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Proc. Natl. Acad. Sci. USA Vol. 78, No. 3, pp. 1920-1924, March 1981 Microbiology Detection of a recombinant murine leukemia virus-related glycoprotein on virus-negative thymoma cells (envelope glycoprotein/lymphoma) PETER J. FISCHINGER*, H. JURGEN THIELt, JAMES N. IHLEt, JOHN C. LEEO, AND JOHN H. ELDER§ *Lboratory of Viral Carcinogenesis, National Cancer Institute, Frederick, Maryland 21701; tDepartment of Surgery, Duke University, Durham, North Carolina 27710; NFrederick Cancer Research Center, Frederick, Maryland 21701; and §Scripps Clinic and Research Foundation, La Jolla, California 92037 Communicated by Charlotte Friend, November 6, 1980 ABSTRACT X-irradiation of outbred Swiss mice resulted in tion-induced C57BL lymphomas do not overtly express MuLV, the development of virus-free thymomas. When put in culture, it has been possible to isolate a transmissible leukemogenic a lymphoblastic cell line (NIXT) expressed neither particles nor agent, radiation leukemia virus (RadLV), which has unique env infectious virus but supported the growth of pure ecotropic mu- rine leukemia viruses (MuLVs) without generating any envelope gene characteristics (14-16). Similarly, we have observed that recombinant (RM) MuLV in more than 20 months of culture. radiation-induced leukemias in Swiss mice do not express type These cells did not support the growth of RM-MuLVs and com- C viruses (17). It was possible that such a system could yield pletely excluded the entry of all RM-MuLV pseudotypes of mu- insights as to how leukemogenesis could take place in the ap- rine sarcoma virus, suggesting specific viral interference. Ra- parent absence of MuLV. We now define such a system and dioimmunocompetition and immunofluorescence assays with demonstrate that, in a virus free x-ray-induced thymic lym- broadly reactive anti-MuLV-p30 and -gp7O antisera were nega- tive. However, in immunofluorescence with antisera specifically phoma in an outbred Swiss mouse, there is expression of a reactive against RM-MuLV gp7O, about 5-20% of the population unique surface MuLV RM-like glycoprotein on thymoma cells. of parental cells or their clones were positive. NIXT cells treated with this antiserum bound protein A and exhibited complement- AND dependent cytotoxicity as assessed by several assays. NIXT cells MATERIALS METHODS could partially absorb neutralizing antibody specific for RM- Mice. Outbred Swiss mice were obtained from the Frederick MuLVs. Based on radioimmunoprecipitation tests, NIXT cells Cancer Research Center colony. They were given four weekly bore, on the cell surface, a glycosylated protein (gp70) reactive 175 R x 102 at 1-2 with RM subgroup as well as some group-specific anti-gp7O anti- doses of (4.5 coulomb/kg) age months. sera. The glycoprotein was also found free in the supernates of Thymomas developed in '=80% of mice (17). NIXT cells. Using affinity chromatography, we determined the Cells, Viruses, and Assays. Tumor cells were initially cul- peptide pattern of the gp7O from NIXT cells to determine its struc- tured on RPMI-1640 medium supplemented with 20% fetal calf tural relationship to gp70s of other MuLVs. NIXT gp7O was found serum. The derivative cell line was adapted to McCoy's 5a me- to be highly related to class III endogenous xenotropic gp70s but, dium with 10% fetal calf serum. Outbred Swiss mouse 3T3 FL in addition, had peptide characteristics of RM-gp70s. Apparently, cells, cat embryo diploid fibroblasts (FEF), normal mink lung NIXT cells code for an unusual gp70 protein in the absence of other MuLV expression. The possible role of this glycoprotein in leu- cells, and murine sarcoma virus (MuSV)-positive, leukemia- kemogenesis is discussed. negative (S + L-) mouse and cat cells were grown on the latter medium as described (18-20). Assays for the presence of ter- Murine leukemia viruses (MuLVs) include a number of repli- minal transferase activity and 20a-hydroxysteroid dehydrogen- cating and defective oncovirus types of varying disease-induc- ase activity were performed as described (21). Ecotropic ing potential and of immunologically distinct protein compo- MuLVs included the Friend Eveline strain (F), the Moloney sition (1-4). The genetic information for MuLVs is generally IC strain (M), and the constitutively produced AKR embryo encoded in the mouse DNA as a family of proviral DNA copie cell-derived MuLV. Polytropic envelope recombinant (RM) some of which have been defined as specific genetic loci (5-7 MuLVs included the prototype RM-MHIX virus, an MCF type MuLVs can be divided into those that grow well in mouse cel RM-AKRTU from Tubingen AKR mouse lymphomas, another (ecotropic), those that are restricted from growth in mouse cel RM-Mp from BALB/c lymphomas from Paris, an RM-Gr de- but not in cells of many other species (xenotropic), and those