Proc. Nat. Acad. Sci. USA Vol. 69, No. 7, pp. 1900-1904, July 1972

Activating and Protective Capacities of a Purified Electrophoretic Fraction of Murine Leukemia for Marine Leukemia Virus Infectivity (virus antigens/group-specific antigen I) PETER J. FISCHINGER*, JENS LANGEt, AND WERNER SCHAFERt * Viral Leukemia and Lymphoma Branch, National Institute, Bethesda, Maryland, 20014; and t Max Planck Institut far Virusforschung, Biologisch-Medizinische Abteilung, Tobingen, Germany Communicated by S. Spiegelman, May 3, 1972

ABSTRACT A highly purified gel electrophoretic frac- MATERIALS AND METHODS tion of murine leukemia virus (MuLV) can confer en- hanced infectivity on MuLV derived from tissue culture and Assays. Gross leukemia virus (GLV) derived and can protect MuLV from neutralization by specific from mouse tumors and passed through rats was adapted for antiserum. Of the known viral tested, a purified growth in mouse 3T3 cells. The IC strain of Moloney leukemia electrophoretic fraction (14,000 daltons) is the only active fraction and seems to consist of the group-specific antigen virus (MLV-IC), isolated from mixtures of murine sarcoma I and an associated lipid moiety. The action of this virus and MuLV, was derived from lymphomas of NIH Swiss fraction obtained from one type of MuLV is group-specific mice or from chronically infected 3T3 cells (11). Stocks of in that it can enhance and protect serologically different Friend leukemia virus (FLV) consisted of homogenates of types of MuLV. The effect of this fraction is exerted on spleens from mice with Friend disease or of supernates from viruses and not on cells. MuLV derived from tumors was not enhanced by the fraction, but became amenable to its long-term infected STU mouse cultured cells. The subgroup action after a single passage through tissue culture. Recon- specificity, neutralization, and antigenic reactivity of the struction experiments suggested that the fraction may above MuLVs have been described in detail (8, 11). All MuLV consist of an exterior associated with some lipids. assays were performed in sarcoma-positive leukemia-nega- This complex, which apparently protrudes on the viral surface, is required for infectivity and shields the type- tive 3T3 cells (S+L- cells), which consisted of a mixture of specific antigen containing the hemagglutinating site. transformed and revertant cells (80/20), by inoculation of a small volume (0.2 ml) of virus directly into a large volume Several of the murine leukemia virus (MuLV) proteins have (5 ml) of supernatant. Lytic-type lesions indicative of MuLV recently been separated, isolated, and identified as distinct replication were scored as focus-inducing units (FIU) as virus antigens (1-7). The availability of relatively specific, described (12, 13). as well as complex, antisera has revealed in complement fixa- tion and immunodiffusion tests several separate group-specific Antisera and Neutralization. A GLV-specific G-serum of (gs) and one type-specific (v) antigen (6, 8). The new nomen- high titer, "Old," obtained from F1 (WFu X BN) rats carry- clature based on purified antigen reactivity recognizes the ing syngeneic lymphomas induced by GLV, and an FLY or following main antigens: IV, the major gs-antigen of the spe- Rauscher leukemia virus (RLV)-specific antiserum produced cies-specific type previously described as gs-1; V, the gs-antigen in monkeys against plasma RLV, RM-serum, "Fink," were also known as gs-3 shared by leukemia viruses of several gifts from Drs. L. Old and M. Fink, respectively (1, 14). mammalian species; III a lesser antigen; II, an antigen com- MuLV was neutralized with concentrations of antiserum re- plex that contains both a gs- and type-specific determinants quired to neutralize about 95% of the virus, and the reduction and hemagglutinating activity; I, a species-specific gs-antigen of FIUs was expressed as the ratio of untreated to antiserum- that can be lost after treatment with neuraminidase and treated MuLV FIUs. Incubation with antiserum lasted for phospholipase C; and finally component X, which could be 1 hr at 200 and 1 hr at 40. identified as a virus-specific structure by complement fixation Isolation of Virus Antigens. Previously described methods (6, 8-10). In attempts to relate structural components to were followed. Briefly, once-banded