SUSHILKUMAR G. DEVARE AND JOHN R. STEPHENSON* ... reactivity by agar gel immunodiffusion analy- ... reactivity detected by agar gel immunodiffu-.
Vol. 23, No. 2 Printed in U.S.A.
JOURNAL OF VIaoLoGY, Aug. 1977, p. 443-447 Copyright 0 1977 American Society for Microbiology
Biochemical and Immunological Characterization of the Major Envelope Glycoprotein of Bovine Leukemia Virus SUSHILKUMAR G. DEVARE AND JOHN R. STEPHENSON* Laboratory of RNA Tumor Viruses, National Cancer Institute, Bethesda, Maryland 20014 Received for publication 8 April 1977
The major envelope glycoprotein of bovine leukemia virus was isolated by lectin-bound Sepharose and DEAE-cellulose column chromatography. This protein was shown to have a molecular weight of about 51,000 and to lack detectable immunological cross-reactivity with glycoproteins of other oncornaviruses. Sera obtained from 100% of cattle examined with clinically diagnosed lymphosarcoma contained high-titered antibody to 125I-labeled bovine leukemia virus glycoprotein, whereas sera from animals in a disease-free herd were antibody negative.
Lymphosarcoma of domestic cattle provides a RNA viruses have revealed these proteins to be unique model system for studies on the role of of about 70,000 in molecular weight and to exoncornaviruses in tumors of their natural hibit type-, group-, and interspecies-specific anhosts. The etiological association of this disease tigenic determinants (1, 7, 19). Whereas the with a horizontally transmissible infectious immunological properties of type B oncornaviagent, bovine leukemia virus (BLV), is well rus glycoproteins have been less well characterestablished (2, 14). Previously, we described the ized, the molecular weight of the major envedevelopment of a radioimmunoassay for the lope glycoprotein of a representative virus of major 24,000-molecular-weight (p24) structural this group, mouse mammary tumor virus protein of BLV (4). Application of this radioim- (MMTV), has been shown to be about 52,000 (5, munological technique to studies of the epide- 13, 15, 16). miology of bovine lymphosarcoma led to the Whereas the purification and development of demonstration of high-titer antibodies to BLV competition immunoassays for glycoproteins of p24 in sera of 100% of animals with clinically several oncornaviruses have been achieved (7diagnosed lymphosarc6ma (4). In a comparison 9, 18, 19), this has been somewhat difficult beof presently available serological tests for de- cause of the labile nature of these proteins. In tection of antibodies to BLV, radioimmunopre- the present study an improved technique for cipitation of 125I-labeled BLV p24 was shown to isolation of oncornavirus glycoproteins was debe much more sensitive than previously de- veloped based upon a combination of affinity scribed procedures (3). A number of sera, how- column chromatography, using lectin-bound ever, were observed that exhibited positive Sepharose (LH-Sepharose), and DEAE-cellureactivity by agar gel immunodiffusion analy- lose chromatography. A 51,000-molecularsis but failed to precipitate 125I-labeled BLV p24 weight envelope glycoprotein of BLV isolated to a significant extent (3). One possible expla- by this means was labeled at high specific activnation for these findings was that the positive ity with 125I, using the recently developed iodoreactivity detected by agar gel immunodiffu- gen reagent, and utilized for the development of sion might be attributable to antibodies di- a radioimmunoprecipitation assay. BLV was concentrated from tissue culture rected against the envelope proteins of BLV. There is accumulating evidence that oncor- fluids of chronically infected fetal lamb kidney naviruses possess high-molecular-weight glyco- cells by density gradient centrifugation. The sylated proteins as constituents of their enve- virus (8 to 10 mg of protein) was disrupted by lopes. As a consequence of their location on the sonic treatment for 20 s in 0.05 M Tris-hydrosurface of the virion, these glycoproteins have a chloride (pH 7.8) buffer containing 1.0% Triton major role in