A affinity chromatography. These glyc0proteins elicited heavy precipitin lines in immunodiffusion test with chicken sera from MDHV-infected birds, although their relation- ship to the already defined A and B antigens were not determined. Analysis of this glyc0protein material by PAGE showed three polypeptide bands in the molecular 3 - 120 x 103 that were not weight range of 100 x 10 present in uninfected cell glyc0protein extracts. This material, when purified by preparative PAGE and then inoculated into chickens, resulted in the production of low titer neutralizing antibody and partial protection against challenge with virulent MDHV. The work demonstrated that HVT can induce production of unique glyc0proteins in infected chicken cells and also 24 demonstrated the possible involvement of these glyc0proteins in eliciting neutralizing activity, although the resulting neutralizing activity induced in vaccinated chickens was of low titer and only a small number of chickens were actually tested. A number of studies have been concerned with the anti-tumor nature of vaccinal immunity. Powell (74) immunized chickens against MD with repeated injections of gluteraldehyde-fixed cells of an MD—lymphoblastoid cell line. The resulting protection was attributed to immunity against MATSA antigen. Using a 51Cr-release assay, Sharma, et al. (101) demonstrated a T-cell- mediated immune response to MD tumor cells in HVT or attenuated MDHV vaccinated chickens. In addition, other studies have shown that HVT inoculated birds developed transient lymphOproliferative lesions (110), MATSA positive cells have been detected in the tissues of HVT and attenuated MDHV vaccinated chickens (91) and a chicken thymus-derived lymphoblastoid cell line has been established from HVT inoculated chickens in which 95% of the cells from this line demonstrated MATSA (39). HVT could be rescued from this cell line by co-cultivation with chicken embryo fibroblasts. Although it has been assumed that the MATSA antigen was the most likely target for an anti-tumor 25 immune response, recent work by Schat and Murthy (92) indicated that the exact role of MATSA in the host immune response is still questionable. MATSA positive cells of a MD-lymphoblastoid cell line were made MATSA negative by treatment with papain. These papain treated cells had become negative for membrane fluorescence, and they did not lyse in the presence of anti—MATSA antibody and complement. These results were not seen if cells were treated with trypsin or mixed glycosidases. When these MATSA negative cells were used as target cells for MD-sensitized spleen cells in a chromium release cyto- toxicity assay, no Significant decrease in the specific release of chromium was observed as compared to MATSA positive cells. The authors concluded that MATSA was not the target antigen in chromium release assay tests, and is probably not an important antigen in cell—mediated immunity to MD. Other suggested antigenic alternatives for MATSA included the possibility of a tumor associated antigen that could not be detected by serological methods such as has been described on Epstein-Barr virus trans- formed B-lymphocytes, and the unlikely possibility of the chicken fetal antigen that is present on all trans- formed cell lines. Although the exact nature of the antigens involved in vaccinal immunity is still unknown, the 26 fact that both anti-viral and anti-tumor responses are important lends support to the theory presented by Payne (73) concerning the mechanism of vaccinal immunity to MD. Payne proposed a two step mechanism of resistance in vaccinated birds. The first step involved both humoral and cell-mediated responses that are stimulated by the vaccinating virus against the replication and spread of infecting MDHV. Thus, by reducing the virus load on the host, there is a reduction in both the immunosuppressive effects of MDHV infection and the probability of neOplastic transformation of lymphoid cells. This permits the host to mount a more effective response to infection. The second step consists of a cell-mediated response directed against tumor cells. This anti-tumor response could be a result of transient lymphOproliferation induced by the vaccinating virus or a result of cell transformation due to the challenging MDHV. This theory is further supported by work done by Powell, et a1. (76). Chickens were immunized with either gluteraldehyde-inactivated, MDHV-infected chicken kidney cells containing virus specific antigens, or with gluteraldehyde-inactivated MDHV-transformed lymphoblas- toid cells containing MATSA. They found that both types of immunizations protected against MD but the mechanisms 27 of protection were different. Immunization with viral antigens was associated with suppression of virus replication after challenge with virulent virus but cytotoxicity to MD tumor cells was not observed. Immunization with tumor antigens did not affect viral multiplication but some evidence of cell-mediated anti-tumor activity was found. The above results were also confirmed by Murthy, et al. (57) who studied early pathological events following immunizations with viral antigens or tumor antigens and subsequent challenge with MDHV. They found that viral antigen vaccine inhibited both replication of challenge virus and tumor formation while tumor antigen vaccine only had an effect on subsequent tumor development. These results probably accounted for the observation that birds given viral antigen vaccines had a higher level of protection against tumor deve10p- ment than birds given tumor antigen vaccine. MATERIALS AND METHODS Cells and Virus Primary duck embryo fibroblast cells (DEF) were prepared and proPagated according to the procedures established by Long, et a1. (50). Cells were seeded in 100 mm diameter plastic tissue culture dishes (1.0 x 107 cells/plate) with 10 ml per plate growth medium consist- ing of standard Medium 199 and nutrient mixture F-10 combination containing 2% calf serum. Growth medium was changed 16-24 hrs after seeding and the pH was adjusted with isotonic NaHCO3 as needed. MDHV strain GA-infected cells in 25th passage and