cultured FLV was treated viral function, we assessed the effects of some individual with sodium dodecyl sulfate and phenol. Viral proteins were purified MuLV fractions on infectious virus and assayed their precipitated from the phenol phase with ethanol (6, 15). activity in MuLV neutralization. The gs-antigen I, even Protein separation was performed on preparative polyacryl- when obtained in a highly purified state by preparative gel amide slab gel electrophoresis apparatus, and the elhting frac- electrophoresis, possessed a surprising biological activity for tions were monitored for 280-nm absorbance (16). The purity MuLV manifested by its activating a majority of otherwise of the isolated fractions was further documented by analytical noninfectious MuLV, and protecting the virion from neutraliz- polyacrylamide gel electrophoresis. Individual eluted fractions ation by specific antisera. were at times again run on the preparative apparatus with or without additional treatment, such as chloroform-methanol Abbreviations: MuLV, murine leukemia virus; GLV, Gross 3:1 (16). Coomassie blue and periodic acid Schiff stains were strain of MuLV; MLV-IC, Moloney strain, IC isolate of MuLV; The FLV, Friend strain of MuLV; gs, group-specific antigen; S+L-, used for proteins and polysaccharides, respectively (6). sarcoma-positive leukemia-negative 3T3 cells transformed by purified FLV hemagglutinating fraction containing IIv and murine sarcoma virus; FIU, focus-inducing units; v, type-spe- IIgs, and antigen X was separated on Sephadex G-150 columns cific antigen. and citrate density gradients as described (6). 1900 Downloaded by guest on September 25, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Activation and Protection of MuLV 1901

Assaysfor Focus Enhancement and Protection from Neutral- ization by Virus Fractions. MuLV grown in tissue culture or extracted from tumors was diluted in medium to give about .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 20-30 FIU/0.2 ml. An increment (20% by volume) of FLV fraction material diluted in phosphate buffered saline (Dulbecco) (pH 7.2) was added to the virus, and the mix- HLJL4U. ture was incubated at 20° for 1 hr and at 40 for an additional ~~~~~~~~~~~~~~~~~~~~~~ hour. The virus (0.2 ml) was then inoculated directly on a dish of 24-hr old S+L- cells containing 5 ml of medium. Alternately, where indicated, a high titer of MuLV was treated with FLV fraction El (gs-antigen I, highly purified by preparative gel electrophoresis; see Results) as above and diluted in medium up to 1000-fold just before infection. Foci were read on the sixth day, and the factor of enhancement was expressed as the ratio of FJUs of treated to control virus, E3W _u which was handled with the same buffer in an identical way. Viruses were neutralized by treatment of MuLV with dilu- tions of specific antisera for 1 hr at 200 and 1 hr at 4°. For determination of the protective effect of FLV fractions, MuLV E2 was first exposed to the virus fraction for 1 hr at 20° and then reacted with the specific antiserum. As a variation, virus frac- E1l tions were first treated with antiserum for 1 hr at 200 and then exposed to infectious MuLV for an additional hour at 20°. Residual, nonneutralized virus content was expressed as the ratio of FIUs after treatment with antiserum to FIUs of the mock-treated control MuLV (VY/Vo). Cells were treated with diethylaminoethyl (DEAE)-dex- tran (Pharmacia) (25 ,ug/ml) for 30 min at 370 followed by two FIG. 1. Concurrent analytical polyacrylamide slab gel elec- washes with medium where indicated, to obtain optimum en- trophoresis of FLV total protein and FLV individual proteins hancement of virus (17). MuLV was treated with DEAE-dex- previously separated by preparative polyacrylamide gel electro- tran in an analogous manner with 25 /g/ml, but the virus phoresis. E3 was examined after a single preparative electro- was then diluted so that the DEAE-dextran concentration phoresis, whereas El and E2 were examined after two preparative in the assay was an ineffectual 0.1Iug/ml or lower. Cells were experiments. For verification of the properties and the purity of treated the same way with El as with DEAE-dextran. FLV proteins derived as preparative electrophoresis eluates, these were analyzed with total FLV proteins on the same gel under RESULTS