Immunochemistry, 1975. Vol. 12, pp. 61-66. Pergamon Press. Printed in Great Britain.
ISOLATION OF MAMMALIAN TYPE C RNA VIRUS CROSS-REACTIVE ANTIGEN AND ANTIBODY BY IMMUNO-AFFINITY CHROMATOGRAPHY S T E P H E N OROSZLAN, DAVID BOVA and R A Y M O N D V. G I L D E N Flow Laboratories, Inc., Rockville, Maryland 20852, U.S.A. (Received 20 M a y 1974)
Abstract--The ca. 30,000 mol. wt internal protein of mammalian Type C viruses, p30, has previously been shown to react with homologous and heterologous p30 antisera. Certain sera, especially those prepared in guinea-pigs, are highly species-specifiC.Absorption in gel experiments strongly indicates that p30s have qualitatively different classes of determinants designated species-specific or interspecific, and that differential serum reactions are not related to potency vs a single class of determinants. The use of immunoadsorbents comprised of hamster Type C virus p30 permitted the purification of interspecies antibody from a polyspecific goat anti-feline leukemia virus antiserum. This antiserum, in turn coupled to an adsorbent, purified from mouse leukemia virus a component of 30,000 mol. wt which possessed both interspecies and species-specific determinants. This clearly indicates the coexistence of both classes of determinants on a single structure.
INTRODUCTION The ~ 30,000 mol. wt internal protein of Type C viruses referred to synonymously as the group-specific (gs antigen; Huebner et al., 1964; Geering et al., 1966; Huebner, 1967) or p30 (August et al., 1974), has proven an important marker for a variety of studies relating to inter-viral relationships (Gilden and Oroszlan, 1972), natural history (Huebner et al., 1970; Hartley et al., 1969), and the relationship of these viruses to cancer (Huebner and Todaro, 1969; Meier et al., 1973). Of signal importance was the finding (Gilden et al., 1971 ; Oroszlan et al., 1971b) that this protein carried both species-specific and interspecific determinants (Geering et al., 1970), thus providing one method for establishing the relationship of new isolates to those already described (Oroszlan et al., 1972c). More recent studies have indicated that the p30s of closely related viruses, i.e. those sharing species-specific determinants, can 'be distinguished by radioimmunoassay (Strand and August, 1974)and quantitative C' fixation (Gilden et al., in press); thus p 30s carry type-specific determinants as well. In addition, the interspecific category of crossreactive determinants has been shown to consist of subgroups as initially found by radioimmunoassay inhibition methods (Parks and Scolnick, 1972), and subsequently shown in gel diffusion (Schafer et al., 1973; Gilden et al., 1974). In this communication we describe an immunochemical method for preparation of interspecies reactive antibody from a polyspecific serum. Using this antibody population, a heterologous p30 was purified. The purified p30 displayed both interspecies and speciesspecific determinants in keeping with previous findings. This general approach has been useful in detect-
ing additional determinants on p30 molecules (see accompanying paper), and should be useful for isolation of type-specific antibody populations as well. MATERIALS AND METHODS Virus
The Rauseher strain of mouse leukemia virus (R-MuLV) was grown in monolayer cultures of chronically infected mouse (BALB/c) bone marrow, JLS-V9 cells (Wright et al., 1967). Rat Type C virus (RaLV) was obtained from the chronically infected MSB-1 cell line (Ting, 1968) derived fro.m a tumor induced by M-MSV in a female rat of the Brown Norway strain. This virus is an interspecies pseudotype with structural proteins common to endogenous rat Type C viruses (Oroszlan et al., 1972a) and nucleic acid sequences of both mouse and rat origin (Okabe et al., 1973). Feline leukemia virus (FeLV, Theilen strain) was obtained from a chronically infected cat lymphocytic cell suspension culture (Theilen et al., 1969). RD-114, an endogenous cat virus was the isolate from the RD rhabdomyosarcoma cell line of McAllister et aL (1972). Hamster Type C virus (HaLV) was the isolate of Kelloff et al. (1970). Virus purification
Viruses were purified by sucrose density gradient centrifugation and radioactive R-MuLV, labeled with ~4C-lysine (> 260 mCi/m-mole, New England Nuclear) was prepared as described previously (Oroszlan et al., 1971h). Puvification of p30s
The isoelectric focusing technique was used as reported previously (Oroszlan et al., 1970). 61
62
S. OROSZLAN, D. BOVA and R. V. GILDEN
l nmmnodiffusion Double immunodiffusion was done in plates that contained 0'8~ agarose, pH 7-4, ionic strength 0.15. In preparing plates, 8 gm agarose, 9.3 gm Tris [-2-amino-2-(hydroxymethyl)l,3-propanediol], 74 ml 1 N HCI, and 7-0gin NaCI were made up to 1 1. with distilled water. Merthiolate was added as a preservative. Plates were kept at room temperature, observed for 72 hr, and photographed when optimum precipitation lines developed.
Complement-fixation Viral group-specific antigens (p30) were titrated in microcomplement-fixation (CF) tests as previously described (Oroszlan et al., 1970).
Sodium dodec yl sulfate-pol yacr ylamide 9el electrophoresis. ( S D ~ P A G E ) was carried out in 10~ polyacrylamide gels (Weber and Osborn, 1969).
Protein determination Protein content of antigen and antibody preparations was determined by the method of Lowry et al. (19511 using crystalline bovine serum albumin as standard.