HVT strain FC—126 infected DEF cells in 16th passage were used to generate stock supplies of MDHV and HVT h and 17th passages, respec- infected DEF cells in 26t tively. Virus infected cells from 100 mm tissue culture dishes were frozen in 1 ml ampules for storage in liquid nitrogen. They were then titered and used in all subsequent experiments as a source of infective virus. DEF monolayers that were approximately 85% confluent, were infected with a 1:8 dilution of a 1 ml ampule of stock infected DEF cells. At this dilution 28 29 of infected cells, cytOpathic effect (CPE) was barely noticeable at 24 hrs post infection (PI) and reached a maximum at approximately 48-72 hrs PI. Infected cell monolayers were maintained in 10 ml of growth medium without calf serum. The medium was changed 24 hrs PI and the pH adjusted with NaHCO3 as needed. Radioactive Labeling Infected cells were labeled at 48-72 hrs PI, when more than 50% of the cells demonstrated CPE. Uninfected cells were handled in the same manner as 35S-methionine infected cells. Labeling of protein with was accomplished by washing cell monolayers three times with warm Hanks Buffered Saline Solution (HBSS, Gibco) followed by a 24 hr incubation period in 5 ml of Dulbecco's Minimal Essential Medium (DMEM, Gibco) containing l/20 the normal concentration of unlabeled 35 methionine and S-methionine (New England Nuclear) at 50 uCi/ml. Glyc0proteins were labeled by incubating cell monolayers for 24 hrs in 5 ml growth medium contain- 14 ing 1%‘calf serum and C—glucosamine (New England Nuclear) at l uCi/ml. 30 Preparation of Labeled Culture Media and Cell Lysates for Immungprecipitation Culture media from infected and uninfected cells were collected and clarified at 5,000 x g for 10 minutes followed by ultracentrifugation at 147,000 x g for 1 hr. Labeled cells were lysed according to the method of Witte, et al. (108). Cell monolayers were washed three times with phosphate buffered saline (PBS) at 4° followed by extraction into a detergent lysis buffer consisting of 0.01 M NaHZPO -NaHPO (pH 7.5)-0.l M NaCl 4 4 containing 1% Triton x-lOO, 0.5% sodium deoxycholate (NaDOC), and 0.1% sodium dodecyl sulfate (SDS). Cell lysates were clarified as described above for culture media. Radioactivity Assays For the determination of radioactive isotOpe incorporation in culture media or cell lysates, 100 pl of 14 3S C-glucosamine labeled material or 10 pl of S-methionine labeled material was Spotted on 2.3 cm diameter Whatman 3 mm filter discs. The discs were oven-dried at 60° and treated on ice with cold 5% Trichloracetic acid (TCA) for 20 minutes followed by a 1 minute wash in acetone. The discs were then dried as above and added to vials containing 5 ml of phosphor scintillation fluid (2,5, Diphenyloxazole [PPO] 22.7 gm; 31 1,4,bis-2—[4—methyl-5-phenyloxazolyl]-benzene (POPOP) 1.9 gm; toluene 8 pints) and counted in a Packard liquid scintillation Spectrometer (Packard Inst. Co.). Protein Determinations Protein determinations were done according to the method established by Lowry, et a1. (51) with crystalline bovine serum albumin (BSA) as the standard. Antisera for Immunoprecipitations All antisera were already available in the laboratory. Immune chicken serum (ICS), which demon- strates a stronger reactivity to A antigen then to B antigen by immunodiffusion analysis, was obtained from birds naturally infected with MDHV. Rabbit anti-A serum (RaA), negative for B antigen by immunodiffusion, and rabbit anti-B antigen sera (RoB), negative for A antigen by immunodiffusion, were prepared by inoculating rabbits with highly purified A and B antigens, respectively (49,105). Rabbit anitsera prepared against infected cell plasma membranes purified by Dextran T—40 gradients (RaPM), and found to be reactive with B antigen by immunodiffusion analysis, was also used. In certain instances, the above immune sera were absorbed with unlabeled, uninfected cell lysates or culture media in order to reduce the appearance of 32 background polypeptides on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The amount of material used for absorption was five times the volume of labeled material used for immunoPrecipitation. After a 24 hr absorption period, on ice at 4°, the sera was centrifuged at 30,000 rpm for 1 hr in a type 50.1 rotor (Beckman) prior to use. In order to block the appearance of either A or B antigens, 40 pl of immune serum was incubated with either 1 unit of A antigen or 1 unit of B antigen (49,106) followed by a centrifuga- tion at 30,000 rpm for 1 hr in a type 50.1 rotor (Beckman) at 4°. Both A and B antigen preparations were donated by Dr. Velicer and were partially purified through isoelectric focusing (106). As a control, normal rabbit serum (NRS) and serum from specific pathogen free birds (NCS) were also used in immunopreci- pitation analysis. Immungprecipitation Infected cell culture medium and lysate material were used as a source of A and B antigens, respectively. Immun0precipitations were performed according to the methods of Witte, et al. (108) with the use of S.aureus Cowan strain I to collect antigen-antibody complexes. This procedure exploits the high specificity and adsorption capacity of the protein A molecule located 33 on the cell wall of the Cowan I strain of the S.aureus bacterium for the Fc region of many IgG subclasses (38). S.aureus Cowan I strain, prepared for use as an immunoadsorbent according to the method of Kessler (38) was provided by Dr. Velicer. Prior to use, the prepared S.aureus was pelleted at 5,000 x g for 10 minutes and resuspended to the original volume with detergent lysis buffer containing 1 mg/ml BSA. This S.aureus immuno- adsorbant was then used at ten times the volume of antiserum used for immunOprecipitation. 