rived from Graffi MuLV stocks, and an RM-MuLV constitui- that grow in both mouse and other cells (1, 8, 9). The last group tively produced by C57BL/6 cells (22). A representative of the is composed of two different subsets: the wild mouse ampho- xenotropic envelope glycoprotein-containing MuLV was the B- tropic, which is different in its envelope properties from eco- MuX isolate (23). Assays for the ml isolate of MuSV and MuLV tropic and xenotropic viruses, and a dual or polytropic group focus-inducing units were as described (18, 19, 23). consisting of recombinant (RM) MuLVs whose env gene se- Immunological Assays. Several rabbit antisera against the quences are derived in part from both ecotropic and xenotropic isolated gp7O and p15 (E) of F-MuLV, and of gp70s of Rauscher MuLV-like sequences (10, 11). The RM group could be onco- (R) MuLV, and the VL-3 isolate of radiation leukemia virus genic in genetically pure form, and it has been suggested that (RadLV) were previously characterized (24, 25). Radioimmu- it is the RM variant that is the actual leukemogenic agent in a noassays for individual MuLV viral proteins were performed number of spontaneous or transmitted lymphocytic leukemias (12, 13). Abbreviations: NIXT, non-virus-producing, X-irradiation-induced Swiss A role for these viruses in primary leu- mouse thymoma cell line; MuLV, murine leukemia virus; F, M, and radiation-induced R, Friend, Moloney, and Rauscher, respectively; RM, recombinant kemias has been less obvious. Although the majority of radia- murine virus; S+L-, sarcoma-positive, leukemia-negative; MuX, mu- rine xenotropic MuLV; MuSV murine sarcoma virus; RM-Mj1jx, RM- The publication costs of this article were defrayed in part by page charge Mp, RM-Gr, and RM-AKR and RM-MuLVs, the first two isolated from payment. This article must therefore be hereby marked "advertise- M-MuLV and the latter, from Graffi and AKR MuLVs, respectively; ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. RadLV, radiation leukemia virus. 1920 Downloaded by guest on September 24, 2021 Microbiology: Fischinger et aL Proc. NatL Acad. Sci. USA 78 (1981) 1921 according to standard procedures (25). Neutralizations and ab- Table 1. Growth of various MuLV isolates in NIXT cells sorption of antibody with infected cells were performed as de- Early harvests Late harvests scribed (24). Two types of cytotoxic assays were performed, the first being a reduction of cloning efficiency of cells in the pres- MULV assayed on assayed on inoculum Mouse S+L- Cat S+L- Mouse S+L- Cat S+L- ence of antiserum and low toxicity to mouse lymphocytes rabbit complement (Cedarlane Labs, Ontario, Canada). 51Cr release Ecotropic: assays were performed by using rabbit (Pel-Freeze) comple- M 5.1 x 104 0 1.2 x 104 0 ment and assorted antisera as described (26). Lactoperoxidase- F 5.0 x 104 0 1.4 x 104 0 catalyzed cell surface iodination and metabolic labeling of cells AKR 2.0 x 103 0 5.0 x 102* generally followed published procedures (27). The precipitates RM: were analyzed on slab gels (28) and processed by fluorography RM-MHa 0 0 0 0 0 0 0 (29). Binding assays of antiserum and "2I-labeled protein A to RM-Mp 0 cells followed published procedures (30). RM-AKRT 0 0 NT NT and RM-C57 0 0 Peptide Patterns. Two-dimensional tryptic chymotryptic Xenotropic: peptide patterns (fingerprints) were determined on the gp7O B-MuX 0 0 present free in the supernates and in Nonidet P40 lysates of NIXT cells as described (31). Data are shown as focus-inducing units per ml of supernate. RM- MuLV- and B-MuX-infected NIXT cells were discarded after 4 weeks of infection when assays in both mouse and cat S+L- cells were neg- RESULTS ative. Negatives represent a minimum of six assays for each set over a 1-year period. NT, not tested. Development of the NIXT Cell Line. A tumor-bearing 4- - Average titers; AKR-MuLV was lost from infected NIXT cells after month-old mouse was sacrificed and the finely minced thymic about 1 year. tumor was put in culture. An adherent feeder layer developed; after several weeks, there was an increase in nonadherent cells. 0.1, with the ml isolate of MuSV pseudotyped with RM-MHIX, Rapid growth of small lymphoblastoid cells and clusters ensued RM-AKRTU, RM-Gr, and RM-C57 envelopes and in a second in the absence of feeder layers. The culture growth and cell set coinfected with ecotropic M-MuLV at a multiplicity of 0.2. characteristics remained unchanged for 18 months. No virus Supernates were harvested daily after infection and assayed in particles were seen in thin sections. The optimal dividing time mink lung, FEF, and mouse and cat S+L- cells. No focus- of cells was -14 hr, and the cells regularly grew in suspension forming MuSV was detected as long as 1 month after infection to =5 X 106 cells per ml with 2% fetal calf serum.

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