JOURNAL OF VIROLOGY, July 1980, p. 211-218 Vol. 35, No. 1 0022-538X/80/07-021 1/08$02.00/0 Characterization of Virus Produced by a Lymphoma Induced by Inoculation of AKR MCF-247 Virus FINN S. PEDERSEN,t DOROTHY L. BUCHHAGEN, C. Y. CHEN,: ESTHER F. HAYS,: AND WILLIAM A. HASELTINE* Sidney Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115 We report the characterization of the virus produced by a lymphoid cell line derived from a lymphoma of an AKR mouse after injection of the polytropic AKR virus MCF-247. The virus displays polytropic host range properties and is indistinguishable from MCF-247 as judged by analysis of the large RNase T1- resistant oligonucleotides of the RNA genome. Restriction enzyme analysis of cellular DNA revealed the presence of sequences homologous to MCF-247 ge- nomic RNA. The EcoRI cleavage fragments were characteristic of MCF-247 DNA provirus cleavage products. Mice of the AKR strain developed thymus some of the viruses produced by cell lines de- lymphoma at a high incidence between 6 months rived from spontaneously occurring thymic lym- and 1 year of age (17). The disease is linked to phomas (SL viruses) (6, 11). For MCF-247 virus, the expression of endogenous retroviruses. The the average age of incidence of thymic leukemia retroviruses which can be derived from tissues is decreased from 269 days to about 135 days (6) of AKR mice constitute a polymorphic group of (see below). agents. Viruses that have a host range restricted Although injection of some strains of virus to mouse cells (ecotropic) and are positive in an accelerates the onset of disease in AKR mice, a XC fusion test can be isolated from AKR mice question remains as to whether the injected any time after 2 weeks of age (18). Viruses ca- virus replicates in the tumor cells. To test this pable ofreplication on cells derived from animals possibility, newborn AKR mice were injected other than mice (xenotropic) can be detected in with the polytropic MCF-247 strain of virus. A older mice (8, 9). A number of other viruses can cell line was established from one of the thymic be isolated from the lymphoid tissues of preleu- tumors which developed 5 months after injec- kemic and leukemic mice. These include viruses tion, and the virus produced by these cells was that are negative in the XC fusion assay and analyzed. that are ecotropic (6, 11, 12, 16) as well as viruses Here we report that the virus produced by the that have a broad host range, including both tumor cells retained its polytropic host range mink cells and mouse cells, and that are also and was indistinguishable from the strain used negative in the XC fusion test (polytropic or for inoculation as judged by a high-resolution MCF viruses) (4, 8). analysis of the RNase T,-resistant oligonucleo- These age-related changes in retrovirus tides of the RNA genome. Restriction enzyme expression may represent early events in the analysis of virus-related sequences in the DNA disease process. To test this hypothesis, viruses of this lymphoma cell line indicates the presence derived from the tissues of preleukemic and of additional DNA cleavage fragments indistin- leukemic AKR mice have been injected into guishable from MCF-247 DNA cleavage prod- newborn animals to determine whether they ucts. accelerated the onset of disease. Results of such tests indicate that whereas the ecotropic XC+ MATERIALS AND METHODS virus that can be isolated from young animals Virus. MCF-247 cloned virus (4) was provided by does not accelerate the onset of disease when Janet Hartley. The virus used for inoculation was injected into newborn animals, several other vi- propagated in CCL-64 mink cells. ruses do (6, 12). These include some of the Cell lines. The cell lines used include a mink lung cloned polytropic isolates such as MCF-247 and fibroblast line (CCL-64), the S+ L- line (1) (kindly supplied by R. H. Bassin), the XC cell line (18), and a t Present address: Laboratory of Nuclear Medicine and line of NIH-3T3 cells. A CCL-64 cell line infected with Radiation Biology, Los Angeles, CA 90024. MCF-247 was used as a source of DNA for the restric- : Present address: Department of Molecular Biology, Uni- tion enzyme analysis of the MCF-247 provirus. The versity of Aarhus, Aarhus, Denmark. line was derived by plating single cells of an infected 211 212 PEDERSEN ET AL. J. VIROL. culture into 96-well Terasaki plates at a dilution of Cells were resuspended in lx SSC (0.15 M NaCl-0.015 less than one cell per well. Wells receiving only a single M sodium citrate), sodium dodecyl sulfate was added cell were grown into