3 4 Aliquots of culture medium containing 10 - 10 35 counts per minute (cpm)/ug.of S-methionine labeled 3 5 cpm/ug l4C-glucosamine labeled 6 protein and 10 - 10 protein, and cell lysates containing 105 - 10 cpm/ug 35 5 7 S-methionine labeled protein and 10 - 10 cpm/ug 14C-glucosamine labeled protein were used for immuno- precipitations. Prior to incubation with immune serum, the antigen containing material was precleared in order to remove material that nonspecifically adsorbs to either IgG or the S.aureus immunoadsorbant. After overnight incubation of the material at 0-40 with 40 ul of normal serum, 400 Ml of the S.aureus immunoadsorbant was then added followed by an incubation period of one hour at 0-4° and centrifuged at 12,000 x g for 3 minutes in an Eppendorf centrifuge. The precleared 34 supernatant was then added to 40 pl of immune serum followed by an overnight incubation at 0-4°, an addition of 400 p1 of S.aureus immunoadsorbant, incubation for 1 hr at 0-4°, and a centrifugation at 12,000 x g for 3 minutes in Eppendorf centrifuge. The supernatant was carefully removed by vacuum and discarded with a pasteur pipet attached to a vacuum flask so as not to disturb the bacterial pellet containing the antigen- antibody complex. The pellet was then washed three times with detergent lysis buffer at 0-4° and the wash supernatant carefully removed by vacuum and discarded as described above. In order to extract all the liquid from the pellet, after the last wash was removed, the pellet was centrifuged once more at 12,000 x g for 1 minute in an Eppendorf centrifuge and any remaining liquid was discarded. The pellet was then prepared for SDS-PAGE as described below. SDS-PAGE and Fluorography Discontinuous stack SDS-Polyacrylamide slab gels were prepared and run according to the methods of Laemmli (41) in a vertical gel apparatus (Hoeffer, model SE-520). Polyacrylamide gels containing a 5% stacking ,gel and a 6.5%, 7.5%, or a 10% separating gel were prepared from a stock solution of a 30:0.8 ratio of acrylamide to bis-acrylamide mixture. Final 35 concentration of the stacking gel was 0.0625 M Tris-HCL, pH 6.8, and 0.1% SDS. The final concentration of the separating gel was 0.184 M Tris-HCL, pH 8.8 and 0.1% SDS. The electrode buffer was 0.05 M Tris, 0.38 M glycine, and 0.1% SDS at pH 8.8. The washed S.aureus pellet containing immuno- precipitates were handled for electrophoresis as described by Witte, et al. (108). Samples were denatured by placing them in 40 pl of Sample Buffer (SB) containing 0.05 M Tris-HCL, pH 6.8, with 1.0% mercapthoethanol, 10% glycerol, 1.0% SDS and 0.001% bromOphenol blue, and heating at 68° for 15-20 minutes. After centrifugation in an Eppendorf centrifuge for 1 minute, one half of the SB supernatant was loaded onto the gel. Acetone precipitates of 14 Ceglucosamine labeled culture media and cell lysates were prepared by adding one volume of culture media or lySate to nine volumes of acetone, and kept overnight at -20°. The precipitate was pelleted at 12,000 x g for 30 minutes and the pellet air-dried before it was dissolved in 40 pl of SB and heat treated as described above, prior to SDS-PAGE 35S-methionine analysis. Direct SDS-PAGE analysis of labeled proteins of culture media and lysates was achieved with a 1:2 dilution of labeled material in 36 double strength SB, followed by heat treatment as described above, prior to gel loading. Standard molecular weight markers included 14C—glucosamine labeled myosin (200,000 daltons), phosphorylase B (92,500 daltons), BSA (69,000 daltons), ovalbumin (46,000 daltons), and carbonic anhydrase (22,000 daltons) (New England Nuclear). Molecular weight estimations were calculated by interpolation between standard proteins, similar to Weber and‘OSborn (107). Electr0phoresis was carried out at a constant voltage of 150 volts (ISCO Electrophorsis Power Supply Model 1493) until the marker dye ran off the gel, usually after 1.5-2 hrs. Gels were fixed overnight in a solution containing 10% acetic; acid and 10% methanol. Detection of labeled proteins and glyc0proteins by fluorography was performed by impregnating the gel with PPO as described by Bonnar and Laskey (6). Gels were then dried under vacuum (Hoeffer Slab Gel Dryer Model ’ SE-540), placed in contact with RP Royal x—OMAT Film RP/R2 (Kodak) and kept at -70° for varying lengths of time. RESULTS Analysis of 35S-methionine and igC—glucosamine Labeled Polypeptides Found in Culture Media Since A antigen has previously been character- ized as a glycoprotein that is shed into the culture medium of MDHV-infected cells (l9,49,50,88), an analysis of culture media from 35 S-methionine and 14C-glucosamine labeled cells was performed on SDS-PAGE (Figure 1). Profiles of culture media from 35S-methionine and 14C-glucosamine labeled infected cells revealed a virus-induced polypeptide with an approximate molecular weight range of 61-65,000 (Figure 1A). Although culture medium from 35S-methionine labeled cells showed apparently virus-specific polypeptides at 40,000 daltons and 67,000 daltons, the 61-65,000 dalton polypeptide appeared to be the major virus-induced polypeptide shed from the cell. Analysis of acetone precipitates of culture medium from 14C-glucosamine labeled cells also demonstrated a virus—specific, 61-65,000 dalton, glycosylated molecule (Figures 1B and 1C) . Acetone precipitates from equal volumes of infected and 37 38 Figure l.--Anal sis of culture media from 35S-methionine and 4C-glucosamine labeled, MDHV-infected (INF) and uninfected (CON) cells. A. Direct analysis, performed as described in materials and methods, of culture medium from 35S-methionine labeled INF and CON cells, containing 4.5 x 105 and 6.0 x 105 Cpm, respectively, on 10% SDS-PAGE, 24 hr fluorographic eXposure. B. Acetoge precipates of culture medium from 4C-glucosamine labeled INF and CON celli, containing 4.0 x 104 and 1.7 x 10 Cpm, respectively, on 10% SDS-PAGE, 16 day fluorographic exposure. C. Acetone precipitates of culture medium from 4C-glucosamine labeled INF and CON cells, containing 6.0 x 104 Cpm each, on 7.5% SDS-PAGE, 10 day fluoro- graphic eXposure. finesse...” in- 532...: .2. ea. .2. ea. _.. as. s. “Tl-h - 3. all. sfl I V sTI . a slut ,_ safn. __ . . _. 3T , ST . . A Input . . ST . . .l . n '13.. S.a-Il _. 