Immunofluorescent Analysis of Expression of the RNA Tumor Virus

Home , Antiserum

(CANCER RESEARCH 37, 922-930, March 1977] Immunofluorescent Analysis of Expression of the RNA Tumor Virus Major Glycoprotein, gp7l , on Surfaces of Virus producing Murine and Other Mammalian Species Cell Lines1

Miles W. Cloyd,2 Dani P. Bolognesi,3 and Darell D. Bigner4

Departments of Pathology (M. W. C., D. D. B.] and Surgery (0. P. B. , D. 0. B.J, Duke University Medical Center, Durham, North Carolina 27710

SUMMARY activity (14), participates in virus neutralization (10, 15), and reacts with natural anti-MuLV antibodies in mice (18, 19). The specificity of a single rabbit antiserum pool raised MuLV gp7l has also been shown to possess multiple anti against the purified major glycoprotein, gp7l , of Friend genic determinants which are classified, according to their munine leukemia virus was determined for a variety of distribution, as type (unique to the particular strain of virus), virus-producing mouse, feline, and gibbon ape cell lines by group (shamedby all munine onconnavinuses), or interspe viable cell membrane immunofluorescence absorption. cies (shared by oncomnavimusesof different species) (1). Among munine cells examined, Friend gp7l type specificity Because of its biological properties and surface location, as was shared only with Rauschemvirus-producingcells, and a well as its availability in pure form and in relatively large group specificity was present for all the mumine leukemia amounts, it has come under consideration as a potential virus-producing cells tested. Friend and Rauschem murine vaccine antigen. Recent investigations have shown that leukemia virus-infected cells shared interspecies cross immunization with F-MuLV gp7l on passive senothemapy reactivity with feline leukemia and gibbon ape lymphoma with heterologous F-MuLV gp7l antisera can influence the virus-producing cells. However, Moloney, Gross, and other course of infection and leukemia development in mice fol virus-producing mumine cells shared some, but not all, of lowing challenge with leukemogenic doses of Friend on these gp7l interspecies determinants with the feline and RauschemMuLV (16, 32). primate cells. Immunofemmitinelectron microscopy localized Since the possible roles of F-MuLV gp7l in immunother these gp7l antigenic determinants on both virus and cell apy of viral leukemia were being investigated, we sought to membranes. define the specificity and/on cross-reactivity of Friend gp7l antisera for various virus-producing cells, and, in addition, INTRODUCTION to ascertain which senologically detectable determinants of gp7l were expressed at the cell surface. Other studies of Recent work has defined many of the characteristics of serological relationships between MuLV gp7l and cell sum structural components of mammalian C-type viruses (Refs. face components have recently provided evidence of a wide 12, 26 to 29, 34, 38, 40; reviewed in Aefs. 4 and 33). These distribution of gp7l in mice, by means of a variety of ap structural antigens and their reagent antisera provide valua proaches such as madioimmunoassay with cell extracts (25, ble tools and approaches for exploring virus-cell interac 39), immunoprecipitation and gel electrophoresis of solubi tions or host response to virus. One of these components, lized cellular components (8, 23), fixed-cell immunofluones the major envelope glycoprotein (gp71) of MuLV,5 is of cence (21, 25), complement-dependent cytolytic assays (7, particular interest since it is involved in a variety of viral 11,14),orIEM (23,35).Nevertheless,discrepancieshave functions and properties. For instance, MuLV gp7l is on the arisen, and the extent of gp7l determinant activity and surfaces of vinions (reviewed in Ref. 4) and murine cells (10, distribution on cell surfaces remains incompletely defined. 14, 35), possesses hemagglutinating and viral interference This and the accompanying report (6) present the results of a study concerning the expression of cell surface gp7l

1 This investigation was supported by National Cancer Institute Grants reactivity using a viable cell membrane indirect immunoflu CA11898 and CA14651 and USPHS Contract NCI NIH CP33308 from the Virus onescence microassay, in which binding of immunoglobulin Cancer Program of the National Cancer Institute. to cell surfaces is directly, reliably, and sensitively detected 2 Predoctoral trainee supported by USPHS Grant l-T32 CAO911 1-01 . To whom requests for reprints should be addressed. (5). 3 Supported by American Cancer Society Faculty Research Award FAA 141. MATERIALS AND METHODS 4 Supported by National Institute of Neurological Diseases and Stroke TIA Fellowship 1F11NS11063 and an American Cancer Society Junior Faculty Clinical Fellowship award. Cells. The cell lines used in this study and their major 5 The abbreviations used are: MuLV, munine leukemia virus; F-MuLV, Friend munina leukemia virus; IEM, immunofernitin electron microscopy; M characteristics are listed in Table 1. 3T3 FL cells, uninfected MuLV, Moloney munine leukemia virus; G-MuLV, Gross murine leukemia and infected with the Eveline strain of F-MuLV (F-3T3FL), virus; STU, Schafer TUbingan mouse strain; Fl, fluorescence index; R-MuLV, Rauschemplasma virus (R-3T3FL), IC isolate of M-MuLV (M Rauscher murine leukemia virus; FaLV, feline leukemia virus; GALV, gibbon ape lymphoma virus. 3T3FL), or mat-passaged G-MuLV (G-3T3FL) (2, 9, 34) were Received September 8, 1976; accepted December 14, 1976. kindly provided by Dr. Peter Fischinger, National Cancer