Antiserum Guinea-pig antisera were prepared against the various gs proteins purified by isoelectric focusing. These sera appear highly species-specific in both gel diffusion and CF assays, although by highly sensitive radioimmunoassay procedures these sera show variable levels of cross-reactive antibody; generally less than 1/20 of the species-specific titer. Goat antisera to FeLV (obtained from Dr. Roger Wilsnack, Huntingdon Labs) and MuLV were prepared against Tween-ether disrupted purified virus. These sera contain sufficient interspecies antibody to produce gel diffusion reactions, but also show an excess of species-specific antibody. MSV-I serum was obtained by immunizing rats with tumor homogenates and 7S antibodies from goat serum were separated on Sephadex G-200 as previously described (Oroszlan et al., 1972b).
lmmunoadsorbent procedures Coupling CNBr to Sepharose. Three ml of Sepharose 4B (Pharmacia) was thoroughly washed with distilled Water. It was then resuspended in distilled water to give a slurry of 5 ml final volume. It was chilled to 0°C (icebath) and in a fume hood, equal volume of cold CNBr (50 mg/ml; Eastman Kodak Co., Rochester, N.Y.) in water was added. The pH was maintained at 11-11-5 by adding as needed 2 N-NaOH while constantly monitoring the hydrogen ion concentration with a Corning Digital l l2pH meter. The reactibn was completed when the pH remained constant without further addition of NaOH. The Sepharose beads were washed on a sintered glass filter with cold distilled water then with cold 0.1 M borate buffer, pH 8.3 (Porath et al., 1967). Separation of interspecies antibody. To prepare immunoadsorbents (Haase and Pereira, 1972) for the separation of interspecies-specificantibody, semi-purified HaLV p30 was used. It was made as follows: sucrose density gradient purified HaLV was pelleted and disrupted by Tween 80-ether and clarified by high speed centrifugation (Oroszlan et al., 1971a). The soluble viral protein mixture was sedimented on a ~20% sucrose gradient in TNE buffer (0.01 M Tris, pH 7.4, 0.1 M NaC1, 0.001 M EDTA) at 40,000 rev/min (IEC Model B-60 centrifuge, SB 283 rotor) for 18 hr. The p30 as detected by CF tests banded in the uppermost part of the gradient together with other nonaggregated lower
mol. wt viral antigcns. Positive fractions were pooled and dialyzed exhaustively against 0-1 M borate buffer, pH 8.3. The CNBr activated Sepharosc was resuspended in 6.5 ml borate buffer (0.1 M, pH 8.3) containing the semipurified HaLV p30 (1;5 mg total protein). The attachment of protein was allowed to proceed for 18 hr at 4r~C in a stoppered glass vial with slow stirring using a magnetic stirrer. After the reaction was completed the beads were allowed to settle and the supernatant was analyzed for total protein and p30 content. Analysis indicated that some protein material remained free and about 90-95 per cent of the p30 was bound to the Sepharose. The solid immunoadsorbent was then washed exhaustively with cold borate buffer (0.1 M, pH 8.3), cold 0-15 M NaC1, 0.1 M acetic acid, 0'15 M NaC1, and finally with borate buffer again. The beads were sucked dry on a sintcred glass filter and were then resuspended in 5 ml borate buffer and packed in a 0.5 cm diameter column (5 ml plastic syringe). With a slow flow rate (0'5 ml/hr), 2 ml of anti-FeLV goat serum (IS-8) was passed through the column and the eluate was monitored with an LKB Uvicord system. The column was washed free of unbound protein (peak 1)with borate until the baseline returned to zero. Elution was effected by 1 M propionic acid and the eluate containing the protein (peak 2) was collected in a single tube into which 0-5 g NaHCO3 was deposited. By the addition of 1 N NaOH, the pH was maintained at or above 7.0 in the collecting tube during elution. Antibodies collected in this fashion (Haase and Pereira, 1972) proved to be more reactive than those left at acidic pH in 1 M propionic acid for longer periods of time. Peaks 1 and 2 were then dialyzed against large volume of Tris-HCl buffer (0'02 M) pH 8"0, containing 0.15 M NaCI. Both fractions were then concentrated to a final volume of 1 ml under negative pressure using S & S collodion membranes (No. 100, Schleicher and Schuell, Inc., Keene, New Hampshire). IgG preparations purified by either DEAE cellulose chromatography or Sephadex G-200 gel filtration were also used instead of whole serum. Subsequent immunodiffusion analysis indicated that peak I contained essentially all the species-specific antibodies, but very little of the interspecies antibody, and as expected peak 2 was free of species-specific and contained only interspecific antibodies. Isolation of p30. To prepare insolubilized antibodySepharose adsorbent for the immunochemical isolation of gs antigen, the antibody purified by affinity chromatography as described above or lgG containing fractions from Sephadex G-200 gel filtration was used. Coupling of antibody to the activated Sepharose beads was carried out in a manner similar to that used for the attachment of antigen. In a typical experiment with 3 ml (see above) Sepharose starting material, 4 ml 7S IgG preparation ( ~ 30 nag proteinj was used. After thorough washing (200ml 0.1 M borate buffer, pH 8.3, 200 ml saline, 100 ml acetic acid, 200 ml distilled water), the antibody-Sepharose immunoadsorbent was reacted in 6ml borate buffer (0.1 M, pH8.3) with Tween-ether disrupted R-MuLV viral protein (3.75 rag) preparation under constant slow stirring overnight at 4"C. The slurry was then poured into a K-09 × 15 Pharmacia column, washed (slow flow rate) with borate buffer until all the unattached protein material was removed (peak l j and the absorbance at 280 returned close to the borate baseline. Washing was continued with 200 ml saline, and finally with 200 ml distilled water. The antibody complexed p30 was dissociated and eluted with 0.1 M acetic acid with a flow rate of 0.2 ml/min (peak 2). The acetic acid eluate was diluted to 1:10 with distilled water, quick frozen, and lyophilized.