40 uninfected cell culture medium revealed a 14 Ceglucosamine labeled, 61-65,000 molecular weight molecule in infected cell culture medium, while uninfected culture medium did 14 not reveal any C-glucosamine labeled molecules (Figure 1B). Since incorporation of 14 C-glucosamine in uninfected cells was 2-4 times less than in infected cells, acetone precipitate samples, containing equal counts per m1 of 14C-glucosamine, were prepared for SDS-PAGE analysis in order to better ascertain the virus-specific nature of the labeled molecules observed (Figure 1C). This analysis confirmed the virus—specific nature of the 61-65,000 dalton molecule. The observation that the 61-65,000 dalton molecule was the major 14C-glucosamine labeled species shed into the culture medium of infected cells, suggested that it may be A antigen. However, in order to confirm rigorously the identity of this molecule as A antigen, highly specific immunological analysis was required. Immunoprecipitatigg Analysis of Culture Medium of S-methionine Labeled Cells Using RaA A 35S—methionine labeled, virus—specific polypeptide with a molecular weight range of 61-65,000, was immunOprecipitated from culture medium using RaA (Figure 2A). This polypeptide was not detected on 41 Figure 2.--Immuno§recipitation analysis of culture media 8 from 3 ~methionine labeled, MDHV-infected (INF) and uninfected (CON) cells with rabbit anti-A serum (RaA). A. 100 pl of culture media from INF and CON cells, containing 4.8 x 105 and 6.6 x 105 Cpm reSpectively, were reacted with RaA. Immun0precipitates were analyzed on 8.5% SDS-PAGE, 48 hr fluorographic exposure. 100 pl of culture medium from INF and CON cells, containing 3.0 x 105 and 4.7 x 105 cpm, respectively, were reacted with RoA and normal rabbit serum (NRS) and immun0precipitates analyzed on 7.5% SDS-PAGE,7 day fluorographic exposure. 100 pl of culture medium from INF cells were reacted with RaA and RdA blocked with A antigen and the immun0precipitates analyzed on 7.5% SDS-PAGE, 48 hr fluoro- graphic exposure. 42 . ‘ 3...; a r: < I. <6: <0: '1..on 2.2.3! one; 1:8 Izod :8 4.3 . I . . ‘ ‘ :00 “.2. a I128 . 2.2.33. amo- 3 28 nlllxmfio “.2. <0: 0.00 o\om.a llxoe. 2.2125 . geese , lilac [loses m. a. zoo uz. <31 41 Figure 2.--Immuno§recipitation analysis of culture media from 3 S-methionine labeled, MDHV-infected (INF) and uninfected (CON) cells with rabbit anti-A serum (RdA). A. 100 pl of culture media from INF and CON cells, containing 4.8 x 105 and 6.6 x 105 opm reSpectively, were reacted with RaA. Immun0precipitates were analyzed on 8.5% SDS-PAGE, 48 hr fluorographic exposure. B. 100 pl of culture medium from INF and CON cells, containing 3.0 x 105 and 4.7 x 105 cpm, respectively, were reacted with RaA and normal rabbit serum (NRS) and immun0precipitates analyzed on 7.5% SDS-PAGE,7 day fluorographic exposure. C. 100 pl of culture medium from INF cells were reacted with RaA and RaA blocked with A antigen and the immun0precipitates analyzed on 7.5% SDS-PAGE, 48 hr fluoro- graphic exposure. 42 Q‘ ‘ 31”, 12. 12. < + <6: <6: 28 $3 22‘ .p».lld§¥ Ewan“: $23.21 . . 23.5 I..l.l X8 . [121.8 2MP ‘ 1.21.3 2.212 <2 a .1 200 o\om..m 200 12. <12... [luv—me A<.>In=$ VET—.0 Ill v30 llxmdm 43 SDS-PAGE analysis of immun0precipitates of infected or uninfected cell culture media with NRS, even with an overexposure of the gel (Figure 2B). In order to con- firm the identity of the 6l-65,000 dalton polypeptide as A antigen, blocking experiments were performed by pre-incubating RoA with A antigen that was purified through isoelectric focusing (49). Figure 2C shows that blocking immune serum with the A antigen prepara- tion selectively eliminated the 61-65,000 dalton polypeptide. Immun0precipitation Analysis of Culture Medium of 3SS-methionine Labeled Cells Using ICS The 61-75,000 dalton polypeptide was immuno- precipitated only from infected cell culture medium using ICS, and was not precipitated by NCS (Figure 3). Confirmation of the identity of this 6l-65,000 dalton polypeptide as A antigen was also achieved by selec- tivity blocking the appearance of the polypeptide on SDS-PAGE with A antigen, purified through isoelectric focusing (49). The gel in Figure 3 was intentionally overexposed to demonstrate the virus-specific nature of the 61-65,000 dalton polypeptide and the thorough- ness with which purified A antigen can block the precipitation of this polypeptide. 44 Figure 3.--Immuno recipitation analysis of culture media from 3 S-methionine labeled, MDHV-infected (INF) and uninfected (CON) cells with immune serum from naturally infected chickens (ICS). 100 pl of culture medium from INF and CON cells, containing 5.0 x 105 and 6.7 x 105 cpm, respectively, were reacted with ICS, normal chicken serum (NCS) and ICS blocked with A antigen (ICS + A). Immun0precipitates were analyzed on 7.5% SDS-PAGE, 5 day fluoro- graphic exposure. 45 $2.55: <.>zos= 23.51% T235 28 ll. . II goo 21.3 I m2. 200 < + m0. mOz 46 Immun0precipitation Analysis of Culture Medium of 14C-glucosamine Labeled Cells A antigen has been previously characterized as a glyc0protein and labeled with 14 C-glucosamine (49,88). In order to confirm further the identity of the 61- 65,000 dalton polypeptide as A antigen, culture medium from 14 C-glucosamine labeled cells was examined by immun0precipitation analysis. A virus-induced molecule, of molecular weight 61-65,000, was observed in immun0precipitates obtained with RaA (Figure 4) and ICS (Figure 5), but was not visible in control precipi- tates obtained with either NRS or NCS. In addition, the appearance of this molecule could be blocked by purified A antigen, as shown in Figure 4 with RaA and in Figure 5 with ICS. Immun0precipitation Analysis of Culture Medium of HVT-Infected Cells Immunodiffusion studies have demonstrated that HVT and MDHV infection results in the appearance of an antigenically related A antigen (49,108). When culture 35S-methionine labeled cells infected with medium from HVT was examined by immun0precipitation analysis with either RaA or ICS, a virus-induced polypeptide was observed with a molecular weight similar to that of the previously identified, MDHV-associated A antigen 47 Figure 4.