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Table 1 Cell lines studied (species or mouse of cul vi of originRef.F-3T3Cell lineOrigin strain, tissue)Type tuneTrans formedÂ°Produce rus'@Laboratory 9M-3T3FLF-MuLV-infected 3T3FLPermanentâ€”F@MuLVrFischinger2, FLPermanentâ€”M-MuLV'Fischinger9R-3T3FLM-MuLV-infected 3T3 FLPermanentâ€”R@MuLVcFischingerG-3T3FLR-MuLV-infected 3T3 FLPermanentâ€”G-MuLV'FischingerEvelineSTUFLG-MuLV-infected 3T3 embryoPermanent+F@MuLVeSchafer36AKR+CAKRmouse embryoPermanentâ€”+rHartley10MCA-10C56BL/6Nmouse MCAd+(Wells11MCA-18C57BL/6N sarcomaPermanent+ MCA+â€˜Wells11GALGibbon sarcomaPermanent+ lymphomaPermanent+GALVKawakami22HF-4MarmosetPermanentâ€”â€”DeinhardtF-422Felineape

leukemiaPermanent+FeLVRickard31FEFNormal cat embryoPermanentâ€”â€”Fischinger

a Morphologically transformed. b All cell lines were checked for C-type virus replication by electron microscopic surveys of both cells and ultracentrifuged culture supernatant pellets.

C Infectious virus was also assayed in the FG-10 S@L murine cell line by the method of Bassin et al. (3). d MCA, 3-methylcholanthrene.

Institute. These chronically infected cells produced 10@to beled Friend gp7l was used for all assays in this study. All 106 infectious units of virus per ml. AKR + C cells, originally sena were heat inactivated for 30 mm at 56Â°and ultracen obtained from Dr. Janet Hartley, NIH, and normal feline tnifuged for 90 mm at 77,700 x g to remove any aggregated embryo cells were also provided by Dr. Fischinger. Normal globulin. marmoset cells were obtained from Dr. Friednich Deinhardt, Fluonescein-conjugated IgG of goat anti-rabbit lgG (0.2 Rush-Presbyterian-St. Luke's, Chicago, Ill. All cell lines mg/mI total protein, fluorescein/protein = 4.3; Meloy Labo were grown in monolayer culture in Richter's improved ratonies, Inc., Springfield, Va.) for use in the immunofluo minimum essential medium zinc option (30), supplemented rescent assay, was preabsombedwith acetone-processed rat with 10% heat-inactivated fetal calf serum, 10 mM N-2-hy liver powder. Femnitin-conjugated lgG of goat anti-rabbit IgG droxyethylpiperazine-N'-2-ethanesulfonicacidbuffer,glu (Cappel Laboratories, Downingtown, Pa.) for use in immu tamine, 584 mg/liter; gentamycin, 50 p@g/mI;penicillin G, noelectron microscopy was also absorbed with rat liver 100 units/mI; and amphotenicin B, 0.5 mg/mI. powder and used at an optimal working dilution of 1:4 with Gibbon ape lymphoma cells, obtained from Dr. Thomas phosphate-buffered saline (in g/litem: CaCI2, 0.10 KCI, 0.20; Kawakami, University of California, Davis (22), and F-422 KH2PO4, 0.20; MgCl2â€¢6H2o,0.10; NaCI, 8.00; and Na2HPO4 feline leukemia cells (31) were grown as suspension cul 7H20, 2.16). tunes in Class III containment facilities of the Duke Univer Absorption of Antisera. Multiple sequential absorption sity Animal and Laboratory Isolation Facility. steps were used to monitor the extent of absorption reac To eliminate variation in cell properties that may occur tions. Antiserum (0.5 ml), diluted within 2 log2 dilutions with long-term culture, large quantities of the cells were above the 50% end-point titer, was incubated with 5 x 10@ viably frozen in 10% v/v dimethylsulfoxide in minimum es cells (total volume, 0.6 ml) for 1 hr at room temperature, sential medium zinc option at a given passage level and then overnight at 4Â°.Additional absorption steps were pen then were thawed for use when needed. formed, 2 to 6 times, in the same manner, and the serum Virus and Purified Viral Components. F-MuLV, grown in was clarified at 8000 x g for 5 mm. Mock absomptions were STU â€œEvelineâ€•mousecells (36), was purified by velocity and done identically but without using cells. Absomptions using equilibrium density gradient centnifugation as described chick embryo cells served as cell absorption controls. previously (34). The major glycoprotein, gp7l, released Immunofluorescence Assay. The indirect live cell mem from the virus by osmotic shock and purified by a procedure brane immunofluorescence assay used has been described comprising density gradient centnifugation, concanavalin A elsewhere (5, 11). Briefly, 5 x 10@mechanically dispensed affinity chromatography, and Sephadex gel filtration (26) viable cells in individual microtitem plate wells were incu was kindly provided by Professor Werner Schafer, Max bated for 20 mm with 50 p1 of various dilutions of primary Planck Institut, Tubingen, Germany. antisera, washed 3 times, and incubated again for 20 mm Antisera. Reagent antiserum was made in New Zealand with 50 Ml of fluorescence-conjugated secondary antise White rabbits against purified Friend gp7l by initial injec rum. The cells were scoredfor membranefluorescence tion of 350 p.g of gp7l with completeH37RAFreund's under dark-field UV illumination with the use of a Zeiss adjuvant (Difco Laboratories, Inc. , Detroit, Mich.) in foot universal microscope with a VZ condenser, HBO-200 men pads, followed by boosts approximately every 2 months cury burner, BG 38 and KP 490 excitation filters, a 50 barrier with gp7l (50 to 150 jtg) administered either i.p., iv., on s.c. filter, and a x25 phase Neofluan objective. Fl's/antiserum Serum from weekly bleedings gave increasing nadioimmu dilution were calculated as follows: Fl = 100 x (A â€”B)/A, noassay titers with nadiolabeled Friend gp7l (7), and a where A is the percentage of nonfluonescing cells with single pool of antiserum from several bleedings with a 50% pneimmune serum and B is the percentage of nonfluoresc titer of 1:5000 in radioimmunoassay against 1 ng of 1251-la ing cells with antiserum (24). The assay has been shown to

