Proc. Natl. Acad. Sci. USA Vol. 78, No. 3, pp. 1920-1924, March 1981

Detection of a recombinant murine leukemia -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 (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 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. These cells with any RM pseudotypes, although the ecotropic cloned efficiently in microwells (e.o.p. 0.38) and in soft agar did grow (Table 2). Control infection with mlMuSV (M-MuLV) (efficiency of plating, 0.3). The supernates of the parental pop- gave rise to ecotropic MuLV and mlMuSV pseudotype. Ac- ulation were negative for reverse transcriptase. These cells cordingly, mlMuSV coated with ecotropic MuLV could enter called NIXT were positive for the Thy. antigen, positive for and replicate in NIXT cells but none of the mlMuSV (RM) vi- terminal deoxynucleotidyltransferase, and negative for 20a-hy- ruses could enter. Immunofluorescence assay with anti-p30 droxysteroid dehydrogenase (21). No infectious virus was ever antiserum was performed to determine whether mlMuSV was detected either by direct infection of normal or S + L- cells of able to translate its gag region-encoded p60. No p60 was de- mouse, rat, cat, mink, or human or by cocultivation with these tected in mlMuSV(RM-MHJx)-infected NIXT cells. It was rea- cells. Initial experiments with iododeoxyuridine also failed to soned that, if entry of RM type MuLVs was prevented because induce replicating MuLV from NIXT cells (32). Radioimmu- of its envelope, RM-MHIX genomically masked with ecotropic nocompetition assays for MuLV p30, AKR MuLV or xenotropic M-MuLV should be able to enter. Using masked RM-MHIX, we MuLV p12, or MuLV gp7O showed that less than 10, 1, or 20 did detect transiently a low level of virus that could replicate ng/mg of cell lysate, respectively, was present (16). in non-mouse cells. Growth of Murine Type C Viruses in NIXT Cells. Ecotropic Presence of Immunological Determinants of RM-MuLV F, M 1869, and AKR MuLVs were used to infect NIXT cells Glycoproteins on NIXT Cells. Because of possible interference after several months in culture (multiplicity of infection, with RM gp7O, NIXT cells were examined by immunofluores- 0.05-0.2). All strains of ecotropic MuLV grew adequately at cence assay with high-titer group-specific anti-F-MuLV gp7O first but in titers significantly lower than in some adherent antisera. Second, we used an antiserum prepared against the mouse cell lines (Table 1). Repeated examination of ecotropic gp7O of the leukemogenic VL-3 RadLV isolate (16). This anti- virus-infected NIXT cell supernates on normal cat or mink VL-3 gp7O antiserum was remarkable in that it was able to neu- S+ L- cells failed to show any extension of host range. Infection tralize, at high dilutions, all RM isolates and it neutralized of mouse S+L- cells with NIXT cell-derived F-, M-, or ecotropic or xenotropic MuLVs at about 1/100th to 1/10th the AKR-MuLV never generated any MuSV pseudotype with a efficiency (unpublished data). Anti-F-MuLV gp7O antiserum polytropic host range. NIXT cells were next infected with RM- did not detectably stain normal NIXT cells but the anti-VL-3 MHIx, RM-Mp,, RM-Gr, RM-AKRTa, or RM-C57 cloned vi- gp7O strongly stained about 5-10% of cells. Clones of NIXT ruses. None of these viruses produced any progeny, even after cells were then examined; of the 29 clones and 10 subclones, several weeks or months. NIXT cells infected with RM-MHIX all were positive with the anti-VL-3 with a minimum of 2% to or ecotropic MuLVs were assessed by immunofluorescence about 25% positive cells. This demonstrated that each cell had with potent group-reactive anti-F-MuLV-p30 and -gp70 anti- the capacity of expressing this determinant. sera. Ecotropic MuLV-infected NIXT cells were positive for Labeled protein A binding assays were also performed on both antigens. In contrast, neither antigen was detected in RM- both live and gluteraldehyde-fixed clone 13 NIXT cells which MHIx-inoculated NIXT cells. were =20%6 immunofluorescence positive. Anti-VL-3, anti-F- Envelope RM-MuLVs Cannot Enter NIXT Cells. Previ- MuLV gp7O, and anti-F-MuLV p15 (E) antisera were used. ously, restriction of mouse xenotropic virus was found to occur Anti-VL-3 gp 70-treated live or fixed NIXT cells bound 'MI-la- at two levels, at the surface of the mouse cell and internally beled protein A much more effectively than did NIXT cells (33). NIXT cells were infected, at a multiplicity of infection of treated with anti-F-MuLV gp7O, anti-pIS (E), or normal rabbit Downloaded by guest on September 24, 2021 1922 Microbiology: Fischinger et at Proc. Nad Acad. Sci. USA 78 (1981)