large cultures, and one such line, to a final concentration of 1.0% and tissues were AKR CL-4B, was used here. MCF-247 was also prop- digested with Proteinase K (10ltg/ml) for 1 h at 37°C. agated in NIH-3T3 cells. Tumor cell lines derived from Phenol saturated with lx SSC was used to extract the spontaneously occurring AKR thymic lymphomas in- tissues twice, followed by four extractions with chlo- clude AKR SL1, SL2, SL3, SL5, SL6, SL7, and SL8 roform-isoamyl alcohol (19:1). Two volumes of cold (10). ethanol were added to the aqueous phase, and the Mice. A strain of AKR/J mice was maintained by DNA was wound out. The DNA was resuspended in continuous, single brother and sister mating. lx SSC and digested with RNase A (Worthington) at Establishment and growth of lymphoma cell 40 ,ug/ml for 30 min at 37°C. The DNA was then line. The donor mouse developed thymic lymphoma redigested with proteinase K as described above and 155 days after neonatal interperitoneal inoculation of re-extracted with phenol and chloroform-isoamyl al- 5 x 102 focus-forming units (FFU) of MCF-247 viruses cohol. Two volumes of cold ethanol were added to the per ml. Cells (5 x 106) from the thymus were placed in aqueous phase, and the DNA was wound out, lyophi- a 35-mm petri dish with Dulbecco-modified Eagle lized, and resuspended in 0.Olx SSC to an approximate medium supplemented with 10% fetal bovine serum, concentration of 1 to 2 mg/ml. 10 jig of asparagine per ml, and 10' M glutathione. Purified DNAs were digested with restriction en- The nonadherent cells were transferred to new dishes donuclease EcoRI (20 U/10 ,g of DNA, New England twice a week. They became independent ofglutathione Biolabs). Digestions were performed at 37°C for 2 h in after 5 weeks in culture. The cells were maintained in the cocktail suggested by the supplier. The DNA was a suspension culture in Dulbecco-modified Eagle me- subjected to electrophoresis on 0.8% agarose gels in a dium-10% total fetal calf serum without added gluta- horizontal plate (14.5 cm by 21 cm by 6 mm) containing thione for 2 months and then were changed to RPMI ethidium bromide at 3 yg/ml. Electrophoresis was at 1640 with 10% fetal bovine serum. Studies with the 10 mA for 3 h followed by 25 mA for 16 h with constant cell line were done after it had been in continuous current with a buffer of 0.04 M Tris-hydrochloride culture for 6 months. (pH 8.0), 0.018 M NaCl, 0.01 M sodium acetate, and Virus assays. The activity of RNA-dependent 0.002 M Na2 EDTA. DNA polymerase in tissue culture supernatant was The DNAs were transferred from the agarose gels determined as described elsewhere (19). The number to nitrocellulose filters (Schleicher & Schuell, BA85) of infectious viruses was determined by assessment of by a modification of the Southern procedure (20). The numbers of foci formed in S+L- (D56) cells as de- gels containing the DNAs were soaked in a denaturing scribed elsewhere (1). The XC cell fusion assay was solution of 1.5 M NaCl-0.5 M NaOH for 20 min and performed by the method of Rowe and Pincus (18). neutralized for 30 min with 3 M NaCl and 0 to 5 M The XC assays were performed on NIH-3T3 cells. Tris-hydrochloride (pH 7.0). After a 5-min soaking in Isolation of virus RNA. Virus particles were iso- 6x SSC, the gels were placed on the nitrocellulose lated from tissue culture supernatants by centrifuga- filters, and DNA transfer was allowed to proceed tion as described previously (13, 14). The viruses were overnight at room temperature. The filters with the disrupted by treatment with proteinase K (25 mg/ml DNA were baked at 80°C for 4 h and stored until used. in sodium dodecyl sulfate), and the 70S RNA was 32P-labeled complementary DNA probes were isolated by velocity sedimentation in sucrose gradient, added to DNA filters that had been preincubated in a followed by chromatography on oligodeoxythymidylic solution containing 3x SSC, 50% formamide, 0.5% acid-cellulose (13, 14). sodium dodecyl sulfate, 0.2% Ficoll, 0.2% polyvinylpyr- Oligonucleotide mapping. The analysis of RNase rolidone, 0.2% bovine serum albumin, 50 mM Tris- T,-resistant oligonucleotides of the viral RNA was hydrochloride (pH 7.5), 1 mM Na2 EDTA, 20 ytg of carried out as described elsewhere (13, 14). Briefly, the bovine rRNA per ml, and 20 mg of yeast RNA per ml. 70S RNA was digested to completion by RNase T1, Approximately 5 x 10' cpm of probe having a specific and the RNase-resistant oligonucleotides were radioa activity of 107 cpm/,ug were added in 0.5 ml of hybrid- actively labeled by [y-32P]ATP and polynucleotide ization buffer.