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1977 American Association for Cancer Research. M. W. Cloyd et aI. be quite reproducible, giving a coefficient of variation of 1:240, and 1:550, respectively (Chart 1). F-MuLV-producing 1.29% with 11 separate determinations of the 50% end-point Eveline STU cells, the homologous virus system, were simi titer of Friend MuLV gp7l antisera for Friend (Eveline) cells larly reactive for F-MuLV gp7l antiserum, having a relatively (5). high 50% titer of around 1:160. An AKA embryo cell line IEM. Cells for IEM were processed in the same manner as (AKR + C), which began to spontaneously replicate AKA in the immunofluorescence assay. Ferritin-conjugated goat virus, had a 50% titer similar to G-MuLV-infected 3T3FL anti-rabbit lgG, preabsorbed on target cells before use, was cells (about 1:30) (Chart 2). Two methycholanthrene-in used as the secondary antiserum. Following the final rinse, duced C57BL/6N sarcoma lines, MCA-10 and MCA-18, rep the cells were fixed in 0.045 N cacodylate-buffened 2.5% licating endogenous C-type viruses, were considerably less glutaraldehyde containing 0.05% CaCI2 (pH 7.3, 390 reactive, with 50% titers around 1:6. Thus, munine cell lines, mOsmole) for 2 hr postfixed in 0.2 Ncacodylate-buffered 1% both productively infected and replicating endogenous mu 0504 for45mm, dehydratedingradedethanol,andembed nineC-type viruses, exhibited significant binding of rabbit F- ded inEpon 812. MuLV gp7l antiserum over that observed with pneimmune serum. The antiserum titers were highest for the Friend and Rauscher virus-producing 3T3FL cell lines, followed by RESULTS those for Friend (Eveline), Moloney, Gross, AKA, and the chemically induced virus-producing lines in decreasing or Specificity of Friend GP71 Antiserum for Virus-produc den. As control cells, normal mouse cells were also exam ing Murine Cell Lines. When tested on several virus-pro ined, and all were found to be reactive except for BALB/3T3 ducing munine cell lines, Friend gp7l antiserum yielded the lines [see accompanying report (6)]. immunofluonescence titers illustrated in Charts 1 and 2. All the virus-producing munine cell lines predominantly Antiserum dilutions where 50% of the cells fluoresced (50% displayed patchy or sectonial membrane fluorescence pat end-point titer) for the 3T3FL derivative lines replicating G temnswith Friend gp7l antiserum and were indistinguisha MuLV, M-MuLV, R-MuLV, and F-MuLV were 1:40, 1:140, ble from each other (Fig. 1). Occasionally, individual cells exhibited complete ring reactions on cap formation. More over, there was a fluorescence intensity difference between cell lines, with the Fmiend-Rauschen virus-producing cell lines displaying much brighten fluorescence than G-3T3FL, AKR + C, MCA-10 lines at low antiserum dilutions. z To determine the specificities of the immunofluomescence 8-z reactions and the nature of the reactive patterns on various LU U U, virus-producing cell lines, multiple montioned absomptions LU were performed. The relationships between the meactivities 0 of Friend, Moloney, Rauschen, AKR + C, and C57BL (MCA U- 10) virus-producing lines are summarized in Table 2. [The expression of senologically detectable gp7l determinants on normal mumine cells and their relationship to those of virus-producing cell lines, multiple monitored absorptions RECIPROCALOFSERUMDILUTION port (6).] The antiserum activity remaining after multiple 24- Chart 1. Titration of Friend gp7l rabbit antiserum on 3T3FL calls produc hr absomptions of 0.5 ml of diluted antiserum with 5 x 10@ tively infected with F-MuLV, R-MuLV, M-MuLV, and G-MuLV with the use of cells is recorded as the percentage of test cells fluorescing. an immunofluorescence assay. Results are expressed as Fl versus serum Absomptions with MCA-10, AKA + C, on Moloney cells elimi dilution based on the percentage of fluorescing cells obtained with antise rum above that obtained with preimmune serum. nated Friend gp7l antiserum activity for the absorbing cell and for each other, but left high levels of antibody activity for Friend and Rauschemcell lines. It required, however, 5 to

