JOURNAL OF VIROLOGY, Mar. 1978, p. 852-859 0022-538X/78/0025-0852$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 25, No. 3 Printed in U.S.A.
Solubilization and Characterization of Herpesvirus saimiriInduced Membrane Antigens LOUIS F. QUALTIERE* AND GARY R. PEARSON Department of Microbiology, Mayo Foundation and Mayo Medical School, Rochester, Minnesota 55901 Received for publication 7 October 1977
Treatment of Herpesvirus saimiri (HVS)-infected owl monkey cells by limited papain digestion removed the HVS-induced membrane antigen (MA) as determined by membrane immunofluorescence and antibody-dependent lymphocyte cytotoxicity (ADLC). Soluble antigenically active HVSMA was detected by inhibition of ADLC and by the decreased binding of "25I-labeled staphylococcus protein A to HVS-infected cells after absorption of an anti-MA-positive serum with papain extracts. Approximately 38% of the inhibitory activity of the papain extracts was sedimentable at 100,000 x g, indicating that the released MA was heterogeneous in size. Preliminary investigations by gel filtration chromatography identified a major peak of MA with a molecular weight between 20,000 and 50,000.
Virus-induced antigens have been detected in the plasma membranes of cells infected with different herpesviruses (5, 8-10, 13, 18, 19, 22, 23). The synthesis of these membrane antigens (MA) appears to depend mainly on late gene functions requiring viral DNA synthesis (13,15), although there is also evidence with some of these viruses for the existence of an "early" MA synthesized in the absence of prior viral DNA replication (3). Investigations on MA induced by herpes simplex virus, Epstein-Barr virus, and Marek's disease herpesvirus have provided evidence that these antigens are also expressed in the envelope of infectious virus particles and are probably the major viral components responsible for the induction of neutralizing antibodies against the specific virus (8, 14, 15, 19, 20). The antigens appear, therefore, to represent the viral proteins of major importance in regard to the host immune response to herpesvirus infection. Herpesvirus-induced MA has only been purified and extensively characterized biochemically for herpes simplex virus (4, 5, 22). There is little information in the literature on the purification and characterization of these antigens induced by other herpesviruses, although encouraging results have recently been reported for Marek's disease herpesvirus (8) and Epstein-Barr virus (2). Because of their apparent role in the induction of immunity to these viruses and their associated diseases and, therefore, their potential importance as a nucleic acid-free viral vaccine, it is important to attempt to isolate and characterize these antigens from other herpesviruses and to precisely determine their biological significance in immunity. In this paper, we report the results of preliminary investigations directed
at the isolation of MA from cells infected with Herpesvirus saimiri (HVS), an oncogenic nonhuman primate herpesvirus (1, 6, 12). MATERIALS AND METHODS Virus. HVS passaged in owl monkey kidney (OMK) cells served as the virus inoculum for these experiments. Cells. OMK cells were cultured in the presence of RPMI 1640 medium containing 10% (vol/vol) heatinactivated fetal calf serum and gentamicin (50 ,ug/ml). MA production. HVS was absorbed for 2 h at 370C at a multiplicity of infection of 0.01 on a 75 to 80% confluent monolayer of OMK cells. Medium was added at the end of the hour, and the cultures were then incubated at 37°C in an atmosphere of 5% CO/95% air for 3 to 5 days. At this time, cultures usually showed extensive cytopathic effects characteristic of HVS. A light trypsinization at this time (0.1% trypsin for 5 min) generally yielded a cell population (90 to 95% viable) containing 45 to 75% MA-positive cells as determined by membrane immunofluorescence (MF) (13). The treatment with 0.1% trypsin