--Immun0precipitation analysis of culture media from C-glucosamine labeled, MDHV- infected (INF) and uninfected (CON) cells with rabbit anti-A serum (RaA). A. 600 pl of culture medium from INF and CON cells, containing 2.4 x 105 and 1.0 x 105 Cpm, respectively, were reacted with RaA and normal rabbit serum (NRS). Immun0precipitates were analyzed on 8.5% SDS-PAGE, 16 day fluorographic eXposure. Same as in A, with RoA blocked with A antigen (RaA + A) on 8.5% SDS—PAGE, 8 day fluorographic exposure. 48 12. ,<_+ . <32 '83 IX: 2.28.: no T28... _ . '28 12. <82 - z w_-fl._ __ -1 1 . ”.11 —w———_ way-T- “w. 8 In‘.“ 4, IL 2 II.. t “LA-‘1 A' _____,_ .K '28 .IullV-OQ. — . w v — w . 2.2.5.5 T233 Ill v.3 Ill. v3.8 49 Figure 5.--Immuno recipitation analysis of culture media from 1 C-glucosamine labeled, MDHV-infected (INF) and uninfected (CON) cells with immune serum from naturally infected chickens (ICS). 600 pl of culture medium from INF and CON cells, containing 4.0 x 105 and 8.0 x 104, respectively, were reacted with ICS, normal chicken serum (NCS) and ICS blocked with A antigen (ICS + A). Immun0precipitates were analyzed on 7.5% SDS-PAGE, 8 day fluorographic exposure. 50 1:. 2+3. see 1:. we: was .1._ 8. A 3. a: I: =3 .8. =2 5.. no: no. 65 A small degree of non-specific trapping of these poly- peptides could be seen with NCS and NRS. DISCUSSION The objective of this work is to further the molecular characterization of the A and B antigens of MDHV-infected cells by SDS-PAGE analysis following S.aureus immunoprecipitation. These antigens have previously been defined only by immunodiffusion (19,49, 50,88), and all previous attempts at biochemical and biOphysical characterization (49,50,87,88,106) were based on this criteria. Molecular characterization and identification of these antigens on SDS-PAGE can sub- sequently serve as a foundation for further analytic studies of entire glyc0proteins and their polypeptide portions. A antigen proved to be the easiest antigen to characterize, probably because it is shed into the culture medium in large quantities. SDS-PAGE profiles, representing the overall 35 S-methionine labeled poly- peptide content of infected and uninfected cell culture media (Figure 1A), Show a major virus-specific poly- peptide with the approximate molecular weight of 61- 65,000. Direct analysis on SDS-PAGE of glyc0proteins 14 found in the culture medium of C-glucosamine labeled 66 67 infected cells also Show that a major_glyc0protein with an approximate molecular weight of 61-65,000 is Shed into the culture medium (Figures 1B and 1C). Results of immun0precipitations with A antigen reactive sera and blocking studies with purified A antigen show unequivocally that A antigen is a glyco- protein with an apparent molecular weight of 61-65,000 on SDS-PAGE. This is demonstrated by the immun0precipi- tation of this polypeptide from the culture medium of infected cells using RoA (Figure 2) and ICS (Figure 3). Furthermore, the immun0precipitation of this polypeptide with both sera could be selectively blocked by incubat- ing the antisera with purified A antigen prior to immun0precipitation. This is observed with both 35S-methionine and l4C-glucosamine labeled material and with all immune sera used (Figures 2,3 and 4). Although early studies initially indicated that avian IgG does not have a high specificity for protein A (40), more recent examinations indicate that immunoglobulin affinity for protein A increases substan— tially when it is bound in an immune complex (38). This is probably the reason immunoprecipitation of the 61— 65,000 dalton polypeptide could be achieved with ICS. This ability of the S.aureus protein A to bind to ICS sera should aide in future immunological studies of MDHV antigens. 68 While this work was in progress, van Zaane and Gielkens (113) reported the detection of a virus- specific glyc0protein of molecular weight 60—65,000 that is secreted into the culture medium of MDHv- infected DEF cells. This glyc0protein was immun0preci- pitated with complex sera obtained from MDHV immunized birds and observed on SDS-PAGE, but no further attempt was made to rigorously confirm its identity as A antigen. While this work provided preliminary data suggesting that this glyc0protein might be A antigen because of its appearance in the culture medium of infected cells, the study done for this thesis proves conclusively that A antigen appears on SDS-PAGE as a glyc0protein of 61- 65,000 molecular weight. The rigorous proof of the identity of A antigen presented here was made possible due to the availability of A antigen-specific antisera and purified A antigen for use in immun0precipitation analysis and blocking studies, respectively. The work reported here is an extension of the initial purification and characterization of A antigen by Long, et al. (49,50). A antigen was purified greater than 200 fold with a 24% recovery on ion exchange column chromatography, isoelectric focusing, and preparative PAGE. However, the analysis of the purified antigen by immunodiffusion and by SDS-PAGE 69 showed that it was not purified to homogeneity. Three precipitin lines were observed in immunodiffusion tests when serum from rabbits inoculated with the highly purified antigen was used. However, absorption of the rabbit serum.with sonicated extracts of uninfected cells, cell culture medium, and calf serum removed antibody to the contaminants, resulting in antiserum monospecific for A antigen in immunodiffusion analysis. When puri- fied A antigen was analyzed on SDS-PAGE, four poly— peptides with approximate molecular weights of 21,000; 52,000; 57,000; and 82,000 were observed (49). A re-examination of the data strongly suggests that the two polypeptides with molecular weights of 52,000 and 57,000 are actually two poorly resolved peaks of a broad band that may represent the 61-65,000 molecular weight A antigen observed in this study. This is significant in light of the observation that under certain gel conditions A antigen sometimes appears as a doublet. The difference in molecular weight values observed in this study and the study by Long, et a1. (49) could be accounted for by the different SDS-PAGE buffer systems used. Long found purified A antigen had an apparent molecular weight of 44,800 by gel filtration in Sephadex G-200 and an apparent molecular