identical conditions. The biological effect of isolated viral fraction on MuLV focus induction Preparative polyacrylamide gel electrophoresis separated the viral proteins into three major peaks whose purity was verified further purified through a second preparative electrophoresis by analytical slab gel electrophoresis (Fig. 1). These are E3 cycle, El did not decrease FIU enhancement. In regular ana- (the largest peak that contained both the principal gs-antigen lytical gel electrophoresis, the El band was often disturbed by IV and the interspecies antigen V of MuLV), E2 (not as yet a nonprotein substance which, however, stained strongly with firmly identified), and El (the smallest protein component periodic acid Schiff stain (6, 16). If purified virus proteins that contained gs-antigen I reactivity) (6, 16, 18). The hemag- were first treated with chloroform and methanol, then the glutinating fraction that contained the type specific HIv and next preparative or analytical run showed a clear and distinct the gs-antigen IIgs were, with antigen X, prepared by Sepha- El band without any periodic acid Schiff staining component. dex G-150 chromatography and density gradient centrifuga- The enhancing property for MuLV, however, was essentially tion (6). Initially, total FLV protein as well as individual frac- lost after chloroform-methanol treatment. Two other purified tions were tested for the absorption of specific neutralizing virus fractions, the hemagglutinating complex, containing antibody. Total FLV proteins, obtained after treatment with the IIgs and IIv reactivities, and X, had no substantial sodium dodecyl sulfate, absorbed anti-FLV neutralizing potentiating effect. The extract of the total virus protein activity only partially, but the isolated FLV hemagglutinating before any electrophoresis did not enhance FIUs despite the complex was, as shown previously, effective in eliminating presence of El-antigen I in the mixture. the neutralizing activity of the "Fink" RM-serum but not of The degree of enhancement was significant for all three the "Old" G-serum (8). During testing of individual FLV frac- strains of MuLV tested and indicated that up to 80% of tions, the control mixture of a purified virus protein El and MuLV derived from tissue culture was not detectable as infectious virus significantly enhanced FIU titers of FLV de- infectious virus in the S+L- cell assay which was as sen- rived from tissue culture (Table 1), whereas the remaining sitive as any other assay for replicating MuLV (13). Dose- virus fractions were unremarkable. The quantities of protein response studies indicated that El activity reached a maxi- of El, E2, and E3 were within a 2-fold range of concentration mum at about a 1:30-1:50 dilution of the original, which of each other. This function of El, gs-antigen I, derived from would contain (based on absorbance at 280 nm) about 50 ,g FLV, was effective for all strains of MuLV tested. If it was of protein/ml. Activity was still manifest at about 1:400 Downloaded by guest on September 25, 2021 1902 Microbiology: Fischinger et al. Proc. Nat. Acad. Sci. USA 69 (1972) dilution of El, which would double the normal FIU counts. TABLE 2. FLV electrophoretic fraction El protects Because FIUs were assayed by inoculation of virus treated MuLV from neutralization by specific antisera with E1 in a small volume into a volume of medium that ElectrohreifractrophoretionMulVDilution tissefrom Specific Ave. N could reduce the extant El concentration 25-fold, it was atnu l nV thought that El interacted with the virus. Removal of medium before inoculation of virus treated with El did not - - FLV 79.0 1.00 FLV Fink 3.0 0.038 show an additional potentiating effect. The total amount of j E3 1:35 FLV 6X5. 