elucidation of host immune re- X-100, clarified by centrifugation at 100,000 x sponses to exogenous virus infection. Moreover, g, and applied to an LH-Sepharose column (1.5 immunoglobulins directed against viral enve- by 5 cm) prepared by the following procedure. lope glycoproteins have been shown to neutral- Activated CH-Sepharose 4B (Pharmacia Fine ize viral infectivity (7, 9, 10, 17). The isolation Chemicals, Inc., Piscataway, N.J.) was washed and immunological characterization of enve- with 1 mM HCl followed by 0.1 M a-methyllope glycoproteins of various type C and type D glucopyranoside and 0.1 M sodium bicarbonate 443
444
NOTES
(pH 8.3). A 20-mg amount of lectin from Lens culinaris (Boehringer Mannheim Biochemicals, Indianpolis, Ind.) was added to 10 g of activated Sepharose in 50 ml of 0.1 M a-methylglycopyranoside, and the mixture was gently stirred for 4 h at 4VC. Excess lectin was removed by washing with 0.1 M a-methylglucopyranoside, and the remaining active groups were blocked with 5 mM ethanolamine, pH 8.4. The LH-Sepharose was washed with three cycles of alternating pH, using 0.1 M sodium bicarbonate (pH 8.3) and 0.1 M sodium acetate (pH 4.0), and stored in 0.01 M sodium phosphate (pH 7.4)-0.15 M NaCl buffer. The column was washed with phosphate-buffered saline at 40C until free of absorbance at 280 nm, and the bound glycoprotein was eluted with 0.1 M amethylmannopyranoside at room temperature. Fractions containing a 51,000-molecular-weight protein, as determined by sodium dodecyl sul-
fate-polyacrylamide gel electrophoresis (12), were pooled, dialyzed against TET (10 mM Tris (pH 7.8), 0.2 mM EDTA, 0.5% Triton X-100) buffer, and applied to a DEAE-cellulose (Whatman, H. Reeve Angle and Co., Clifton, N.J.) column (1.5 by 5 cm) equilibrated with the same buffer. The column was washed with TET buffer, and bound protein was eluted with a linear gradient of 0.0 to 0.5 M NaCl. Fractions containing a single protein of about 51,000 in molecular weight (BLV gp5l) were pooled, aliquoted, and stored under liquid nitrogen. Following purification, BLV gp5l was labeled with 125I at high specific activity (5 to 20 ,4Ci/,g) by a newly developed procedure utilizing the iodogen reagent (Pierce Chemical Co., Rockford, Ill.). For this purpose 10 mg of iodogen was dissolved in 1 ml of chloroform and aliquoted (20 ul) into several vials. After removal of chloroform by evaporation, 10 to 15 ,ug of BLV gp5l in 0.05 ml of TET buffer and 1 mCi of 125I (Amersham/Searle, Arlington Heights, Ill.) were added. The reaction mixture was allowed to stand at room temperature for 60 s, and the iodinated glycoprotein was separated from free 125I by P-10 (Bio-Rad Laboratories, Richmond, Calif.) column chromatography. Fractions containing 125I-labeled glycoprotein were pooled, aliquoted, and stored at -20°C. A determination of radiochemical purity, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicated that 1251_ labeled BLV gp5l migrated as a single radioactive peak with a molecular weight of about 51,000 relative to protein standards (Fig. 1). Antisera produced in goats by active immunization with detergent-disrupted BLV as well as sera from cattle with clinically diagnosed lymphosarcoma were tested for immunoprecipi-
J. VIROL. 4
1
§ § T-
3
§~ I
0
x
IL
a-
z C z
w
2-
a uJ -j 'U
co
I0l
1
20 30 10 40 GEL LENGTH (mm)
50
FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of 125I-labeled BLVgp51. About 300,000 cpm 125I-labeled BLV gp5l was subjected to electrophoresis on 55-mm sodium dodecyl sulfate-polyacrylamide gels (8.75%) at 2.5 mA per gel for 3 h. After electrophoresis, the samples were either stained with Coomassie blue or sliced into 1mm fractions and tested for radioactivity in a Searle gamma counter model 1285. Molecular weight standards used for calibration included P-galactosidase (130,000), bovine serum albumin (69,000), aldolase (40,000), carbonic anhydrase (29,000), and 9&lactoglobulin (18,500).
tation of 125I-labeled BLV gp5l. As shown in Table 1, each of the anti-BLV sera tested bound 125I-labeled BLV gp5l at high titer (