weight of 70 53,160 by calibration from sedimentation coefficient. The differences in the molecular weight values observed by Long using gel filtration and those found in this study by SDS-PAGE following immun0precipitation are probably a consequence of the technical differences involved in these different analytical procedures. It is also difficult to achieve an accurate assessment of the molecular weight of glycosylated proteins by SDS- PAGE since glycosylated polypeptides may bind SDS anomalously, resulting in migration rates not strictly inversely pr0portional to the logarithm of the molecular weight (103). Thus, as indicated above, the size estimates resulting from this molecular characteriza- tion study must be considered apparent molecular weights. It has been previously demonstrated by immunodiffusion that HVT and MDHV infected cells share an immunologically similar A antigen (49,109). SDS- PAGE immun0precipitation analysis of culture medium from HVT infected cells with sera specific for MDHV-A antigen also demonstrates a 61-65,000 polypeptide similar to that of MDHV (Figure 6). This result also agrees with the preliminary immun0precipitation studies of van Zaane and Geilkens (112). The finding that both HVT and MDHV infected cells share an immunologically similar A antigen is Significant in light of 71 hybridization studies which indicate that HVT and MDHV Share only a 2-5% DNA homology. This would allow MDHV and HVT infected cells to share coding potential to only 4 daltons three or four proteins of approximately 5 x 10 (47). Presumably, MDHV~A antigen, or at least a portion of the polypeptide responsible for its antigenic identity, would be encoded by this region of homology. Since vaccination with HVT confers a highly effective immunity against Marek's disease, these common proteins maybe of major importance when considering anti-viral immunity to Marek's disease. Because A antigen is a common antigen, it would be of major interest to determine the functional role of A antigen in the host's response to Marek's disease. Attempts by many laboratories have been hampered by poor purification of the antigen, but preliminary work cited by Velicer, et al. (106) suggested that the monospecific rabbit anti-A serum did not neutralize cell-free MDHV. It also appeared that both A and B antigens were associated with the plasma membrane of infected cells. A similar procedure and general approach used for the identification of A antigen on SDS-PAGE, was applied in an attempt to detect B antigen. Due to the cell-associated nature of B antigen, detergent lysates 72 of infected cells were examined. Direct analysis of 35S-methionine labeled infected polypeptides found in and uninfected cell lysates on SDS-PAGE Show a very complex pattern. The most noticeable virus induced polypeptides have molecular weights of 83,000 and 150,000 (Figure 7A). SDS-PAGE analysis of acetone precipitates from 14 C-glucosamine labeled cell lysates did not Show the presence of any major virus-specific glyc0proteins, except for the 60-65,000 dalton molecule which could be A antigen (Figures 7B and 7C). The presence of A antigen in cell lysates probably represents cell-associated A antigen (106). It could also be a result of contamina- tion of cell lysates with culture medium, despite the fact that cell monolayers were washed several times before lysis. Immun0precipitation analysis of 35S-methionine labled lysates most consistently reveal virus-specific polypeptides of molecular weight 83,000 and 150,000 on SDS-PAGE (Figure 8). These bands are evident in infected cell lysates immun0precipitated with RaPM, RoB, and ICS. The 83,000 and 150,000 molecular weight polypeptides are also visible after reaction of infected cell lysates with NCS and NRS, but to a much lesser degree than with immune sera. The appearance of these polypeptides at low levels with nonimmune sera is 73 probably due to nonspecific binding of these polypep- tides to the IgG molecule or to the S.aureus immuno- adsorbant. Since the 83,000 and 150,000 molecular weight polypeptides are much more intensely visible after immun0precipitation with all three sera known to have specificity for B antigen, these polypeptides appear to be immun0precipitated and can be considered major candidates for B antigen. SDS-PAGE analysis of immun0precipitates prepared from lysate material with B antigen specific sera could not conclusively identify B antigen. The fact that infected cell lysates contain a large number of normal cell components, as well as the tendency of immune sera to react nonspecifically with normal cell components, are factors that hindered the identifica- tion of B antigen by contributing to the complexity and variability of the SDS-PAGE profiles of infected cell lysates. Due to the cell-associated nature of MDHV, it is impossible to achieve a synchronized infection, and consequently infected cell lysates always contain an array of infected and uninfected cell polypeptides. The overall appearance of the immun0precipitation profile of infected cell lysates on SDS-PAGE is therefore influenced by the extent in which the monolayer is infected. If the monolayer is 74 lysed when the bulk of the cells are well advanced in the infectious cycle, then most of the labeled proteins will be virus-induced. If cells are lysed when only a fraction of the monolayer is infected, virus-induced precursor polypeptides, including proteins in different stages of glycosylation, will be more apparent. Results of blocking experiments with purified B antigen are not conclusive enough to determine the relationship of either of the candidate B antigen polypeptides to B antigen. The results shown in Figure 8 demonstrate the prior incubation of rabbit anti-B sera with B antigen purified through isoelectric focusing not only eliminated the appearance of both the 83,000 and 150,000 molecular weight polypeptides from the immun0precipitation profile on SDS-PAGE, but eliminated many of the background polypeptides as well. It was, therefore, difficult to assess the ability of the B antigen preparation to specifically block B antigen related