0.83 the virus used for the reaction with a single concentration of FLV Fink 2.3 0.029 j El was assessed for the degree of enhancement attained. E2 1:35 FLV - 86.5 1.09 FLV Fink 2.0 0.025 More than a 10-fold variation in the virus inoculum did not ( El 1:35 FLV -- TMTC Enhanced (one cycle) FLV Fink 109.0 1.38 modify FIU titers if a single concentration of El was used for El 1:35 FLV - TMTC Enhanced all virus inocula. (Two cycles) FLV Fink 109.3 1.39 Eltwo cycles, 1:35 FLV - 975 1.23 Protection of MuLV after treatment with FLV-protein Chloroform methanol FLV Fink 4.7 0.059 fractions from neutralization by specific antisera Selected electrophoretic fractions of FLV were reacted first The second salient effect of El, but not of other protein with the "Fink" anti-RLV serum (RM-serum) at a serum con- electrophoretic fractions, was a direct protection of the centration that would neutralize about two logarithm units of treated virus from neutralization by specific antibody. In a FLV. FLV was then exposed to the mixture of "Fink" serum and homologous system of FLV electrophoretic fractions reacting El, and the total was inoculated onto S+L- cells. Virus neu- with FLV derived from infected tissue culture, only El tralization is expressed as the ratio of FIU after neutralization afforded excellent protection from antibody (Table 2). A to the original virus titer (V,,/Vo). Ave, average; TMTC, too degree of enhancement was clearly found in the control virus many to count. preparations not exposed to antiserum. If both factors are taken into account, namely about 4-fold enhancement with a 95% neutralization, it is evident that only a teraction of FLV with El must be faster than that of FLV slight residual neutralization was still manifest. The pro- and its antibody. Again the state of El purification was tective effect is remarkable specifically because, under the commensurate with its protective capacity; analogous to above conditions, El and neutralizing antibody were ex- virus-enhancing capability, the effect of El persisted through posed to the virus at the same time. Thus, the rate of in. two cycles of preparative electrophoresis but was lost after treatment of El by chloroform and methanol. Because antigen I contained in El is gs in nature and be- TABLE 1. Enhancement of MuLV focus induction in cause it also enhanced serotypically different GLV, El derived S + L-cells some by purified fractions of FLU from FLV was examined for a potential protective effect on

Final Indicatort Enhancement* GLV. FLV fraction W dilution antigen content MuLV of FIU A fairly high dilution of E1 was first reacted with GLV Preparative electrophoresis derived from infected 3T3 cells and then exposed to GLV E3 (1:35) IV, V FLV 0.83 In E3 (1:200) fV V MLV 0.97 specific antiserum. the controls, a good degree of enhance- E2 (1:35) FLV 1.09 ment was seen as expected and the GLV treated with El and El 4l:36) I FLV 4.6 exposed to antibody was eminently protected. In this case 2.El (1:35)1 I GLV 4.6 CM-El (1:35)A I FLV 1.23 residual neutralization was even lower, possibly because of El (1:200) I MLV 3.4 previous treatment with E1 as opposed to simultaneous Purified hemagglutinin (1:50M Ilg, lv MLV 1.3 addition of E1 and antibody. As a corollary, GLV was also Purified fraction "X" (1:50)' X MLV 1.0 first neutralized with the "Old" G-serum and the neutralized virus was treated with El to determine whether virus already Total FLV protein before All known in eleacrophoresis (1:25) antigens FLV 0.95 neutralized could be reactivated by El. A slight rise foci was observed in the GLV preparation treated with antibody * The antigen composition of the individual preparative and subsequently reacted with El, but the magnitude of the electrophoretic fractions of MuLV was assessed by immuno- effect is compatible with an enhancement of the residual diffusion and complement fixation by various reference antisera nonneutralized GLV titer rather than a reactivation of (6,8). previously neutralized GLV. t Representative laboratory strains of MuLV were all derived from supernates of chronically infected mouse 3T3FL cells, for Biological activity of El compared by alternate treatment MLV and GLV, and from STU embryo cells, for FLV. of MuLV or target cells + Enhancement of MuLV focus induction is expressed as the Neither El nor other isolated FLV fractions by themselves ratio of FIUs in S+L- cells after treatment with the isolated had any observable effect on the S+L- cells used in the virus-protein fraction to FIUs of mock-treated virus. virus assays. Because surface and charge-mediated effects § 2-El denotes two cycles of preparative electrophoresis before could have been active in some of the above phenomena, the assay. CM-El describes the final El fraction after the following effect of a well-known virus potentiating agent, DEAE- treatment sequence: a preparative run, exposure to a mixture of