for 5 min had no measurable effect on the number of MA positive cells or the relative intensity of the fluorescent staining. In fact, treatment of HVS-OMK cells for up to 20 min with 0.1% trypsin had no apparent effect on MA expression (unpublished observations). MF assay. The indirect MF assay was performed as previously described in detail (13), using a fluorescein-conjugated goat anti-human immunoglobulin G (IgG) (H and L chain specific) reagent (Hyland Laboratories, Los Angeles, Calif.). Sera. The main serum used in these experiments was obtained from an owl monkey that developed lymphoma after HVS infection. The antibody titers for this serum against HVS-induced antigens (11) were 640 against late antigen, early antigen, and MA, as determined by MF. An antibody-negative control serum pool was prepared from uninfected monkeys. The
852
VOL. 25, 1978
HERPESVIRUS SAIMIRI-INDUCED MEMBRANE ANTIGENS
fractions were heat inactivated at 560C for 30 min and stored at -20°C until used. ADLC assay. The antibody-dependent lymphocyte cytotoxicity (ADLC) assay was performed with 51Cr-labeled HVS-OMK cells as previously described in detail (16, 17), using baboon lymphocytes separated on a Ficoll-Hypaque gradient (LSM solution, Litton Bionetics, Inc., Kensington, Md.) as effector cells. Cytotoxicity was calculated by subtracting the background (or spontaneous) release from the 5'Cr release induced by the serum/lymphocyte mixture divided by initial 5'Cr incorporation minus spontaneous release times 100. ADLC was calculated by subtracting the cytotoxicity value for lymphocytes incubated in the presence of a negative serum from the cytotoxicity figure in the presence of lymphocytes and immune serum. When papain-treated HVS-OMK cells were used as target cells in the ADLC, the final cytotoxicity value was normalized to correct for the higher spontaneous release observed in the target cells after papain digestion. In the inhibition assays, the standard immune serum (OM 981; ADLC titer = 64,000) was first incubated with different concentrations of papain extracts overnight at 40C before assay in the ADLC
serum
test.
Radiolabeling of staphylococcal protein A. Protein A (Staphylococcus aureus), obtained from Pharmacia Fine Chemicals, Piscataway, N.J., was dissolved in 0.1 M phosphate buffer, pH 7.4. The method of "2I labeling was a modification of the chloramine T method (7), using a chloramine T-to-protein ratio of 1:10 (wt/wt) and a 15-min incubation period at 25°C. The reaction was terminated by the addition ofsodium metabisulfite. This mixture was then passed through a 1.5-ml column of Bio-Gel A containing covalently coupled human IgG (the active protein A will bind to the Fc region of human IgG). The column was washed exhaustively to remove all the unbound "2I as well as any inactive radiolabeled protein A before the specifically bound radiolabeled protein A was then eluted by 3.5 M potassium thiocyanate (KSCN). The eluate was dialyzed exhaustively to remove the KSCN, diluted to 0.01 phosphate-0.15 M NaCl, pH 7.2 (PBS)-1% bovine serum albumin (BSA), and stored at -20°C until used. Specific activities of 1 x 106 cpm per jg of protein A were obtained at a 40% labeling efficiency. Papain treatment. Uninfected OMK well as HVS-OMK cells were removed by trypsinization (0.1% for 5 min) and then washed three times with Hanks buffered salt solution. Only when the viability was greater than 90% were the cells utilized for papain digestion. To 0.600 ml of cells (1 x 107 to 2 x 107) in Hanks buffered salt solution was added 0.025 ml of papain (10 mg/ml) (Sigma Chemical Co., St. Louis, Mo.; 2x crystallized, 20 U/mg of solid) followed by 0.100 ml of 10 mM L-cysteine (Sigma) to start the reaction. The papain-cell mixture was then incubated at 370C with slow mixing for the appropriate length of time. The reaction was terminated by the addition of 0.090 ml of 40 mM iodoacetamide, after which a check on cell viability usually revealed less than a 5% decrease when compared with the pre-papain-treated cells. The cells were then removed by centrifugation at 1,000 x g for 15 min, and the supernatant fluid was as