Isolation of Mammalian Type C RNA Virus
63 RESULTS
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Fig. 1. Precipitation of internally labeled mouse (R-MuLV) p30 by species-specific guinea-pig antiserum to R-MuLV p30 and interspecies reactive goat antiserum to Tween 80ether disrupted FeLV (IS-8) antisera. The upper figure shows the purification of t~C-lysine labeled p30 by isoelectric focusing. The main radioactive (O------O) peak (pl 6.7) corresponds to the gs protein measured by complement-fixation. The lower figures show the precipitation of the peak fraction and subsequent sedimentation in sucrose gradients using species-specific (gs-1) and interspecies-specific (gs-3) sera. See text.
Immunophysicochemical characterization of cross-reaction between mammalian group-specific antigen (p30) and goat interspecies antibody In order to characterize the cross-reactive antiserum (IS-8) used in the present studies for immunoadsorption in a quantitative manner, the following experiments were carried out. 14C-lysine labeled R-MuLV gs protein was purified by isoelectric focusing (Fig. 1, upper graph). This radioactive protein, homogeneous by isoelectric focusing (pI 6.7), was reacted both with species-specific guinea-pig antiserum as well as the cross-reactive antiserum prepared in goats against FeLV (IS-8). Antigen-antibody reactions were allowed to take place at 37°C,for I hr, then the reaction mixtures were centrifuged through 5-20% sucrose gradients (Fig. 1, lower graph). In the presence of normal guinea-pig or goat antisera the radioactivity remained in the upper part of the gradient, in the presence of either antisera no radioactivity remained in the free antigen zone while all is found in the pellet (P).
Absorption in gel When guinea pig sera which showed only speciesspecific reactions and a heterologous goat serum prepared against FeLV containing cross-reactive antibodies were placed in adjacent wells and allowed to react with the appropriate antigen, single fused precipitin lines were formed. The same reaction pattern was obtained if either unfractionated disrupted virus or highly purified gs protein was used. As shown in the left hand panel of each set (A, B, C, D) in Fig. 2, the
(A)
(B)
(C)
(D)
Fig. 2. Gel diffusion analysis of species-specific and cross-reactive antigenic determinants of several mammalian gs proteins (p30s). In each pattern purified p30s (MuLV, RD 114, RaLV and HaLV) are placed in the upper well. Species-specific antisera (guinea pig antiserum to mouse gs: MgsS; to RD 114 gs: RDgsS; rat gs: RgsS; and to hamster gs: HagsS) are placed in the lower wells and interspecies reactive serum, IS-8, in the right hand well of the left hand patterns of each set (A, B, C, D). Note reactions of identity between the two sera with each protein. On the right hand side of each set, an absorption experiment is shown. Both antiserum wells on each individual pattern are first filled with a heterologous antigen as indicated prior to the addition of antiserum. In each case the heterologous antigen completely absorbed all reactivity of IS-8 serum while not affecting the species-specific reactions,
64
S. OROSZLAN, D. BOVA and R. V. GILDEN
identity reaction was obtained with each of the viruses tested. With this data alone it would seem reasonable to conclude that the homologous and cross-reactive sera were detecting a single molecular structure. To show the qualitative distinction between species and interspecies determinants, absorption-in-gel experiments were performed. These results are shown in the right hand panels of Fig. 2. In each seL the reaction of the goat anti-FeLV serum (interspecies reaction) was completely removed by absorption with a heterologous antigen while the reactions of species-specific sera were not affected by similar concentration of absorbing antigen. In the figure, absorptions with HaLV, MuLV, and RD 114 are shown; in other patterns RaLV was equally effective in absorbing interspecies reactivity. We would emphasize that even under conditions of large antigen excess the precipitin line formed by the species-specific guinea pig sera against homologous antigen were undiminished in intensity and appeared in the same relative position in the gels. From the viewpoint of virus distinction it is noteworthy that the goat anti-FeLV serum showed only the interspecies pattern of reactivity against the RD 114 virus (now known to be an endogenous cat virus), and thus in conjunction with the available specific guinea-pig serum against each p30 allows the immunological distinction of this second family of cat Type C viruses and the conventional cat Type C viruses (upper right panel). In other experiments (not shown) heterologous antigens were not able to inhibit the reaction of the goat anti-FeLV serum with its homologous antigen.
hmmmoadsorption Using CNBr activated Sepharose, two types of immunoadsorbents were prepared as described in Materials and Methods. One utilized the p30 of HaLV to
purify interspecies antibody from an anti-FeLV (IS-8) serum and the second utilized the interspecies antibody to purify MuLV p30. The reaction patterns of goat anti-FeLV before and after passage over the HaLV p30 column are shown in Fig. 3. The left hand panel shows that this serum reacts clearly with two (also faintly with a third) components in disrupted FeLV and produces strong spurs with heterologous antigens placed in wells adjacent to FeLV. When heterologous antigens are adjacent to one another, only a single fused precipitin band is seen. This represents the interspecies reaction, while the strong spurring indicates the excess of species-specific antibodies present in this serum. After absorption and elution from the column, the interspecies reactivity was recovered in good yield, while the other antibody reactivities found in unfractionated serum were no longer detectable (Fig. 3, right). A slight spur was still seen when FeLV and MuLV p30s were compared; however, this was not seen with FeLV and HaLV. It seems likely that the use of HaLV p30 as the adsorbent, selected for a population of antibodies with a greater affinity for this antigen than for MuLV p30. This would indicate some degree of heterogeneity of interspecies determinants among the p30s from various species. When interspecies antibody coupled to Sepharose was used to purify MuLV proteins, further supportive evidence for the presence of species-specific and interspecies determinants associated with the same structure was obtained. This is illustrated in Fig. 4. As shown (upper panel), goat anti-FeLV reacted with the input disrupted virus and the cluted fraction (peak 2) which initially bound to the column giving a single line of identity. This fraction showed the presence of species determinants also since it reacted with a species-specific guinea-pig serum (lower left)and showed a spurring pattern when FeLV was placed in an adjacent well and .