Table 2. Entry into NIXT cells of MuSV pseudotyped with ecotropic, RM, or xenotropic MuLV envelopes Assay* of progeny virus on Pseudotype Added ecotropic or Mouse Mink Mouse Cat virus RM helper, 3T3 FL,t lung,t S+L-, S+L-, inoculum multiplicity FFU/ml FFU/ml FIU/ml FIU/ml m1MuSV (M-MuLV) 1.5 x 103 0 1.3 x 104 0 mlMuSV(AKR) 1 x 102 0 7 x 102 0 mlMuSV(RM-Mmx) None 0 0 0 0 RM-MHIX, 0.1 0 0 0 0 F-MuLV, 0.2 0 0 1.5 x 104* 0 mLMuSV(RM-AKRT) None 0 0 0 0 RM-MHIX, 0.1 0 0 0 0 F-MuLV, 0.2 0 0 1.5 x 104t 0 mLMuSV(RM-GR) 0 0 0 0 mLMuSV(RM-C57) 0 0 0 0 mLMuSV(B-MuX) 0 0 0 0 * Virus was harvested every 24-48 hr and assayed on normal cells to detect MuSV and on S+L- cells to detect helper viruses. FFU, focus-forming units; FIU, focus-inducing units. t Helper virus was added to each assay system to detect total MuSV; 2 x 104 focus-inducing units of M-MuLV and 2 x 104 of feline leukemia virus was added to mouse and mink cells, respectively. Negatives represent a minimum of three tests for each in mink or FEF or cat S+L- cells. t At day 26 after inoculation. serum (Fig. 1). Two cytotoxicity tests were used. Control clon- Oligopeptide Maps of gp7O Found in Supernates of NIXT ing efficiency with normal sera and low-toxicity guinea pig or Cells. gp70s from various sources were isolated by immunoaf- rabbit complement was 0.40. The addition of anti-VL-3 anti- finity chromatography (31). The peptide structure of NIXT gp7O serum at 1:30 (final dilution) reduced cloning efficiency to 0.02. (Fig. 3A) was highly related to class III endogenous xenotropic In the absence of complement, anti-VL-3 antiserum had no virus gp7O (31, 35), such as AT-124 gp7O. However, NIXT gp7O effect. Normal mouse serum, normal rabbit serum, or anti-rab- was not identical to AT-124 gp7O and differed primarily in a bit F-MuLV gp7O antiserum with or without complement had configuration of peptides in the lower right quadrant of the map no effect. Additionally, several antisera were used in 51Cr re- (indicated by brackets). The most extensive homology among lease assays. Anti-MuLV p15 (E) antiserum had no effect on the endogenous gp70s was observed between NIXT gp7O and NIXT cells (compared to control serum), whereas a standard a gp7O of serum or thymocytes from the 129 mouse strain (Fig. anti-F-MuLV gp70 antiserum killed about 20%, and anti-VL-3 3B). This gp7O had similarities with NIH Swiss xenotropic virus gp7O antiserum killed about 50% of cells at a 1:50 dilution. To gp7O and also shared with NIXT gp7O the configuration of pep- assess whether NIXT cells could specifically absorb RM tides shown in Fig. 3A (brackets) which was absent from NIH subgroup-specific neutralizing antibody, RM-MHIx