was examined in a manner. El and chloroform and methanol, and a second preparative run. dextran, comparative Purified virus fraction with hemagglutinating activity and DEAE-dextran were compared in their effects on virus or on the antigen X preparation were obtained from FLV after Tween- the S+L- cells. In the S+L- cell preparation, repeated ether treatment, Sephadex G-150 chomatography, and potassium assays of MuLV on cells previously treated with DEAE- citrate density gradient centrifugation as described (6). dextran showed remarkably little enhancement (2-fold) over Downloaded by guest on September 25, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Activation and Protection of MuLV 1903

untreated S+L- cells. DEAE-dextran had such a minimal TABLE 4. El fraction fails to enchance MuLV derived effect on MuLV FIUs (Table 3). MuLV of high titer was directly from tumors treated with DEAE-dextran at the same concentration as that used for cells. Subsequently, this MuLV of high titer Virus Source dilutionEl Enhancementof FlU was diluted so that the concentration of residual DEAE- dextran would be insignificant (<0.1 iug/ml). No enhance- FLV Mouse Spleen, 1:200 1.1 ment of FIUs was observed with MuLV treated with DEAE- Friend disease dextran. Treatment of target S+L- cells with El under GLV Rat lymphoma 1:200 1.3 variables of time and concentration had no effect several on GLV 3T3 cells P4 1:200 3.9 MuLV titers. Treatment of MuLV with El followed by up to a 1:3200 dilution of the inoculum still retained the El- MLV-IC Mouse lymphoma 1:150 1.0 mediated enhancing effect. Over a 100-fold dilution range of MLV-IC 3T3 cells P1 1:150 3.2 MuLV treated with El, the degree of enhancement was MLV-IC 3T3 cells P2 1:150 3.8 independent of virus dilution. Accordingly, biological activity of El seems to be mediated by a direct effect on viruses rather Selected MuLV strains were obtained from mouse or rat tumors than on target cells. and tested with El for enhancement. The same virus isolates were then inoculated into 3T3FL cells, and progeny virus was ex- amined after one or more passages for whether it could be en- Nature of MuLV suitable for El enhancement hanced by El. Although all strains of MuLV passed through tissue culture were enhanced to a fairly similar degree by FLV El electro- phoretic fraction, comparative studies with MuLV obtained of viruses derived from tissue culture were neutralized about as tumor extracts showed no potentiation of virus titer at all. 10-fold better than their parental virus extracted from tumors. All three MuLV types assessed, GLV, FLV, and MLV, did It is possible that MuLV derived from tumors had adequate not show any enhancing virus in their stocks although the antigen I content and accordingly had better protection from GLV stock used for most experiments was derived from the specific antibody, while MuLV derived from tissue culture same tumor GLV after only 10-15 passages through tissue had less antigen I, was more easily neutralized, and corre- culture (Table 4). To see how rapidly the capacity for MuLV spondingly was protected by an excess of gs-antigen I. potentiation develops with tissue culture passage of the Reconstruction of participating entities and events leading virus, a MLV-IC virus stock derived from Swiss mouse to MuLV enhancement by El tumors was passed for one, two, or more passages in 3T3 cells To determine whether MuLV from tissue culture was sen- and the virus progeny were assessed for El enhancement at sitive to the El effect because it contained heat-inactivated each step. The increase in foci observed after El treatment virus, FLV derived from tumors was incubated slowly at 200 became immediately apparent in the first harvest of MLV-IC or 370 for various lengths of time, and the surviving virus 5 days after infection and was maintained at about the was assayed for El enhancement. Neither parental tumor same efficacy at least up to the fortieth passage. Another MuLV stock nor its derived partially heat-inactivated virus strain of GLV (Joachim) obtained from rat cells, which did was potentiated. not form overt foci in S+L- cells but which had all the The loss of activity of El after exposure to chloroform and known GLV antigens including IIv, did not produce foci also methanol was interesting because, antigenically, some El was