(viral transformation/viral-coded antigens/tumor immunity)

Feline sarcoma virus (FeSV) rescued from transformed nonproducer mink or rat cells contains two FeSV-specific antigens (p15 and p12), and the feline oncornavirus-associated cell membrane antigen (FOCMA). All three antigens are helper virus-independent and are encoded by the FeSV genome. FOCMA, pl5, and p12 antigens cochromatograph as phosphorylated molecules of 85,000 molecular weight (pp85), adsorb to immunoadsorbant columns prepared with antibodies to feline leukemia virus (FeLV), and are precipitated with antisera to FeLV or FOCMA. Antibodies to FOCMA can be adsorbed with fractions containing pp85 but not with FeLV proteins, including p15 and p12. Thus, a virus-coded tumor antigen which immunizes cats against tumors induced by feline type C viruses is packaged in FeSV particles and is linked to viral structural protein. The feline oncornavirus-associated cell membrane antigen (FOCMA) is found on cat cells transformed by feline leukemia (FeLV) or sarcoma (FeSV) viruses (1-3). Under experimental or field conditions, titers of antibodies to FOCMA in virusexposed cats correlate inversely with the rate of tumor progression (1, 4-6), and cells expressing FOCMA can immunize animals against viral-induced leukemias and sarcomas (7, 8). Antibodies to FOCMA, then, are important in determining whether cats can successfully resist tumors caused by oncogenic feline type C viruses. Adsorption of cat antisera reactive to FOCMA with disrupted FeLV (3, 9) or with two proteins (gp70 and p30) purified from virions (10) fails to reduce the anti-FOCMA titers. Surveys of cat sera have shown no concordance between titers of antibodies to FOCMA and those to viral gp7O, p30 (3, 10), or reverse transcriptase (unpublished observations). Thus, FOCMA appears to be distinct from the major FeLV structural proteins. "Nonproducer" mink cells transformed by FeSV also express FOCMA on their cell surfaces, suggesting that FOCMA is encoded by the feline sarcoma virus genome (11). In this report, we show that viral pseudotypes obtained from FeSV-transformed mink or rat nonproducer cells contain FOCMA as well as FeLV-related p15 and p12 antigens. MATERIALS AND METHODS Cells and Viruses. MvlLu mink cells (CCL 64) obtained from the American Type Culture Collection were used to prepare "nonproducer" clones transformed by FeSV (64F1 CL1Q, 64F3 CL7, and 64F2) or by Kirsten sarcoma virus (64J1) (12). A nonproducer rat cell clone (F3-NRK CL2) containing FeSV was similarly derived (12). Dog kidney cells (MSV/DK) transformed by Moloney [S+L-] mouse sarcoma virus (MSV) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1505 were obtained from Paul Peebles. Mink cells productively transformed with the HT-1 strain of Moloney MSV (13) were obtained by infection with MSV (M7) pseudotype stocks. Other lines included the human rhabdomyosarcoma A204 (14), canine thymus cells (FCf2Th), rat kidney cells (NRK) (15), and cat kidney cells (FEC). Type C viruses included the endogenous baboon viruses M7 and M28 (16, 17),.the simian sarcoma-associated virus (SSAV) (18), the xenotropic mouse virus AT-124 (19), the endogenous cat virus RD-114 (20), the endogenous mink virus MiLV (21), and the Rickard strain (F422) of FeLV (22). Isopycnically banded (p = 1.16 g/ml in sucrose) virions containing FeSV were obtained by infection of nonproducer mink or rat cells with helper viruses (12). Radioimmunoassays. Purification of viral p30, p15, and p12 proteins, preparation of antisera, and techniques for radioimmunoassays were as described (23-25). Gel Filtration. Viruses disrupted with detergent were filtered on 90 X 1.5 cm columns of Sephadex G-200 (23, 26). Fractions were concentrated by Iyophilization and assayed for viral proteins by radioimmunoassay. Gel filtration in Bio-Gel A5M containing 6 M guanidine hydrochloride and 20mM dithiothreitol was performed as described (25). Cells producing virus were labeled with 32p (27). Extracellular virions were concentrated, disrupted, labeled with 125I (28), and denatured in the presence of 0.1 M 2-mercaptoethanol (25). Regions of the effluent containing viral proteins were dialyzed and lyophilized (25). Preparation of Immunoadsorbant Columns. FeLV (40 mg of protein) was disrupted with detergent, filtered over Sephadex G-25, and Iyophilized. Viral protein suspended in 0.2 M Na citrate, pH 6.5, was conjugated to CNBr-activated Sepharose 4B (29); 70 mg of CNBr was used per ml Sepharose, and protein (4 mg/ml of Sepharose) was conjugated for 18 hr at 4°. Goat antiserum to Tween/ether-disrupted FeLV was fractionated with 50% ammonium sulfate. The precipitate was suspended and dialyzed against 0.1 M Tris buffer, pH 8.0, containing 0.1 M NaCl and adsorbed to the column containing FeLV proteins. Antiviral antibodies were eluted with 0.5 M acetic acid, neutralized, dialyzed, and Iyophilized. Antibodies at a concentration of 8 mg/ml had radioimmunoassay titers (24) of 15,000,000 against FeLV p30, 80,000 against FeLV p15, and 7000 against FeLV p12 proteins. An immunoadsorbant containing purified antiviral antibodies was prepared as described above. Serum Adsorptions and Immunofluorescence. Cat sera Abbreviations: FOCMA, feline oncornavirus-associated cell membrane antigen; FeLV, feline leukemia virus; FeSV, feline sarcoma virus; MSV, mouse sarcoma virus; SSAV, simian sarcoma-associated virus; NaDodSO4, sodium dodecyl sulfate; KiSV, Kirsten sarcoma virus; MiLV, endogenous mink type C virus. Proc. Nati. Acad. Sd. USA 75 (1978)