and F- Swiss xenotropic virus gp7O (not shown), as well as standard MuLV were neutralized, each with its own type or subgroup- endogenous class I and II viral gp70s, here represented by specific serum, at levels such that .95% of the homologous ecotropic BALB virus 1 (Fig. 3D), and by xenotropic BALB virus was inactivated. Control NIXT cells and F-MuLV-prein- virus 2 (Fig. 3C). This latter group of peptides was found in fected NIXT cells, either live or acetone-fixed, were used for several recombinant virus gp70s including AKR 247 recombi- absorption. NIXT cells in the absence of antibody did not re- nant virus (Fig. 3E), Graffi recombinant virus (Fig. 3F), and duce the titer of virus by interference. Both normal and F- RadLV (not shown). In contrast, prototype xenotropic BALB MuLV-infected cells could abrogate the anti-VL-3 neutraliza- virus 2 gp7O and ecotropic BALB virus 1 gp7O isolates lacked tion of RM-MHIX by two-thirds. However, F-MuLV-infected the bracketed peptides. Additionally, peptides denoted by ar- NIXT cells could completely abrogate the specific neutralizing rows at the baseline of the maps in Fig. 3 E and F have been effect of the anti-R-MuLV gp70 antiserum (data not shown). Based on this, only a partial relationship appears to exist be- tween the VL-3 MuLV gp7O determinant and the reactive mol- ecule found on NIXT cells. 15- Radioimmunoprecipitation of a gp7O from NIXT Cells. Ra- dioimmunoprecipitation was performed with surface-iodinated clone 13 NIXT cells. Antisera directed against MuLV gp70s precipitated a molecule of approximately 70,000 daltons (Fig. X 10 2). Anti-F-MuLV and anti-VL-3 rabbit antisera were equally Ei potent whereas an anti-F-MuLV p15 (E) or normal rabbit serum 0 had no effect. To determine that the molecule was glycosylated, clone 13 NIXT cells were pulse labeled for 7 hr with - 5 [3H]glucosamine. An analogous pattern of immunoprecipita- tion was observed (Fig. 2B). Both anti-VL-3 gp7O and anti-F- MuLV gp7O precipitated a glucosamine-labeled component in the position of gp7O, whereas anti-pl5(E) was not reactive. Su- 1/5 1/25 1/125 1/625 1/3125 pernates of clone 13 NIXT cells were also examined; antigens Serum dilution from cells pulse labeled with glucosamine were immunopre- FIG. 1. Binding assays with fixed NIXT cells (clone 13) and anti- cipitated (Fig. 2C). Again, the anti-VL-3 and anti-F-MuLV gp7O VL-3 gp7O antiserum (o), anti-F-MuLV gp7O antiserum (o), anti-F- antisera reacted specifically with a gp7O protein. MuLV p15 (E) antiserum (A), and normal rabbit serum (x). Downloaded by guest on September 24, 2021 Microbiology: Fischinger et aL Proc. Natl. Acad. Sci. USA 78 (1981) 1923