.
.
.
Fig. 3. Immunodiffusion reactions of IgG fraction of IS-8 serum (left hand panel) and peak 2 (right) fraction eluted from HaLV p30-Sepharose immunoadsorbent. Viral antigens in peripheral wells as indicated. See text. Avian myoblastosis virus (AMV) is included as negative control. The yield of interspecies antibody obtained from the immunoabsorbent was estimated at - 50 per cent based on gel diffusionendpoints and correction for dilution factors.
Isolation of Mammalian Type C RNA Virus
65
Fig. 4. Precipitin reactions of interspecies antibody-Sepharose immunoadsorbent purified MuLV p30 (peak 2) with various anti~ra as indicated. See text. The yield of MuLV p30 from the immunoabsorbent was ~ 80 per cent of the input based on activity in complement-fixation tests. both antigens were diffused against a rat serum containing both species-specific and interspecies antibodies. Thus, whcn MuLV interspecies reactivity was purified by a heterologous interspccies reactive serum, MuLV species-specific reactivity was co-purified. To indicate the efficacy of the immunoabsorbent SDSpolyacrylamide gel electrophoresis was made on the eluted fraction. As shown (Fig. 5), a single viral component corresponding to a mol. wt of - 30.000 was specifically bound to and eluted from the column. Additionally, some physically adsorbed gamma-globulin co-eluted from the columns as evidenced by protein bands corresponding to heavy and light chains.
DISCUSSION Previous studies have demonstrated that antisera with species-specific properties and heterologous antisera with interspecific properties were reactive with the same viral protcin, p30 (Gilden et al., 1971: Oroszlan et al., 1971h). While one could argue that differential reactivity was a function of potency rather than reactions with distinct determinants, the data presented here strongly support the latter hypothesis. Thus: ability of heterologous p30s to remove all reactivity of heterologous antisera without effect on species-specific antisera effectively defines two qualitatively distinct
Fig. 5. SDS-polyacrylamide gel electrophorcsis analysis of acetic acid elution peak 2 (A) from the interspecies antibody-Sepharose immunoadsorbent. The main band has an estimated mol. wt of 30,000. The other two bands represcnt heavy and light chains of gamma-globulin which occasionally co-eluted with the viral protein. These bands were identified on the lower gel (Bt in which 0.1 M acetic acid eluate was electrophoresed which was obtained from a Sepharosc column to which only goat gamma-globulin was coupled. This is similar to previous cxperienccs reported by others (Pihko et al., 1973) and the amount of contaminating gamma-globulin could be reduced or almost completely eliminated by more drastic washing of the adsorbent before coupling the antigen. Migration is from right to left.
66
S. OROSZLAN, D. BOVA and R. V. GILDEN
Groves W, E., Davis F. O. C., Jr. and Sells B. H. (1968) Anacategories of reaction. Identity reactions in gel diffulyt. Biochem. 22, 195. sion must then result from occurrence of both deterHaase A. T. and Pcreira H. G. (1972) J. Imm,n. 108, 633. minants on a single molecule. The use of immunoadHartley J. W., Rowe W. P., Capps W. and Huebncr R..I. sorbent techniques further emphasizes this conclusion. (1969) J. Virol. 3, 126. Interspecies reactive antibodies, purified by this proHuebner R. J. (1967) Prec. natn. Acad. Sci. U.S.A. 58, 835. cedure and attached to the adsorbent, detected a single Huebner R. J., Armstrong D., Okuyan M., Sarma P. S. and molecular species identified as p30 by acrylamide gel Turner H. C. (1964) Prec. hath. Acad. Sci. U.S.A. 51,742. electrophoresis. This purified protein demonstrated Huebner R. J., Kelloff G. J., Sarma P. S., Lane W., Turner both interspecies and species-specific determinants. H. C., Gilden R. V., Oroszlan S, Mcicr H., Mycrs D. D. Immunoadsorption techniques similar to those deand Peters R. L. (1970) Prec. hath. 4cad. Sci. ~ .S..,I. 67, 366. scribed here have been successfully used in combinaHuebner R. and Todaro G. (1969) Prec. mat,..toad. Sci. tion with chemically modified p30s to isolate different U.S.A. 64, 1087. antibodies belonging to species-specific class (see KelloffG., Huebner R. J., Lee Y. K., Toni R. and Gilden R. accompanying paper). Experiments are in progress to V. (1970) Prec. natn. Acad. Sci. U,S.A. 65, 310. determine whether similar procedures could be used to Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. purify type-specific antibody populations from antiJ. (1951) J. biol. Chem, 193. 