polypeptide(s). This could be due to contaminating proteins in the partially purified B antigen preparation which might be reacting with components in the immune sera. As a result, both background polypeptides and candidate B antigen poly- peptides may be blocked. Alternatively, due to a high carbohydrate content, B antigen might be a glyc0protein 75 which adheres to other polypeptides or to immune sera, thus entirely blocking the immun0precipitating capabili- ties of the sera. B antigen has been characterized as a common antigen between HVT and MDHV infected cells (106,109). For this reason, HVT infected cell lysates were also subject to immun0precipitation analysis with B antigen reactive sera (Figure 10). The fact that both the 83,000 and the 150,000 molecular weight polypeptides were visible on SDS-PAGE is significant in light of the minimal amount of DNA homology between the two viruses (26,35,47). This observation also supports the possibility that the two polypeptides are related to an antigen(s) common to HVT and MDHV infected cells, which may include B antigen. 14C-glucosamine labeled lysates by Analysis of immun0precipitation with B antigen reactive sera followed by SDS-PAGE did not provide any evidence for the glycosylation of either the 83,000 or the 150,000 candidate B antigen polypeptides. Figure 9 shows that the only major virus-induced, 14C-glucosamine labeled molecule that is immun0precipitated by both RaPM and RoB has a molecular weight of approximately 230,000. However, a virus-induced polypeptide of 230,000 molecular weight is not visible on SDS-PAGE 76 35S-methionine labeled immun0precipitation profiles of material, although it is possible that its presence is obscured by background polypeptides, especially if methionine constitutes only a small fraction of the glyc0peptide. Alternatively, the SDS-PAGE conditions used may not have maximized resolution in that region of the gel, and further analysis on a 5% gel, rather than a 78% gel, may be necessary. 1 35 The preliminary finding that the S-methionine and 14C-glucosamine labeled lysates yield very different immun0precipitation profiles on SDS-PAGE suggest a number of possibilities concerning the relation of the 355-methionine labeled polypeptides, the 230,000 dalton molecule, and B antigen. One possibility is that the 230,000 dalton molecule represents B antigen while the 83,000 and 150,000 molecular weight polypeptides are virus-induced polypeptides reacting to antibody in the immune sera which is specific for viral proteins other than B antigen. However, this would imply that the different antisera with anti-B activity would, in every case, have this same antibody present. This interpretation can only be substantiated by comparative tryptic peptide analysis of the 83,000 and 150,000 dalton polypeptides and the polypeptide portion of the 230,000 dalton molecule. 77 Another interpretation of the different SDS-PAGE 35 immun0precipitation profiles of S-methionine and l4C-glucosamine labeled lysates involves the existence of glycosylated intermediates. Most viral glyc0proteins, including herpesvirus glyc0proteins, are membrane proteins (104) and current theories of membrane glyc0protein synthesis lend support to the existence of partially glycosylated intermediates. There also appear to be many biosynthetic and structural similari- ties among membrane glycoproteins of infected and uninfected cells (20,29). Current studies indicate that membrane glyc0proteins are composed of a core carbohydrate chain linked to asparagine residues of a backbone polypeptide chain as depicted below (20): Man n GlcNAC FucoseiGlcNAC -Asn- Membrane glyc0protein structures tend to diverge from this point. Mannose-linked side chains containing vary- ing amounts of mannose, galactose, n—acetylglucosamine, Sialic acid, and fucose are added to this asparagine linked carbohydrate core (20,30,84). 78 Extensive studies concerning the biosynthesis of viral glyc0proteins, such as the G glyc0protein of vesicular stomatitis virus (VSV), have been important in elucidating the important steps involved in the growth and maturation of membrane glyc0proteins. The first step in the glycosylation of the VSV G glyco- protein occurs when the core carbohydrate chain is transferred from a lipid-linked oligosaccaride carrier to the G polypeptide being synthesized on polyribosomes of the rough endoplasmic reticulum. The partially glycosylated polypeptide is then inserted into smooth internal membranes of the Golgi apparatus where additional carbohydrate chains are both added and trimmed to yield the final virus-Sized G glyc0protein (29,30,84). In addition, proteolytic cleavage of the VSV G polypeptide also appears to be associated with membrane insertion (37). The glyc0protein is then transported to the plasma membrane where it is involved in envelopment of the virion (37). Although current knowledge of the structure and biosynthesis of herpesvirus glyc0proteins is not as extensive as that of the VSV G glyc0protein, studies of herpes simplex virus indicate that the biosynthesis of herpesvirus glyc0proteins and VSV G glyc0protein share many similarities. The initial site of glycosylation 79 of herpes simplex virus glyc0proteins occurs in the rough endOplasmic reticulum, either before completion or immediately after synthesis of the polypeptide, while the bulk of carbohydrate addition is associated with smooth membranes (27). There appears to be a large pool of nonglycosylated precursor polypeptides in cells infected at high multiplicities (27). Immun0precipita- tion analysis of herpes Simplex type 1 infected cells with antisera specific for viral glyc0proteins indicate that glycosylation of precursor polypeptides occur in two discreet steps, yielding partially glycosylated intermediates and fully glycosylated products (103). There is no existing evidence of proteolytic cleavage of the polypeptide chain, or of any processing or alteration of the carbohydrate chains. In view of the current knowledge of glycoprotein synthesis, the 83,000 molecular weight polypeptide might represent the unglycosylated polypeptide backbone; the 150,000 