853
dialyzed exhaustively against PBS at 4°C. The insoluble material that formed during dialysis was removed by centrifugation at 10,000 x g fot 30 min followed by centrifugation at 100,000 x g for 60 min. This final supernatant fluid was operationally defined as "soluble" papain extract and was the extract used in all the blocking assays and in the gel chromatography studies. IPAT. The isotopic staphylococcal protein A test (IPAT) utilized in this study for measuring antibody bound to MA was a modification of the isotopic antiglobulin test originally described by Sparks et al. (21). With this assay, III-labeled protein A was substituted to detect membrane-bound antibody instead of 125Ilabeled antiglobulin. Target cells were divided into aliquots of 2 x 105 and placed into Kimble disposable tubes (6 by 50 mm) followed by the addition of 0.100 ml of serum diluted in PBS-1% BSA. The cell-serum mixtures were incubated at 370C for 60 min and then washed four times with a total of 4 ml of PBS-1% BSA. The final cell pellet was resuspended in 0.100 ml (1 x 105 cpm) of protein A diluted in PBS-1% BSA and incubated at 370C for 1 h. The cells were again washed four times with PBS 1%-BSA and counted in a Beckman autogamma spectrophotometer. All tubes were set up in duplicate. Corrections were made for nonspecifically bound protein A bound in the presence of normal pooled owl monkey serum at the same dilution as the immune serum tested. Inhibition studies were performed by first incubating the immune serum with different quantities of the papain extracts overnight at 40C. Specific inhibition was measured as a reduction of the lII-labeled protein A bound in comparison with appropriate controls. Column fractionation. Gel filtration chromatography was carried out on a column (6 by 90 cm) of Bio-Gel A-5m (Bio-Rad, Richmond, Calif.) packed under a head pressure of 12 cm. The column was equilibrated with PBS, pH 7.2, and run at a flow rate of 7.6 ml/h at 40C.
RESULTS Solubilization of HVS-OMK-induced MA. To determine whether HVS-MA could be solubilized from the cell surface, viable HVS-OMK celis were treated with different enzymes to determine whether one or more of these enzymes might cleave or promote the release of HVSinduced membrane proteins from the cell surface without cell damage and significant leakage of intracellular HVS antigens into the reaction supernatant. Of the three enzymes tested (neuraminidase, trypsin, and papain), only papain was able to effectively remove HVS-OMK membrane proteins. Neuraminidase was completely unsuccessful in stripping the virally induced MA off HVS-OMK cells, and even 2.5% trypsin was only partially successful (
Vol. 25, No. 3 Printed in U.S.A.
Solubilization and Characterization of Herpesvirus saimiriInduced Membrane Antigens LOUIS F. QUALTIERE* AND GARY R. PEARSON Department of Microbiology, Mayo Foundation and Mayo Medical School, Rochester, Minnesota 55901 Received for publication 7 October 1977
Treatment of Herpesvirus saimiri (HVS)-infected owl monkey cells by limited papain digestion removed the HVS-induced membrane antigen (MA) as determined by membrane immunofluorescence and antibody-dependent lymphocyte cytotoxicity (ADLC). Soluble antigenically active HVSMA was detected by inhibition of ADLC and by the decreased binding of "25I-labeled staphylococcus protein A to HVS-infected cells after absorption of an anti-MA-positive serum with papain extracts. Approximately 38% of the inhibitory activity of the papain extracts was sedimentable at 100,000 x g, indicating that the released MA was heterogeneous in size. Preliminary investigations by gel filtration chromatography identified a major peak of MA with a molecular weight between 20,000 and 50,000.