after El treatment. The GLV and FLV, derived from tumors, still detectable and the polysaccharide components were ap- used above were also compared with their tissue culture parently present in the initial fraction eluting before El, as counterparts for sensitivity to neutralization at a given seen by periodic acid Schiff staining. The now separated dilution of respective specific antisera. In each case the FIUs periodic acid Schiff staining peak did not enhance MuLV. A recombination of the inactive El, after treatment with chloroform and methanol, with the periodic acid Schiff stain- TABLE 3. El exerts its MuLV enhancing effect on virus ing peak did not restore enhancement normally seen with El. and not on cells Thus, the biological activity of El seemed to depend on an interaction of antigen I and some lipid moiety. Initial reaction of Added virus or Enhancement Antigen I reactivity of MuLV was also lost after treatment enhancer and target initial reaction' mixture of FlU with phospholipase C and neuraminidase. The virus treated with enzymes only then acquired the ability to hemagglutinate El 1:200+ GLV 1:40 same 3.8 erythrocytes, while its antigen II complex, including type El 1:200+3T3cells GLV 1:40 0.99 specificity, remained intact (6). A high titer FLV derived DEAE-D + 3T3 cells GLV 1:40 2.0 from tumors and not at all enhanced by El was exposed to El 1:200 + MLV-IC 1:1 (MLV-IC + El) 1:200 2.7 various concentrations of both enzymes and treated for 30 (MLV-IC + El) 1:3200 2.7 min at 37°. The surviving virus was checked for enhance- DEAE-D + FLV 1:4 (FLV + DEAE-D) 1:400 1.0 ment by El after the virus-enzyme mixture was greatly El or DEAE-dextran (DEAE-D) was used to treat either diluted (1:1000). A combined enzyme treatment of FLV viruses or cells. If cells were treated with either enhancer, then derived from tumors with concentrations of 500 or 50 units/ml virus was added to treated cells. If virus was treated, then the of neuraminidase and 0.1 or 0.01 mg/ml of phospholipase C virus-enhancer mixture was diluted sufficiently so that the resi- reduced the virus titer 1.5 and 0.5 logarithmic units, re- dual concentration of the enhancer would be without effect. spectively. After treatment with several concentrations of Downloaded by guest on September 25, 2021 1904 Microbiology: Fischinger et al. Proc. Nat. Acad. Sci. USA 69 (1972) both enzymes, the residual infectious FLV was enhanced 2- to Accordingly, MuLV derived from tumors could be vis- 3-fold. Although this result suggested that exogenous El ualized as being more fully equipped with an adequate El replacement led to a gain of infectivity, careful variation of complement. The less prominent IIv component would be enzyme concentration, time, and mode of treatment are re- relatively more protected from antibody and would function quired to magnify and confirm this effect. as a less adequate immunogen. The MuLV group is notorious The treatment of MuLV derived from tissue culture with for the difficulty of obtaining specific antisera of high titer to total FLV proteins before electrophoresis did not enhance, the viruses. Interestingly, the antisera that best detected although its El complement was present. Accordingly, a re- antigen I in immunodiffusion were prepared against RLV association of El and other FLV proteins was done. MuLV derived from plasma from leukemic mice (8). Secondly, the potentiation by El alone was compared to enhancement by virus derived from tumors could be more pathogenic because El reacted with one or more fractions. No single of a surface rich in El. This supposition gained credence when electrophoretic fraction of FLV, nor the hemagglutinating it was shown that a highly pathogenic RLV derived from entity or antigen X alone, eliminated the biological activity of plasma lost a disproportionate amount of leukemogeinc El. The cumulative combination of all the virus fractions, potential on a single passage through tissue culture when however, did eliminate the biological activity of El completely leukemogenic potential was related to the titer of helper so that the loss of activity of the artifactual amalgam of virus activity of RLV derived from tumors or tissue culture (21). fractions mirrored the lack of enhancement shown by the Analogously, MLV from lymphomas was enhanced by El original unseparated