265. serums made against the various murine Type C virus McAllister R. M., Nicolson M., Gardner M. B., Rongcy R. p30s. In addition to the immunological data (Strand W., Rasheed S., Sarma P. S., Huebner R. J., Hatanaka M., and August, 1974; Gilden et al., 1974), we have recently Oroszlan S., Gilden R. V,, Kabigtin A. and Vernon L. obtained biochemical evidence for the existence of . (1972) Nature N e w Biol. 235, 3. type-specific differences in the primary structure of Meier H., Taylor B. A., Cherry M. and Huebner R. J. (1973) Prec. natn. Acad. Sci. U.S.A. 70, 1450. these viral proteins (Oroszlan et al., 1974). Okabe H., Gilden R. J. and Hatanaka M. (1973) Prec. ~latn. Acad. Sci. U.S.A. 70, 3923. Acknowledgement,s~This work was supported by Contract Oroszlan S., Bova D., Huebner R. J. and Gilden R. V. NOI-CP-3-3247 from the Virus Cancer Program of the (1972a) J. I/irol. 10, 746. National Cancer Institute, National Institutes of Health, Oroszlan S., Bova D., Toni R. and Gilden R. V. (1972ht Bethesda, Maryland 20014. The authors wish to thank Mrs. Science 176, 420. Carol Foreman and Mr. Larry Masters for excellent techni- Oroszlan S., Bova D.. White M. H. M., Toni R., Foreman cal assistance. C. and Gilden R. V. (1972c) Prec. natn. Acad. Sci. U.S.A. 69, 121 I. Oroszlan S., Fisher C, F., Stanley T. B. and Gilden R. V. (1970) J. gen. Firol. 8, 1. REFERENCES August T.. Bolognesi D. P., Fleissncr E., Gilden R. V. and Oroszlan S., Foreman C., Kelloff G. and Gilden R. V. ( 197 la) Virology 43, 665. Nowinski R. (1974) Virology, 60, 595. Davis J., Charman H.. Oroszlan S. and Gilden R. V. (1974) Oroszlan S., Huebner R. J. and Gilden R. V. (1971h) Prec. hath. Acad. Sci. U.S.A. 68, 901. lntervirol. (in press). Oroszlan S., Summers M., Foreman C. and Gilden R. V. Davis J., Gilden R. V. and Oroszlan S. (1973) Virology 56, (1974) J. Virol. (in press). 411. Geering G., Aoki T. and Old L. J. (1970) Nature, Lend. 226, Parks W. P. and Scolnick E. M. (1972) Prec. natn. Acad. Sci. U.S.A. 69, 1766. 265. Pihko H., Lindgren J. and Ruoslahti E. (1973) hmmmocheGeering G.. Old L. J. and Boyse E. A. (1966) J. exp. Med. mistry 10, 381. 124, 753. Gilden R. V. and Oroszlan S. (1972) Prec. hath. Acad. Sci. Porath J., Axen R. and Ernback S. (1967) Nature, Lend. 215, 1491. U.S.A. 69, 1021. Gilden R. V., Oroszlan S. and Hatanaka M. (1974) Virus, seh/ifer W., Pister L., Hunamann G. and Moinnig V. (1973) Nature New Biol. 245, 75. E~,olution and Cancer (Edited Maramorosch K. and Kurstak E.) from Second Int. Congress of Comparative Viro- Strand M. and August J. T. (1974) J. Virol. 13, 171. logy, 1973, Montreal: Academic Press, New York. (in Theilen G. H., Kawakami T. G., Rush J. D. and Mann K. J. (1969) Nature, Lend. 222, 589. press). Ting R. C. (1968) J. |irol. 2, 865. Gilden R. V., Oroszlafl S. and Hucbncr R. J. (1971} Nature Weber K. and Osborn M. (1969) J. hiol. Chem. 244, 4406. New Biol. 231, 107. Gildcn R. V., Toni R., Hanson M., Bova D., Charman H. Wright B. S., O'Brien D. A., Shibley G. P,, Mayyasi S. A. and Las Fargues J. C. (1967) Cancer Res. 27, 1672. P. and Oroszlan S. (1974) J. hnmun. 112, 1250.
ISOLATION OF MAMMALIAN TYPE C RNA VIRUS CROSS-REACTIVE ANTIGEN AND ANTIBODY BY IMMUNO-AFFINITY CHROMATOGRAPHY S T E P H E N OROSZLAN, DAVID BOVA and R A Y M O N D V. G I L D E N Flow Laboratories, Inc., Rockville, Maryland 20852, U.S.A. (Received 20 M a y 1974)
Abstract--The ca. 30,000 mol. wt internal protein of mammalian Type C viruses, p30, has previously been shown to react with homologous and heterologous p30 antisera. Certain sera, especially those prepared in guinea-pigs, are highly species-specifiC.Absorption in gel experiments strongly indicates that p30s have qualitatively different classes of determinants designated species-specific or interspecific, and that differential serum reactions are not related to potency vs a single class of determinants. The use of immunoadsorbents comprised of hamster Type C virus p30 permitted the purification of interspecies antibody from a polyspecific goat anti-feline leukemia virus antiserum. This antiserum, in turn coupled to an adsorbent, purified from mouse leukemia virus a component of 30,000 mol. wt which possessed both interspecies and species-specific determinants. This clearly indicates the coexistence of both classes of determinants on a single structure.