Virus-induced antigens have been detected in the plasma membranes of cells infected with different herpesviruses (5, 8-10, 13, 18, 19, 22, 23). The synthesis of these membrane antigens (MA) appears to depend mainly on late gene functions requiring viral DNA synthesis (13,15), although there is also evidence with some of these viruses for the existence of an "early" MA synthesized in the absence of prior viral DNA replication (3). Investigations on MA induced by herpes simplex virus, Epstein-Barr virus, and Marek's disease herpesvirus have provided evidence that these antigens are also expressed in the envelope of infectious virus particles and are probably the major viral components responsible for the induction of neutralizing antibodies against the specific virus (8, 14, 15, 19, 20). The antigens appear, therefore, to represent the viral proteins of major importance in regard to the host immune response to herpesvirus infection. Herpesvirus-induced MA has only been purified and extensively characterized biochemically for herpes simplex virus (4, 5, 22). There is little information in the literature on the purification and characterization of these antigens induced by other herpesviruses, although encouraging results have recently been reported for Marek's disease herpesvirus (8) and Epstein-Barr virus (2). Because of their apparent role in the induction of immunity to these viruses and their associated diseases and, therefore, their potential importance as a nucleic acid-free viral vaccine, it is important to attempt to isolate and characterize these antigens from other herpesviruses and to precisely determine their biological significance in immunity. In this paper, we report the results of preliminary investigations directed
at the isolation of MA from cells infected with Herpesvirus saimiri (HVS), an oncogenic nonhuman primate herpesvirus (1, 6, 12). MATERIALS AND METHODS Virus. HVS passaged in owl monkey kidney (OMK) cells served as the virus inoculum for these experiments. Cells. OMK cells were cultured in the presence of RPMI 1640 medium containing 10% (vol/vol) heatinactivated fetal calf serum and gentamicin (50 ,ug/ml). MA production. HVS was absorbed for 2 h at 370C at a multiplicity of infection of 0.01 on a 75 to 80% confluent monolayer of OMK cells. Medium was added at the end of the hour, and the cultures were then incubated at 37°C in an atmosphere of 5% CO/95% air for 3 to 5 days. At this time, cultures usually showed extensive cytopathic effects characteristic of HVS. A light trypsinization at this time (0.1% trypsin for 5 min) generally yielded a cell population (90 to 95% viable) containing 45 to 75% MA-positive cells as determined by membrane immunofluorescence (MF) (13). The treatment with 0.1% trypsin for 5 min had no measurable effect on the number of MA positive cells or the relative intensity of the fluorescent staining. In fact, treatment of HVS-OMK cells for up to 20 min with 0.1% trypsin had no apparent effect on MA expression (unpublished observations). MF assay. The indirect MF assay was performed as previously described in detail (13), using a fluorescein-conjugated goat anti-human immunoglobulin G (IgG) (H and L chain specific) reagent (Hyland Laboratories, Los Angeles, Calif.). Sera. The main serum used in these experiments was obtained from an owl monkey that developed lymphoma after HVS infection. The antibody titers for this serum against HVS-induced antigens (11) were 640 against late antigen, early antigen, and MA, as determined by MF. An antibody-negative control serum pool was prepared from uninfected monkeys. The
852
VOL. 25, 1978
HERPESVIRUS SAIMIRI-INDUCED MEMBRANE ANTIGENS
fractions were heat inactivated at 560C for 30 min and stored at -20°C until used. ADLC assay. The antibody-dependent lymphocyte cytotoxicity (ADLC) assay was performed with 51Cr-labeled HVS-OMK cells as previously described in detail (16, 17), using baboon lymphocytes separated on a Ficoll-Hypaque gradient (LSM solution, Litton Bionetics, Inc., Kensington, Md.) as effector cells. Cytotoxicity was calculated by subtracting the background (or spontaneous) release from the 5'Cr release induced by the serum/lymphocyte mixture divided by initial 5'Cr incorporation minus spontaneous release times 100. ADLC was calculated by subtracting the cytotoxicity value for lymphocytes incubated in the presence of a negative serum from the cytotoxicity figure in the presence of lymphocytes and immune serum. When papain-treated HVS-OMK cells were used as target cells in the ADLC, the final cytotoxicity value was normalized to correct for the higher spontaneous release observed in the target cells after papain digestion. In the inhibition assays, the standard immune serum (OM 981; ADLC titer = 64,000) was first incubated with different concentrations of papain extracts overnight at 40C before assay in the ADLC
serum
test.