LU z 8-z U :-@Cla(:@ â€˜I) LU 0 D lb RECIPROCALOFSEftUNDILUTIOU Chart 2. Immunofluonescent titration of Friend gp7l antiserum on virus Fig. 1. Membrane immunofluorescenca patterns on viable murine cells producing munine cell lines. Evelina is productively infected with F-MuLV, treated with Friend gp7l antiserum. (a) Eveline cells (antiserum, 1:10), and whereas AKR + C, MCA-10, and MCA-18 replicate endogenous viruses. (b) MCA-10cells(antiserum,1:4). x 400.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1977 American Association for Cancer Research. gp7l Determinants on Virus-producing Cells Table 2 Summary of the specificity of Friend gp7l antiserum for the major MuLV-producing cell lines as defined by immunofluorescence absorption Sequential 24-hr absorptions were performed 1 to 5 times with 5 x 10@cells/0.5 ml diluted antiserum. Cells tested

Moloney (M- Rauscher (A- Friend (Eve AKR+C 3T3) 3T3) line) Cells usedfor anti :101:100Unabsorbed64r9212953596549769Mockserum absorption.MCA-10(1 :lO@)b1 :101 :1001 :101 :1001 :101 :1001

absorbed6291NTâ€•90NT96NT95NTChickembryo61878912894479563MCA-1000NT0NT90NT92NTAKR+C0003089248040Moloney

(M-3T3)0000086258048Rauscher (R-3T3)000000000Friend (Eveline)000000000

a Serum dilution. b MCA-10 cells not reactive with antiserum diluted 1 :100.

,. Percentage of cells fluorescing; representing the lowest reactivity observed of the sequentially absorbedantisera. d NT, not tested.

6 sequential absomptions with MCA-10 cells to eliminate this TARGET CELLS I â€¢F-422(F.LV) activity for AKR + C and Moloney cells; whereas only 1 to 2 2' GAL absorptions with AKR + C or Moloney cells completely 3.I*-@ 4, removed the antiserum activity for MCA-10 cells. Mock ab â€˜C sorptions, using no cells, and chick embryo cell absomp z tions removed little antiserum activity, but 1 on 2 absomp 8- tions with Rauscher and Friend virus-producing cells elimi z nated all the activity, not only for the homologous target U U,LU cells, but also for AKR + C, MCA-10, and Moloney cells. The 0 antiserum thus contained high specific activity for Friend :D and Rauscher virus-producing cells that was not removed by exhaustive sequential absorption with MCA-10, AKA + C, on Moloney virus-producing cells, and a 2nd class of activity for all other MuLV-pmoducing munine target cells tested that 2 8 32 20 512 2040 could be removed by absorption with Friend, Aauschen, RECIPROCALOFSERUMDILUTION Chart 3. Friend gp7l antiserum immunofluorescent titration curves for (1) Moloney, AKR + C, on MCA-10 virus-producing lines. Infer FeLV- and GALV-producing cell lines and (2) nonvinus-producing normal entially, these represent the gp7l type and group specifici feline embryo (FEF) and marmoset (HF-44 call lines. GAL, gibbon ape lym ties,respectively. phoma. In addition, the cell lines replicating viruses of the Gross and AKR classes (G-3T3FL and AKR + C) were completely cross-reactive, each able to absorb totally the gp7l antise mumactivity for the other. The 2 Friend virus-producing cell b I'.â€˜[email protected]. lines (F-3T3FL and Eveline) were likewise completely cross reactive. VI Friend gpll Interspecies Cell Surface Cross-reactions. V The expression of cross-reactive gp7l determinants was ;â€˜ . ,., explored also on FeLV (F-422) and gibbon ape lymphoma @4â€¢ â€˜, virus-producing cells, using Friend gp7l antiserum in im â€˜Is ..-@ munofluorescence. Both F-422 and gibbon cells were found V.â€¢ to bind significant amounts of this antiserum above the level 2aâ€˜ -â€˜2b @@ of preimmune serum (Chart 3), but the reactivity was greater c .1@ Fig. 2. Viable cell membrane immunofluorescence patterns on (a) F-422 for F-422 than for the gibbon cells. Normal feline embryo (FaLV) and (b) gibbon ape Iymphoma cell lines. Friend gp7l antiserum dilu (FEF) and marmoset (HF-4) cell lines were not significantly tion, 1:8.a, x 400;b, x 96@, reactive (Chart 3). Nearly all F-422 cells, which are thymus derived, displayed capping of membrane fluorescence, The serological relationship between GALV, FeLV, and whereas gibbon ape lymphoma presented predominantly MuLV-pnoducing cells with respect to Friend gp7l was de patch or sectomial fluorescence patterns, with occasional temmined by cnoss-absorptions. Friend gp7l antiserum ab capping (Fig. 2). sorbed with F-422 on gibbon ape lymphoma cells was not