molecular weight polypeptide might represent a partially glycosylated intermediate; and the 14C-glucosamine labeled, 230,000 molecule could be the fully glycosylated antigen. Problems with this interpretation include the fact that the polypeptide portion of the 230,000 molecule should also be present in SDS-PAGE immun0precipitation analysis of 80 35 S-methionine labeled lysates. In addition to the reasons cited above, the apparent nonexistence of a 35S-methionine virus-induced 230,000 polypeptide in labeled lysates might be a consequence of extensive proteolytic cleavage of the polypeptide portion of the antigen during the course of glycosylation, which might subsequently reduce its intensity on SDS—PAGE. Following this same line of reasoning, if the 150,000 polypeptide actually is a partially glycosylated B antigen intermediate, it would be expected that this 150,000 molecular weight polypeptide show some incor- poration of l4C-glucosamine. The apparent absence of a 150,000 glyc0protein on SDS-PAGE analysis of 14C-glucosamine labeled material might be a consequence of poor 14C-glucosamine incorporation, and a much longer exPosure of the gel might ultimately identify the 150,000 polypeptide as a glyc0protein. In addition, labeling with another carbohydrate may also be required. The exact relationship between the 230,000 dalton molecule and the 83,000 and 150,000 polypeptides can ultimately be delineated by further characterization of both the protein and carbohydrate components of these molecules. Tryptic peptide analysis of the polypeptide chain can be accomplished by first removing the carbohydrate portion of the glyc0protein with 81 endonuclease H (108), while further characterization of the carbohydrate chain can be accomplished after removing the polypeptide chain with proteases. These eXperiments, however, are beyond the scepe of this master's thesis. APPEND IX 82 APPENDIX ATTEMPTED IDENTIFICATION OF B ANTIGEN BY PURIFICATION WITH CONCANAVALIN A AFFINITY CHROMATOGRAPHY AND ISOELECTRIC FOCUSING Materials and Methods Concanavalin A (Con A) Affinity Chromatography Isolation of glyc0proteins from labeled cell extracts by Con A affinity chromatography was performed as described by Velicer, et al. (106). Sonically dis- 35S—methionine rupted MDHV-infected cells, labeled with and clarified at 147,000 x g for 1 hr, were loaded at a protein concentration of 1 mg/ml onto a Con A column with a packed volume of 10 ml. Fractions of 0.5 ml each were collected and assayed for radioactivity, and peak fractions that eluted with a-methly-mannoside were pooled and dialyzed for isoelectric focusing. Isoelectric Focusing Pooled fractions containing labeled glyc0pro- teins that eluted from Con A columns with a-methly- mannoside, were dialyzed and isoelectric focused, as described by Velicer, et al. (106) for the purification 83 84 of B antigen. At the end of the focusing period, 1 ml fractions were collected and assayed for pH and radioactivity. Fractions at the isoelectric point of B antigen (pH 4.5) were pooled and used for SDS-PAGE analysis. Results and Conclusions As an alternative approach, the identification of B antigen was attempted using the methods of B antigen purification involving Con A affinity chromatog- raphy and isoelectric focusing, as established by Velicer, et al. (106). The rationale was to determine which polypeptide(s) could be found at the pI of B antigen after isoelectric focusing. 35S-methionine labeled cell extracts were applied to a Con A column and glyc0proteins eluting with a-methly-mannoside were pooled and applied to an isoelectric focusing column. Profiles of both the Con A and the isoelectric focusing columns are shown in Figures 11 and 12, respectively. The major difficulty encountered with these procedures involved the loss of large amounts of labeled material with each step of the purification process so that an insufficient amount of labeled material was available for SDS-PAGE analysis. Approximately 55% of the total amount of labeled material that was applied to the Con A column remained irreversibly bound to the column 85 even after elution with 2 M NaCl. Only 2% of the labeled material applied to the column eluted as glyc0protein with o-methyl-mannoside and only 2% of this material eluted at the B antigen isoelectric point of 4.5. As a result, an insufficient amount of labeled protein remained for immunoprecipitation analysis on SDS-PAGE following these purification procedures, and no conclusions could be drawn concerning the nature of the B antigen. In future attempts at the identification of B antigen by these methods of purification, the loss of large amounts of labeled protein might be minimized by the addition of unlabeled cell extracts with the labeled cell extracts. This should result in a decreased percentage of labeled protein lost at each step of the purification process, although it might reduce the Specific activity of the B antigen in the mixture. 86 Figure ll.--Con A affinity chromatography of 35S—methionine labeled cell sonicates. The indicated frac- tions eluting with a—methyl-mannoside were pooled, dialyzed and used for isoelectric focusing. The arrows indicate the addition of 0.1 M o—methyl-mannoside in PBS and 2.0 M NaCl in PBS, respectively. 87 mmmZDz ZOHBUQNh OOH ova ONH 00H om ow ov 0H i AUtz EN 2220 Lon OI X TN/WdD SNINOIHLLSW-SSE S- 88 Figure 12.--Isoelectric focusing analysis. Pooled and dialyzed fractions eluting from a Con A column with o-methly-mannoside were subject to isoelectric focusing. Fractions at the isoelectric point of B antigen (pH 4.5) were pooled and used for immun0precipitation analysis on SDS-PAGE. ma e mi mwn I v 89 X 0 N mV 3 m / mm T. O Q .7 m ... . .o . . . . . .. .~ . ~ . s- a. ... all E. E. .....::.-.... -..--.......:..-.:.:.......+. x . m.» we zoohsz< m . . Hd Q ‘ - Q - - REFERENCES 90 LIST OF REFERENCES Adldinger, H. K., and B. W. Calnek. 1973. Pathogene- sis of Marek's disease: Early distribution of virus and viral antigens in infected chickens. J. Natl. Cancer Inst. 50:1287-1298. Ahmed, M., and G. Schidlovsky. 1972. 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