INTRODUCTION The ~ 30,000 mol. wt internal protein of Type C viruses referred to synonymously as the group-specific (gs antigen; Huebner et al., 1964; Geering et al., 1966; Huebner, 1967) or p30 (August et al., 1974), has proven an important marker for a variety of studies relating to inter-viral relationships (Gilden and Oroszlan, 1972), natural history (Huebner et al., 1970; Hartley et al., 1969), and the relationship of these viruses to cancer (Huebner and Todaro, 1969; Meier et al., 1973). Of signal importance was the finding (Gilden et al., 1971 ; Oroszlan et al., 1971b) that this protein carried both species-specific and interspecific determinants (Geering et al., 1970), thus providing one method for establishing the relationship of new isolates to those already described (Oroszlan et al., 1972c). More recent studies have indicated that the p30s of closely related viruses, i.e. those sharing species-specific determinants, can 'be distinguished by radioimmunoassay (Strand and August, 1974)and quantitative C' fixation (Gilden et al., in press); thus p 30s carry type-specific determinants as well. In addition, the interspecific category of crossreactive determinants has been shown to consist of subgroups as initially found by radioimmunoassay inhibition methods (Parks and Scolnick, 1972), and subsequently shown in gel diffusion (Schafer et al., 1973; Gilden et al., 1974). In this communication we describe an immunochemical method for preparation of interspecies reactive antibody from a polyspecific serum. Using this antibody population, a heterologous p30 was purified. The purified p30 displayed both interspecies and speciesspecific determinants in keeping with previous findings. This general approach has been useful in detect-
ing additional determinants on p30 molecules (see accompanying paper), and should be useful for isolation of type-specific antibody populations as well. MATERIALS AND METHODS Virus
The Rauseher strain of mouse leukemia virus (R-MuLV) was grown in monolayer cultures of chronically infected mouse (BALB/c) bone marrow, JLS-V9 cells (Wright et al., 1967). Rat Type C virus (RaLV) was obtained from the chronically infected MSB-1 cell line (Ting, 1968) derived fro.m a tumor induced by M-MSV in a female rat of the Brown Norway strain. This virus is an interspecies pseudotype with structural proteins common to endogenous rat Type C viruses (Oroszlan et al., 1972a) and nucleic acid sequences of both mouse and rat origin (Okabe et al., 1973). Feline leukemia virus (FeLV, Theilen strain) was obtained from a chronically infected cat lymphocytic cell suspension culture (Theilen et al., 1969). RD-114, an endogenous cat virus was the isolate from the RD rhabdomyosarcoma cell line of McAllister et aL (1972). Hamster Type C virus (HaLV) was the isolate of Kelloff et al. (1970). Virus purification
Viruses were purified by sucrose density gradient centrifugation and radioactive R-MuLV, labeled with ~4C-lysine (> 260 mCi/m-mole, New England Nuclear) was prepared as described previously (Oroszlan et al., 1971h). Puvification of p30s
The isoelectric focusing technique was used as reported previously (Oroszlan et al., 1970). 61
62
S. OROSZLAN, D. BOVA and R. V. GILDEN
l nmmnodiffusion Double immunodiffusion was done in plates that contained 0'8~ agarose, pH 7-4, ionic strength 0.15. In preparing plates, 8 gm agarose, 9.3 gm Tris [-2-amino-2-(hydroxymethyl)l,3-propanediol], 74 ml 1 N HCI, and 7-0gin NaCI were made up to 1 1. with distilled water. Merthiolate was added as a preservative. Plates were kept at room temperature, observed for 72 hr, and photographed when optimum precipitation lines developed.
Complement-fixation Viral group-specific antigens (p30) were titrated in microcomplement-fixation (CF) tests as previously described (Oroszlan et al., 1970).
Sodium dodec yl sulfate-pol yacr ylamide 9el electrophoresis. ( S D ~ P A G E ) was carried out in 10~ polyacrylamide gels (Weber and Osborn, 1969).
Protein determination Protein content of antigen and antibody preparations was determined by the method of Lowry et al. (19511 using crystalline bovine serum albumin as standard.
Antiserum Guinea-pig antisera were prepared against the various gs proteins purified by isoelectric focusing. These sera appear highly species-specific in both gel diffusion and CF assays, although by highly sensitive radioimmunoassay procedures these sera show variable levels of cross-reactive antibody; generally less than 1/20 of the species-specific titer. Goat antisera to FeLV (obtained from Dr. Roger Wilsnack, Huntingdon Labs) and MuLV were prepared against Tween-ether disrupted purified virus. These sera contain sufficient interspecies antibody to produce gel diffusion reactions, but also show an excess of species-specific antibody. MSV-I serum was obtained by immunizing rats with tumor homogenates and 7S antibodies from goat serum were separated on Sephadex G-200 as previously described (Oroszlan et al., 1972b).