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1977 American Association for Cancer Research. M. W. Cloyd et aI. appreciably less active for munine cell lines (Charts 4 and 5), although F-422 cells removed some activity for Friend (Eve line) cells, whereas gibbon cells did not. Chart 6 shows the effects of multiple sequential absomptions with various vi â€˜C mus-producing cell lines on the Friend gp7l antiserum activ z ity for F-422 cells. Eveline cells efficiently absorbed this 8â€”z U activity, nearly eliminating it with a single absorption, and 2 U) absorptions with Rauschem cells were similarly effective. 0 Similar absomptions with F-422 cells removed the antiserum :D activity for F-422 cells, but this required 4 absomptions. Chick embryo and gibbon ape lymphoma cells did not ap preciably diminish Friend gp7l antiserum activity for F-422 cells, even after 5 sequential absonptions, while MCA-10, NUMBEROFABSORPTIONS AKA + C, and Moloney cells absorbed only a small amount 11THCALCELLS of the antiserum activity. Chart 5. The effects of sequential absorptions with GAL cells on the Friend gp7l antiserum neactivities for Eveline, AKR + C, MCA-10 and gibbon When tested on gibbon ape lymphoma, the Friend gp7l ape lymphoma (GAL) calls (antiserum dilution, 1:10). antiserum activity was completely removed by absonptions with Eveline and Aauscher cells and was partially removed F-422 (FeLV)TARGETCELLS by absorptions with Moloney, AKR + C, and chemically activated MCA-10 virus-producing cells (Chart 7). F-422

cells were able to absorb completely the antiserum activity â€˜C for the gibbon cells (Chart 7). These results are consistent z with other studies which document variability of the inter z species region of oncomnavirus gp7l , not only among differ U ent species, but also within the mumine C-type viruses (17) (See â€œDiscussionâ€•). 0 Immunoferritin Localization of Cell Surface GP71 Reac tivity. Precise localization of the various gp7l antiserum specificities on cellular surface structures was accom plished by IEM. Friend gp7l antiserum, diluted 1:10 and 1 2 3 4 5 NUMBEROFA8SORPTIONS absorbed with chick embryo cells, labeled both virus-free 11THIND)CAT(DCELLLINES membrane segments and virions (budding and free) on Chart 6. The effects of sequential absorptions with chick embryo (CEF), gibbon ape lymphoma (GAL), F-422, and various MuLV-pnoducing munine Friend virus-producing 3T3FL cells (Fig. 3a), AKR + C cells cell lines on the reactivity of Friend gp7l antiserum for F-422 (FeLV) calls (Fig. 3b), F-422 cells (Fig. 3c), and gibbon ape lymphoma (antiserum dilution, 1:20). cells (Fig. 3d). Unabsonbed preimmune serum at the same dilution did not label any of these cells lines (not illustrated). AKA + C-absorbed antiserum, type-specific for Friend Rauscher munine cells in immunofluorescence, similarly

labeled F-3T3FL cells in IEM, binding to both vimions and â€˜C virus-freemembranesegments(Fig.3e,1:10antiserumdi z lution; Fig. 3f, 1:100), but AKR + C cells remained unlabeled z (Fig. 3g). Antiserum absorbed with normal STU embryo U mouse cells was also reactive with F-422 cells, labeling virus 0 â€˜cc EVELSNETARGETCELLS

8C â€˜C LU AKR-C TARGETCELLS NUMBEROFABSOIPTIONS z V)THINDICATEDCELLLINES @6C @ 8- MCA-1O TARGET CELLS Chart 7. The effects of sequential absonptions with various cell lines (as in z Chart 6) on the Friend gp7l antiserum reactivity for gibbon ape lymphoma (GAL)cells (antiserumdilution, 1:10).

@ â€˜ â€˜ I F@422TARGET CB_LS and virus-free cell membrane (Fig. 3h). Antiserum absorbed with Eveline cells was unmeactive for all cells tested by IEM (notillustrated).

1 2 3 4 NUNIEROFABSORPTIONS DISCUSSION 11THF-422(FeLV)CELLS Chart 4. The effects of sequential absorptions with F-422 (FaLV) cells on the Friend gp7l antiserum neactivities for Eveline, AKR + C, MCA-10, and F- As measured by immunofluomescence, the major specific 422 cells (antiserum dilution, 1:10). ity of rabbit antiserum against Friend gp7l was a distinct