Radiolabeling of staphylococcal protein A. Protein A (Staphylococcus aureus), obtained from Pharmacia Fine Chemicals, Piscataway, N.J., was dissolved in 0.1 M phosphate buffer, pH 7.4. The method of "2I labeling was a modification of the chloramine T method (7), using a chloramine T-to-protein ratio of 1:10 (wt/wt) and a 15-min incubation period at 25°C. The reaction was terminated by the addition ofsodium metabisulfite. This mixture was then passed through a 1.5-ml column of Bio-Gel A containing covalently coupled human IgG (the active protein A will bind to the Fc region of human IgG). The column was washed exhaustively to remove all the unbound "2I as well as any inactive radiolabeled protein A before the specifically bound radiolabeled protein A was then eluted by 3.5 M potassium thiocyanate (KSCN). The eluate was dialyzed exhaustively to remove the KSCN, diluted to 0.01 phosphate-0.15 M NaCl, pH 7.2 (PBS)-1% bovine serum albumin (BSA), and stored at -20°C until used. Specific activities of 1 x 106 cpm per jg of protein A were obtained at a 40% labeling efficiency. Papain treatment. Uninfected OMK well as HVS-OMK cells were removed by trypsinization (0.1% for 5 min) and then washed three times with Hanks buffered salt solution. Only when the viability was greater than 90% were the cells utilized for papain digestion. To 0.600 ml of cells (1 x 107 to 2 x 107) in Hanks buffered salt solution was added 0.025 ml of papain (10 mg/ml) (Sigma Chemical Co., St. Louis, Mo.; 2x crystallized, 20 U/mg of solid) followed by 0.100 ml of 10 mM L-cysteine (Sigma) to start the reaction. The papain-cell mixture was then incubated at 370C with slow mixing for the appropriate length of time. The reaction was terminated by the addition of 0.090 ml of 40 mM iodoacetamide, after which a check on cell viability usually revealed less than a 5% decrease when compared with the pre-papain-treated cells. The cells were then removed by centrifugation at 1,000 x g for 15 min, and the supernatant fluid was as
853
dialyzed exhaustively against PBS at 4°C. The insoluble material that formed during dialysis was removed by centrifugation at 10,000 x g fot 30 min followed by centrifugation at 100,000 x g for 60 min. This final supernatant fluid was operationally defined as "soluble" papain extract and was the extract used in all the blocking assays and in the gel chromatography studies. IPAT. The isotopic staphylococcal protein A test (IPAT) utilized in this study for measuring antibody bound to MA was a modification of the isotopic antiglobulin test originally described by Sparks et al. (21). With this assay, III-labeled protein A was substituted to detect membrane-bound antibody instead of 125Ilabeled antiglobulin. Target cells were divided into aliquots of 2 x 105 and placed into Kimble disposable tubes (6 by 50 mm) followed by the addition of 0.100 ml of serum diluted in PBS-1% BSA. The cell-serum mixtures were incubated at 370C for 60 min and then washed four times with a total of 4 ml of PBS-1% BSA. The final cell pellet was resuspended in 0.100 ml (1 x 105 cpm) of protein A diluted in PBS-1% BSA and incubated at 370C for 1 h. The cells were again washed four times with PBS 1%-BSA and counted in a Beckman autogamma spectrophotometer. All tubes were set up in duplicate. Corrections were made for nonspecifically bound protein A bound in the presence of normal pooled owl monkey serum at the same dilution as the immune serum tested. Inhibition studies were performed by first incubating the immune serum with different quantities of the papain extracts overnight at 40C. Specific inhibition was measured as a reduction of the lII-labeled protein A bound in comparison with appropriate controls. Column fractionation. Gel filtration chromatography was carried out on a column (6 by 90 cm) of Bio-Gel A-5m (Bio-Rad, Richmond, Calif.) packed under a head pressure of 12 cm. The column was equilibrated with PBS, pH 7.2, and run at a flow rate of 7.6 ml/h at 40C.
RESULTS Solubilization of HVS-OMK-induced MA. To determine whether HVS-MA could be solubilized from the cell surface, viable HVS-OMK celis were treated with different enzymes to determine whether one or more of these enzymes might cleave or promote the release of HVSinduced membrane proteins from the cell surface without cell damage and significant leakage of intracellular HVS antigens into the reaction supernatant. Of the three enzymes tested (neuraminidase, trypsin, and papain), only papain was able to effectively remove HVS-OMK membrane proteins. Neuraminidase was completely unsuccessful in stripping the virally induced MA off HVS-OMK cells, and even 2.5% trypsin was only partially successful (