lmmunoadsorbent procedures Coupling CNBr to Sepharose. Three ml of Sepharose 4B (Pharmacia) was thoroughly washed with distilled Water. It was then resuspended in distilled water to give a slurry of 5 ml final volume. It was chilled to 0°C (icebath) and in a fume hood, equal volume of cold CNBr (50 mg/ml; Eastman Kodak Co., Rochester, N.Y.) in water was added. The pH was maintained at 11-11-5 by adding as needed 2 N-NaOH while constantly monitoring the hydrogen ion concentration with a Corning Digital l l2pH meter. The reactibn was completed when the pH remained constant without further addition of NaOH. The Sepharose beads were washed on a sintered glass filter with cold distilled water then with cold 0.1 M borate buffer, pH 8.3 (Porath et al., 1967). Separation of interspecies antibody. To prepare immunoadsorbents (Haase and Pereira, 1972) for the separation of interspecies-specificantibody, semi-purified HaLV p30 was used. It was made as follows: sucrose density gradient purified HaLV was pelleted and disrupted by Tween 80-ether and clarified by high speed centrifugation (Oroszlan et al., 1971a). The soluble viral protein mixture was sedimented on a ~20% sucrose gradient in TNE buffer (0.01 M Tris, pH 7.4, 0.1 M NaC1, 0.001 M EDTA) at 40,000 rev/min (IEC Model B-60 centrifuge, SB 283 rotor) for 18 hr. The p30 as detected by CF tests banded in the uppermost part of the gradient together with other nonaggregated lower
mol. wt viral antigcns. Positive fractions were pooled and dialyzed exhaustively against 0-1 M borate buffer, pH 8.3. The CNBr activated Sepharosc was resuspended in 6.5 ml borate buffer (0.1 M, pH 8.3) containing the semipurified HaLV p30 (1;5 mg total protein). The attachment of protein was allowed to proceed for 18 hr at 4r~C in a stoppered glass vial with slow stirring using a magnetic stirrer. After the reaction was completed the beads were allowed to settle and the supernatant was analyzed for total protein and p30 content. Analysis indicated that some protein material remained free and about 90-95 per cent of the p30 was bound to the Sepharose. The solid immunoadsorbent was then washed exhaustively with cold borate buffer (0.1 M, pH 8.3), cold 0-15 M NaC1, 0.1 M acetic acid, 0'15 M NaC1, and finally with borate buffer again. The beads were sucked dry on a sintcred glass filter and were then resuspended in 5 ml borate buffer and packed in a 0.5 cm diameter column (5 ml plastic syringe). With a slow flow rate (0'5 ml/hr), 2 ml of anti-FeLV goat serum (IS-8) was passed through the column and the eluate was monitored with an LKB Uvicord system. The column was washed free of unbound protein (peak 1)with borate until the baseline returned to zero. Elution was effected by 1 M propionic acid and the eluate containing the protein (peak 2) was collected in a single tube into which 0-5 g NaHCO3 was deposited. By the addition of 1 N NaOH, the pH was maintained at or above 7.0 in the collecting tube during elution. Antibodies collected in this fashion (Haase and Pereira, 1972) proved to be more reactive than those left at acidic pH in 1 M propionic acid for longer periods of time. Peaks 1 and 2 were then dialyzed against large volume of Tris-HCl buffer (0'02 M) pH 8"0, containing 0.15 M NaCI. Both fractions were then concentrated to a final volume of 1 ml under negative pressure using S & S collodion membranes (No. 100, Schleicher and Schuell, Inc., Keene, New Hampshire). IgG preparations purified by either DEAE cellulose chromatography or Sephadex G-200 gel filtration were also used instead of whole serum. Subsequent immunodiffusion analysis indicated that peak I contained essentially all the species-specific antibodies, but very little of the interspecies antibody, and as expected peak 2 was free of species-specific and contained only interspecific antibodies. Isolation of p30. To prepare insolubilized antibodySepharose adsorbent for the immunochemical isolation of gs antigen, the antibody purified by affinity chromatography as described above or lgG containing fractions from Sephadex G-200 gel filtration was used. Coupling of antibody to the activated Sepharose beads was carried out in a manner similar to that used for the attachment of antigen. In a typical experiment with 3 ml (see above) Sepharose starting material, 4 ml 7S IgG preparation ( ~ 30 nag proteinj was used. After thorough washing (200ml 0.1 M borate buffer, pH 8.3, 200 ml saline, 100 ml acetic acid, 200 ml distilled water), the antibody-Sepharose immunoadsorbent was reacted in 6ml borate buffer (0.1 M, pH8.3) with Tween-ether disrupted R-MuLV viral protein (3.75 rag) preparation under constant slow stirring overnight at 4"C. The slurry was then poured into a K-09 × 15 Pharmacia column, washed (slow flow rate) with borate buffer until all the unattached protein material was removed (peak l j and the absorbance at 280 returned close to the borate baseline. Washing was continued with 200 ml saline, and finally with 200 ml distilled water. The antibody complexed p30 was dissociated and eluted with 0.1 M acetic acid with a flow rate of 0.2 ml/min (peak 2). The acetic acid eluate was diluted to 1:10 with distilled water, quick frozen, and lyophilized.
Isolation of Mammalian Type C RNA Virus
63 RESULTS
161 pH -12 12
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Fraction No. Guinea pig sera Goo'r sera
p 2468
p 246
8
p 2468
P 2468
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Fig. 1. Precipitation of internally labeled mouse (R-MuLV) p30 by species-specific guinea-pig antiserum to R-MuLV p30 and interspecies reactive goat antiserum to Tween 80ether disrupted FeLV (IS-8) antisera. The upper figure shows the purification of t~C-lysine labeled p30 by isoelectric focusing. The main radioactive (O------O) peak (pl 6.7) corresponds to the gs protein measured by complement-fixation. The lower figures show the precipitation of the peak fraction and subsequent sedimentation in sucrose gradients using species-specific (gs-1) and interspecies-specific (gs-3) sera. See text.
Immunophysicochemical characterization of cross-reaction between mammalian group-specific antigen (p30) and goat interspecies antibody In order to characterize the cross-reactive antiserum (IS-8) used in the present studies for immunoadsorption in a quantitative manner, the following experiments were carried out. 14C-lysine labeled R-MuLV gs protein was purified by isoelectric focusing (Fig. 1, upper graph). This radioactive protein, homogeneous by isoelectric focusing (pI 6.7), was reacted both with species-specific guinea-pig antiserum as well as the cross-reactive antiserum prepared in goats against FeLV (IS-8). Antigen-antibody reactions were allowed to take place at 37°C,for I hr, then the reaction mixtures were centrifuged through 5-20% sucrose gradients (Fig. 1, lower graph). In the presence of normal guinea-pig or goat antisera the radioactivity remained in the upper part of the gradient, in the presence of either antisera no radioactivity remained in the free antigen zone while all is found in the pellet (P).