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1977 American Association for Cancer Research. gp7l Determinants on Virus-producing Cells type-reaction for Friend and Rauscher virus-producing Table 3 cells, not detected on Moloney, Gross, or other MuLV Presenceoncell or absence of Friend gp7l interspecies determinants producing cells or on cells infected with FeLV or GALV. The absorptionRabbitsurfaces as detected by immunofluorescence antiserum also contained group-reactivity for all MuLV-pro sequentiallyabsorbedantiserum against Friend MuLV gp7l was ducing cells tested, as well as an interspecies cross-reactiv andother with Friend, AKR, feline, gibbon ape lymphoma, feline(F-422)virus-producing cell lines and tested for activity with ity for GALV and FeLV-producing cells. All 3 of the major 7).CellsGp71and gibbon ape lymphoma cells (see Charts 6 and gp7l determinant categories (type, group, interspecies) were localized on both budding virus and virus-free areas of cell surface membrane by immunoelectron microscopy. Gibbondeterminants@'interspecies MuLV gp7l intenspecies cross-reactions have been sub on Friend Feline ape lym stantially documented in other investigations. With the use cell surfacephomaa (Eveline) AKR+C (F-422) of complement fixation (15), antisera against F-MuLV gp7l â€”b +â€˜ â€” + reacted with FeLV, feline sarcoma virus, rat leukemia virus, +c + â€” + + + + + and hamster leukemia virus, but not endogenous cat, PLCP (pig), simian sarcoma virus, GALV, or avian viruses. With aArbitrarysymbolstodesignatedistinctgp7l interspeciessub competition radioimmunoassay (37), MuLV gp7l cross-re specificities or determinants. 11 Ã· indicates presence and â€” indicates absence of interspecies actions were noted with FeLV and feline sarcoma virus, but determinantsa, b, orc. primate viruses did not inhibit the reaction of anti-FeLV antibody with R-MuLV gp7l . Similarly, with chromium shared among onconnavimuses of small rodents and eco release cytolytic and immunoelectron microscopic assays, tropic FeLV. Another gp7l intenspecies determinant subset, gp7l intenspecies cross-reactions were restricted to cells â€œb,'has been described as a common determinant among producing oncomnaviruses of small rodents and ecotropic almost all onconnaviruses so fan tested (32). The latter may FeLV only (14, 35), a pattern similar to a determinant subset be analogous to the interspecies specificities b plus c in (designated â€˜â€˜a'â€˜)ofthe major internal protein P30(reviewed Table 3. The distinction between b and c relates only to in Ref. 32). However, other studies with radioimmunoassay the presence on absence of certain interspecies determi found gp7l interspecies relationships between MuLV, nants on AKR and Friend MuLV-pnoducing cells as deter GALV, simian sarcoma virus, and pig viruses but not en mined by their capacity to absorb antiserum activity for dogenous cat or baboon viruses (13, 32). This reactivity feline (F-422 and gibbon ape lymphoma cells. This is pattern is similar to determinant â€˜bâ€•ofP)( (32). Although consistent with the serological characterization of purified another cellular immunofluonescence study found no MuLV gp7l from AKR MuLV, which showed that this glycoprotein gp7l cross-reaction to FeLV-pmoducing cells (21), our me contained only some of the interspecies antigenic detenmi suIts with cell membrane immunofluorescence support and nants shared by FeLV and F-MuLV or A-MuLV gp71 mole extend observations documenting broad gp7l interspecies cules (17). cross-reactions and indicate further that gp7l interspecies A final point in need of clarification concerns the lack of reactions are even more complex, since different viruses Friend gp7l antiserum activity for FeLV on GALV-producing within the mumine species can vary considerably in their cells in another recent IEM study (35). We found IEM, pen gp7l interspecies determinants. formed in an analogous fashion to the immunofluonescence In this study, at least 3 subspecificities were noted in assay, was not nearly as sensitive as the latter in detecting Friend gp7l cross-reactions with feline and primate virus antibody binding to cell surfaces. This was probably due to producing cells. The effects of absomptions with various the extremely limited view of the cell surface obtained in cells on the Friend gp7l antiserum activities for feline (F- thin sections (approximately 1/300 of the surface of a sphen 422) and gibbon ape lymphoma cells (Charts 6 and 7) ical cell 20 @mindiameter). In an attempt to resolve this showed that: (Point a) AKA + C cells absorbed only a small apparent discrepancy between our immunofemnitin results amount of the activity for both F-422 and gibbon cells (me and those of Schwamzet al. (35), we preabsorbed our anti ductions in Fl of around 20 and 30, respectively); (Point b) serum with normal STU cells as the latter authors did, and absomptions with gibbon cells removed only a small amount retested it on F-422 cells using IEM. We also tested some of of the activity for F-422 cells (Fl reduction of around 10); their antiserum (diluted 1:8) on these cells. No discernible and (Point c) F-422 cells absorbed all activity for gibbon decrease in reactivity was noted in either case, indicating cells. These results demonstrate that 1 major Friend gp7l that the discrepancy may be due to sensitivity differences interspecies specificity (designated a in Table 3) reacts with between our IEM methods. That all 3 of the major gp7l determinantspresenton FeLV-pmoducingF-422cells,and determinant categories were localized on virus-free areas of not with AKR or gibbon cells (from Points a, b, and c cell surface membrane, as well as on vimions, corresponds above). Another major specificity (designated b in Table 3) to other electron microscopic results (20, 35) and indicates is foundon bothgibbonandF-422cells,but is not present no differential expression of any gp7l determinants on cell on AKA + C cells (Point a above). We have designated as c surface structures. (Table 3) the small but significant reduction in activity of The expression of various gp7l determinants on the sun Friend gp7l antiserum for F-422 and gibbon cells by ab faces of munine and hetenologous species virus-producing sorption with AKR + C cells (Charts 6 and 7). cells indicates that selective viral gp7l antigenic determi The gp7l interspecies specificity designated a in Table 3 nants are available as targets for humoral or cellular immu may correspond to a gp7l interspecies determinant subset, nological reactions. Whether on not these are involved in also designated â€œaâ€•bySchafer and Bolognesi (32), that is immunological recognition in the animal, particularly in