A 2 3 4 5 2 3 4 C 2 3 4

gp7O-~ _._.,.: 970_ gp7O-..-i-p7

p30-

pl2 -

FIG. 3. Chymotryptic peptides of gp7O isolated from the NIXT cell line and from endogenous and recombinant retrovirus gp70s. (A) gp7O purified from tissue culture supernatant of the radiation-induced NIH FIG. 2. (A) Immunoprecipitation from surface-iodinated NIXT Swiss thymoma line, NIXT. (B) Class III type xenotropic gp70 isolated cells (clone 13). Cells were extracted and 5 x 106 cpm was incubated from serum of the virus-negative 129 mouse strain. (C) gp7O of the with 1 ,ul of antiserum. The molecular weight markers on this 12% slab class II BALB virus 2 xenotropic virus. (D) gp7O of class I BALB virus gel are [3H]leucine-labeled F-MuLV structural proteins (lane 1). 1 ecotropic virus. (E) gp7O of the recombinant RM-AKR virus. (F) gp7O Lanes: 2, NIXT cells with normal rabbit serum; 3, NIXT cells with anti- of Graffi recombinant virus. Class I, II, and III terminology is accord- VL-3 gp7O; 4, NIXT cells with anti-F-MuLV p15 (E); 5, NIXT cells with ing to Stephenson et al. (34). Brackets in A, B, E, and F are to draw anti-F-MuLV gp7O. (B) Immunoprecipitation from [3H]glucosamine- attention to a conformation of peptides common among NIXT gp70 labeled NIXT cells (clone 13). Cells were pulse labeled for 7 hr and then (A), endogenous xenotropic virus-like gp7O of serum (B), and two re- extracted; and 2 x 106 cpm was incubated with 1 ,ul of antiserum. combinant retrovirus gp7O (E and F) that are absent from prototype Lanes: 1, NIXT cells with anti-VL-3 gp7o; 2, NIXT cells with normal xenotropic and ecotropic virus gp70s (C and D, respectively). Arrows rabbit serum; 3, NIXT cells with anti-F-MuLV p15 (E); 4, NIXT cells in E and F indicate cluster of peptides common to recombinant virus with anti-F-MuLV gp70. (C) Immunoprecipitation of gp7O from su- gp7Os and absent from others. pernates of NIXT cells (clone 13). Cells were pulse labeled with [3H]glucosamine for 7 hr and chased for another 12 hr. The cell-free supernatant was extracted, and 3 x 106 cpm was incubated with 1 ,ul of antiserum. Lanes as in B. ently had the capacity to express this antigen. NIXT cells could in turn also absorb some but not all of the neutralizing activity of this antiserum. Labeled protein A and cytotoxicity studies found to be a general feature of recombinant MuLVs and not showed that the VL-3 gp7O antibody was bound to the NIXT found in ecotropic or xenotropic isolates. These peptides may cell surface. More direct attempts at detection of gp7O by im- represent glycosylated peptides that are not resolved in this munoprecipitation showed that a gp7O was detected at the sur- chromatography system. From other work, we know that this face of NIXT cells and that it could also leach off into the su- configuration of peptides is a constituent of a 32,000-dalton pernates. In contrast to cytotoxicity and binding assays, in this COOH-terminal fragment (p32) of gp7O. By tryptic peptide test both anti-VL-3 gp7O and anti-F-MuLV gp7O antisera were analyses, p32 contains a xenotropic peptide which is the hall- about equally active. It may be that in its normal membrane mark of these recombinant viruses (unpublished data). position the gp7O molecule may present only its RM subgroup- specific determinants but, when isolated, may also present its DISCUSSION more broadly reactive sites. This situation may have analogies The high efficiency of lymphoma induction in the outbred Swiss to the GIX antigen which apparently is a gp7O with type-specific mouse by x-rays in the apparent absence of virus expression was determinant(s) (1). Analysis of peptides of the isolated gp70 of interest because of extensive biological and molecular knowl- from NIXT cells showed extensive similarity to class III endog- edge of various families of murine leukemogenic viruses (1). enous xenotropic gp7O but, in addition, contained peptides that The well-investigated radiation-induced leukemogenesis in the have been found to be a general component of RM-MuLVs. inbred C57BL mouse was thought to serve as a model system; Although MuLV gp70s are known to exist on most mouse cell it was clear that, although several viruses were detected with surfaces in the absence of complete virus expression, all other virus passage, many primary radiation-induced tumors had low mouse cells tested were susceptible to entry of the above RM- or undetectable levels of virus expression (15). Swiss mouse x- MuLVs (36). It is not known whether the origin or the gp7O or ray-induced thymomas as well as the derived NIXT line did not NIXT cells is encoded by an intact or defective provirus or even express virus or detectable viral antigens (17, 32). Infection of a cellular gene or whether it resulted from internal recombi- NIXT cells with several ecotropic MuLV strains resulted in a nation subsequent to x-irradiation. productive infection. Attempts at infection of NIXT cells with Such an autonomously produced RM gp7O could play a role wide host range polytropic RM-MuLVs which could infect other in leukemogenesis. It is attractive to postulate that chronic im- mouse cells were negative. By using several different mune stimulation with this altered gp7O could result in blas- MuSV(RM)s, it was clear that these pseudotypes could not enter togenesis. The hypothesis of Weissman is feasible in that it dem- NIXT cells. A lack of entry could have been due to surface in- onstrates in the AKR system the existence of receptors for RM- terference with an RM type glycoprotein because RM viruses MuLV gp70s in a specific T-cell subpopulation so that the gen- share an interference group that is different from ecotropic eration of RM-MuLVs results in continued blastogenesis (37). MuLVs (35). If the above is true, then the present system could demonstrate The anti-RadLV VL-3 gp7O antiserum, which specifically that expression of whole RM-MuLV is not necessary and that recognized the RM-MuLV subgroup by neutralization, was able leukemia could occur in an analogous manner in a virus-free to detect a related molecule on NIXT cells. Every cell appar- state. Downloaded by guest on September 24, 2021 1924 Microbiology: Fischinger et aL Proc. Nad. Acad. Sci. USA 78 (1981)

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