viral proteins. after a single passage through tissue culture. Attenuation, leukemogenicity, and immunogenicity of MuLV, as well as DISCUSSION enhancement of several marine sarcoma virus pseudotypes, are being investigated in terms of El content. The isolated El fraction of FLV had a dual biological effect We thank Mrs. Irma Schneider for excellent technical as- on several strains of MuLV grown in tissue culture: the sistance. realization of an infectious potential of otherwise undetec- 1. Fink, M. A., Sibal, L. R., Wivel, N. A., Cowles, C. A. & table MuLV and the conferring of protection to MuLV from O'Connor, T. E. (1969) Virology 37, 605-614. specific antibody. Both effects were group specific in that any 2. Gregoriades, A. & Old, L. J. (1969) Virology 37, 189-202. strain of MuLV was enhanced by E1 of FLV, and GLV or 3. Schafer, W., Anderer, F. A., Bauer, H. & Pister, L. (1969) reactions with their respective Virology 38, 387-394. FLV specific antibodies did 4. Duesberg, P. H., Martin, G. S. & Vogt, P. K. (1970) not result in the expected virus neutralization after previous Virology 41, 631-646. treatment with FLV-El. Treatment of El, which destroyed 5. Oroszlan, S., Foreman, C., Kelloff, G. & Gilden, R. V. one of the above activities, also negated the second, and (1971) Virology 43, 665-674. residual recoverable gel electrophoresis fractions(s) of El did 6. Schafer, W., Lange, J., Fischinger, P. J., Frank, H., Bolog- nesi, D. P. & Pister, L. (1972) Virology 47, 210-228. not affect singly or in combination either biological activity. 7. Moroni, C. (1972) Virology 47, 1-7. We propose the following descriptive model, which is com- 8. Schafer, W., Fischinger, P. J., Lange, J. & Pister, L. (1972) patible with the above observations. Murine RNA tumor Virology 47, 197-209. viruses have close to or on their surface a complex structure 9. Geering, G., Hardy, W. D., Old, L. J., de Harven, E. & (El) that consists of a group-specific protein antigen I and Brodey, R. S. (1968) Virology 37, 678-707. 10. Geering, G., Aoki, T. & Old, L. J. (1970) Nature 226, 265- an associated lipid or polysaccharide fraction (6, 16). The 266. contiguous position of El derived from MuLV and a glucos- 11. Fischinger, P. J., Moore, C. 0. & O'Connor, T. E. (1969) amine-containing fraction in polyacrylamide gels has been J. Nat. Cancer Inst. 42, 605-622. confirmed (7). Both antigen I and an attending, possibly 12. Bassin, R. H., Tuttle, N. & Fischinger, P. J. (1970) Int. J. Cancer 6, 95-107. glycolipid, structure are necessary for activity of El. This 13. Bassin, R. H., Tuttle, N. & Fischinger, P. J. (1971) Nature complex protrudes in such a way that the neighboring type- 229, 564-566. specific antigen, IIv, is partially protected from accessibility 14. Geering, G., Old, L. J. & Boyse, E. A. (1966) J. Exp. Med. by antibody. Enzyme treatment of the whole virus particle 124, 753-772. removes I and exposes with its 15. Bolognesi, D. P. & Bauer, H. (1970) Virology 42, 1097-1112. antigen IIv-antigen, which, 16. Schafer, W. (1972) in Comparative Leukemia Research, IIgs-antigen, represents a polyvalent array of sites capable of ed. Dutcher, R. M. (S. Karger, Basel, Muinchen, Paris, reacting with erythrocytes and functioning as a hemag- New York), in press. glutinin (19). Some El is probably required for infectivity 17. Duc-Nguyen, H. (1968) J. Virol. 2, 643-644. and may be related to some surface "spike" glycoprotein 18. Schafer, W., Bauer, H., Bolognesi, D., Fischinger, P., Frank, H., Gelderblom, H., Lange, J. & Nermut, M. (1972) material of that is needed for infection in The 25th Annual Symposium on Fundamental Cancer (20). The requirement of El activity is quantitative and Research, "Molecular Studies in Viral Neoplasia" (Houston, relative so that a minimal amount of El is necessary for Texas 1972), in press. infectivity and the addition of maximal amounts of El pro- 19. Schafer, W. & Szanto, J. (1969) Z. Naturforsch. B 24, tects most sites from In this context 1324-1331. IIv-antigen antibody. 20. Rifkin, D. B. & Compans, R. W. (1971) Virology 46, the lack of enhancement of MuLV derived from tumors was 485-489. commensurate with its unexplainably greater resistance to 21. Schlom, J., Moloney, J. B. & Groupe, V. (1971) Cancer specific antiserum. Res. 31, 260-264. Downloaded by guest on September 25, 2021