MARCH 1977 927

relationship to immunotherapy studies with viral reagents, Properties of Mouse Leukemia Viruses. X. Occurrence of Viral Structural Antigens on the Call Surface as Revealed by a Cytotoxicity Test. Vinol awaits further studies. ogy, 69: 157-168, 1976. Normal virus-negative munine cells, initially serving as 15. Hunsmann, G., Moennig, V., Pister, L., Seifert, E., and Schafer, W. Properties of Mouse Leukemia Viruses. VIII. The Major Viral Glycopro control cells, were also tested for Friend gp7l antiserum tamofFriendLeukemiaVirus.Senoimmunological,InterferingandHem reactivity. A low but significant level of antigenic reactivity agglutinating Capacities. Virology, 62: 307-318, 1974. was observed with most normal muninecells, and repeated 16. Hunsmann, G. , Moennig, V., and Schafer, W. Properties of Mouse Leu kemia Viruses. IX. Active and Passive Immunization of Mice against absorptions with normal cells completely eliminated the Friend Leukemia with Isolated GP71 Glycoprotein and Its Corresponding gp7l antiserum activity for all virus-producing munine cell Antiserum. Virology, 66: 327-329, 1975. lines except Friend or Rauscher. This suggested that a 17. IhIe, J. N., Denny, T. P., and Bolognasi, D. P. Purification and Serologi cal Characterization of the Major Envelope Glycoprotein from AKR Mu component of the normal cell antigenicity was group spe rine LeukemiaVirus and Its Reactivitywith AutogenousImmuneSara cific in nature, which is elaborated upon in the accompany from Mice. J. Virology, 18: 727-736, 1976. ingpaper(6). 18. IhIe, J. N., Domoton, J. J., Jr., and Bengali, K. M. Characterization of the Type and Group Spacificities of the Immune Response in Mice to Munine Leukemia Viruses. J. Virol., 18: 124-131 , 1976. 19. IhIe, J. N., Hanna, M. G., Jr., Roberson, L. E., and Kennay, F. T. ACKNOWLEDGMENTS Autoganous Immunity to Endoganous RNA Tumor Virus: Identification of Antibody Reactivity to Select Viral Antigens. J. Exptl. Med., 136: 1568- 1581,1974. We are grateful to Professor Werner Schafer, Max-Planck-Institut fÃ¼r 20. IhIe, J. M., Lee, J. C., Longstreth, J. , and Hanna, M. G., Jr. Characteriza Virusforschung, TUbingen, West Germany, for the generous gifts of purified tion of Virion and Cell SurfaceReactivitiesof NaturalImmuneSarato Friend gp7l and STU mouse cells, and Dr. John Grant, Department of Sun Munine Leukemia Viruses. In. A. Knowell, J. Friedman, and J. Prier (ads.). gery, Duke University, Durham, N. C. , for providing several munine cell lines. Tumor Virus Infections and Immunities, pp. 197-214. Baltimore: Univar We extend special gratitude to Dr. Peter Fischinger, National Cancer Insti sity Park Press, 1976. tute, Bethesda, Md., for his generous gifts of many cell lines and helpful 21. Ikeda, H., Pincus, T., Yoshiki, T., Strand, M., August, J. T., Boyse, E. A. criticism and review of the manuscript. The excellent technical assistance of and Mellors, A. C. Biological Expression of Antiganic Determinants of Carl Bishop. Bill Boyarsky, and Jessie Calder for photographic support, Murine Leukemia Virus Proteins GP71 and P30. J. Virol., 14: 1274-1280, Bernard Lloyd for electron microscopic preparation, Don Powell for chart 1974. Illustration, and Tim Denny for the radioimmunoassay is gratefully acknowl 22. Kawakami, T. G., Huff, S. D., Buckley, P. M., Dungworth, D. L., Snyder. edged. S. P., and Gilden, A. V. C-type Virus Associated with Gibbon Lymphoma Sarcoma. Nature New Biol., 235: 170-171, 1972. 23. Kennel, S. J. , and Feldman, J. D. Distribution of Viral Glycoprotein GP69/71 on Cell Surfaces of Producer and Nonproducer Cells. Cancer REFERENCES Res., 36: 200-208, 1976. 24. Klein, E., and Klein, G. Antigenic Properties of LymphomasInduced by the Molonay Agent. J. NatI. Cancer Inst., 32: 547-568, 1964. 1. August, J. 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Technique for Munina Leukemia Viruses. Nature, 229: 564-566,1971. 27. Nowinski, R. C., Flaissnar, E., Sarkar, N. H., and Aoki, T. Chromato 4, Bolognesi, D. P. Structural Components of RNA Tumor Viruses. Advan. graphic Separation and Antigenic Analysis of Proteins of the Onconnavi Virus Res., 19: 315â€”359,1974. ruses. II. Mammalian Leukemia-Sarcoma Viruses. J. Virol., 9: 359-366, 5. Cloyd, M. W., and Bignar, D. D. A Contained Indirect Viable-Cell Mem 1972. brane Immunofluorescence Microassay for Surface Antigen Analysis of 28. Oroszlan, S., Foreman, C., Kelloff, G., and Gilden, A. V. The Group Cells Infected with Hazardous Viruses. J. Clin. Microbiol. , in press. Specific Antigen and Other Structural Proteins of Hamster and Mouse C- 6. Cloyd. M. W., Bolognesi, D. P., and Bignen, D. D. Immunofluorescent Type Viruses. Virology, 43: 665-674, 1971. Analysis of Expression of the RNA Tumor Virus Major Glycoprotain, 29. Oroszlan, S., Summers, M. R.. Foreman, C., and Gildan, A. V. 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Schafer, W., and Bolognasi, D. P. Mammalian C-Type Oncornaviruses. 9. Fischinger, P. J., Moore, C. 0., and O'Connor, T. E. Isolation and Relationship between Viral Structural and Cell Surface Antigens and Identification of a Helper Virus Found in the Molonay Sarcoma-Leukemia Their Possible Significance in Immunological Defense Mechanisms. Virus complex. J. NatI. Cancer Inst., 42: 605-622, 1969. Contemporary Topics Immunobiol. , in press. 10. Fischingar, P. J. , Schafer, W., and Bolognesi, D. P. Neutralization of 33. SchAfer, W., Damsey, A., Frank, H., Hunsmann, G., Lange, J. . Moennig, Homologous and Heterologous Oncornavinuses by Antisera against the V., Pister, L., Bolognesi, D. P., Green, A. W. , Luftig, R. B., Shaper, J., P15(E) and GP71 Polypeptidesof Friend Murina Leukemia Virus. Virol and HUpen,G. Morphological, Chemical and Antiganic Organization of ogy, 71: 169-184, 1976. Mammalian C-Type Viruses. In: Y. Ito, and A. M. Dutchar (ads.), Sixth 11. Grant, J. P., Bigner, D. D., Fischinger, P. J., and Bolognasi, D. P. International Symposium on Comparative Leukemia Research and Re Expression of Munine Leukemia Virus Structural Antigens on the Surface latad Diseases, pp. 497-515. Basal, Switzerland: S. Kargen AG, Basal, ofChemicallylnduced Munine Sarcomas. Proc. NatI. Acad. Sci. U. S., 71: 1973. 5037-5041 â€¢1974. 34, Schafer, W. , Lange, J. , Fischingen, P. J. , Frank, H., Bolognasi, D. P., and 12. Green, R. W., Bolognesi, D. P., Schafer, W., Pister, L., Hunsmann, G., Pisten, L. Properties of Mouse Leukemia Viruses. II. Isolation of Viral and de Noronha, F. Polypeptides of Mammalian Oncornaviruses. I. Isola Components. Virology, 47: 210â€”228,1972. tion and Serological Analysis of Polypeptides from Munine and Feline C- 35. Schwarz, H., Hunsmann, G., Moennig, V., and Schafer, W. Properties of Type Viruses. Virology, 56: 565-579, 1973. Mouse Leukemia Viruses. Xl. Immunoelectron Microscopic Studies on 13. Hino, 5. , Stephenson. J. A., and Aaronson, S. A. 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Substantial Amounts of Friend Leukemia Virus by a Suspension Tissue Protein and Envelope Glycoprotain. J. Virol., 13: 171-180, 1974. Culture Line (Eveline Suspension Cells). Z. Naturforsch., 30c: 698-700, 39. Strand, M., Lilly, F., and August, J. T. Host Control of Endoganous 1975. Murine Leukemia Virus Gene Expression: Concentrations of Viral Pro 37, Strand, M. , and August, J. T. Structural Proteins of Oncoganic Ribonu teins in High and Low Leukemia Mouse Strains. Proc. NatI. Acad. Sci. U. cleic Acid Viruses. Intarspec II, a New Interspacies Antigen. J. Biol. S., 71: 3682-3686, 1974. Chem., 248: 5627-5633, 1973. 40. Tronick, S. R., Stephenson, J. A. , and Aaronson, S. A. Immunological 38. Strand, M., and August, J. T. Structural Proteins of Mammalian Onco Characterization of a Low Molecular Weight Polypeptide of Munine Leu genic RNA Viruses: Multiple Antigenic Determinants of the Major Internal kemia Virus. Virology, 54: 199â€”206,1973.

MARCH 1977 929

M. W. Cloyd et al.

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Fig. 3. Immunofarnitin electron microscopic localization of call surface antigens reactive with antibodies of Friend gp7l antiserum (diluted 1:10). Fernitin outlined bybrackets. a, F-3T3 cells, x 39,000; b, AKA + C cells, x 45,000; c, F-422 (FeLV) cells, x 78,000; d, gibbon ape lymphoma cells, 68,000x. e, F-3T3FL cells reacted with AKR + C absorbed antiserum, x 66,000; f, F-3T3FL cells reacted with AKR + C-absorbed antiserum diluted 1:100, x 74,000; g AKR + C and AKR + C-absorbed antiserum (1:10), x 72,000; h, Friend gp7l antiserum (1:10) absorbedwith normal STU mouseembryo cells applied to F-422 (FeLV) target cells,x51,000.

930 CANCER RESEARCH VOL. 37

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1977 American Association for Cancer Research. Immunofluorescent Analysis of Expression of the RNA Tumor Virus Major Glycoprotein, gp71, on Surfaces of Virusproducing Murine and Other Mammalian Species Cell Lines

Miles W. Cloyd, Dani P. Bolognesi and Darell D. Bigner

Cancer Res 1977;37:922-930.

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