Absorption in gel When guinea pig sera which showed only speciesspecific reactions and a heterologous goat serum prepared against FeLV containing cross-reactive antibodies were placed in adjacent wells and allowed to react with the appropriate antigen, single fused precipitin lines were formed. The same reaction pattern was obtained if either unfractionated disrupted virus or highly purified gs protein was used. As shown in the left hand panel of each set (A, B, C, D) in Fig. 2, the
(A)
(B)
(C)
(D)
Fig. 2. Gel diffusion analysis of species-specific and cross-reactive antigenic determinants of several mammalian gs proteins (p30s). In each pattern purified p30s (MuLV, RD 114, RaLV and HaLV) are placed in the upper well. Species-specific antisera (guinea pig antiserum to mouse gs: MgsS; to RD 114 gs: RDgsS; rat gs: RgsS; and to hamster gs: HagsS) are placed in the lower wells and interspecies reactive serum, IS-8, in the right hand well of the left hand patterns of each set (A, B, C, D). Note reactions of identity between the two sera with each protein. On the right hand side of each set, an absorption experiment is shown. Both antiserum wells on each individual pattern are first filled with a heterologous antigen as indicated prior to the addition of antiserum. In each case the heterologous antigen completely absorbed all reactivity of IS-8 serum while not affecting the species-specific reactions,
64
S. OROSZLAN, D. BOVA and R. V. GILDEN
identity reaction was obtained with each of the viruses tested. With this data alone it would seem reasonable to conclude that the homologous and cross-reactive sera were detecting a single molecular structure. To show the qualitative distinction between species and interspecies determinants, absorption-in-gel experiments were performed. These results are shown in the right hand panels of Fig. 2. In each seL the reaction of the goat anti-FeLV serum (interspecies reaction) was completely removed by absorption with a heterologous antigen while the reactions of species-specific sera were not affected by similar concentration of absorbing antigen. In the figure, absorptions with HaLV, MuLV, and RD 114 are shown; in other patterns RaLV was equally effective in absorbing interspecies reactivity. We would emphasize that even under conditions of large antigen excess the precipitin line formed by the species-specific guinea pig sera against homologous antigen were undiminished in intensity and appeared in the same relative position in the gels. From the viewpoint of virus distinction it is noteworthy that the goat anti-FeLV serum showed only the interspecies pattern of reactivity against the RD 114 virus (now known to be an endogenous cat virus), and thus in conjunction with the available specific guinea-pig serum against each p30 allows the immunological distinction of this second family of cat Type C viruses and the conventional cat Type C viruses (upper right panel). In other experiments (not shown) heterologous antigens were not able to inhibit the reaction of the goat anti-FeLV serum with its homologous antigen.
hmmmoadsorption Using CNBr activated Sepharose, two types of immunoadsorbents were prepared as described in Materials and Methods. One utilized the p30 of HaLV to
purify interspecies antibody from an anti-FeLV (IS-8) serum and the second utilized the interspecies antibody to purify MuLV p30. The reaction patterns of goat anti-FeLV before and after passage over the HaLV p30 column are shown in Fig. 3. The left hand panel shows that this serum reacts clearly with two (also faintly with a third) components in disrupted FeLV and produces strong spurs with heterologous antigens placed in wells adjacent to FeLV. When heterologous antigens are adjacent to one another, only a single fused precipitin band is seen. This represents the interspecies reaction, while the strong spurring indicates the excess of species-specific antibodies present in this serum. After absorption and elution from the column, the interspecies reactivity was recovered in good yield, while the other antibody reactivities found in unfractionated serum were no longer detectable (Fig. 3, right). A slight spur was still seen when FeLV and MuLV p30s were compared; however, this was not seen with FeLV and HaLV. It seems likely that the use of HaLV p30 as the adsorbent, selected for a population of antibodies with a greater affinity for this antigen than for MuLV p30. This would indicate some degree of heterogeneity of interspecies determinants among the p30s from various species. When interspecies antibody coupled to Sepharose was used to purify MuLV proteins, further supportive evidence for the presence of species-specific and interspecies determinants associated with the same structure was obtained. This is illustrated in Fig. 4. As shown (upper panel), goat anti-FeLV reacted with the input disrupted virus and the cluted fraction (peak 2) which initially bound to the column giving a single line of identity. This fraction showed the presence of species determinants also since it reacted with a species-specific guinea-pig serum (lower left)and showed a spurring pattern when FeLV was placed in an adjacent well and .
.
.
.
Fig. 3. Immunodiffusion reactions of IgG fraction of IS-8 serum (left hand panel) and peak 2 (right) fraction eluted from HaLV p30-Sepharose immunoadsorbent. Viral antigens in peripheral wells as indicated. See text. Avian myoblastosis virus (AMV) is included as negative control. The yield of interspecies antibody obtained from the immunoabsorbent was estimated at - 50 per cent based on gel diffusionendpoints and correction for dilution factors.
Isolation of Mammalian Type C RNA Virus
65
Fig. 4. Precipitin reactions of interspecies antibody-Sepharose immunoadsorbent purified MuLV p30 (peak 2) with various anti~ra as indicated. See text. The yield of MuLV p30 from the immunoabsorbent was ~ 80 per cent of the input based on activity in complement-fixation tests. both antigens were diffused against a rat serum containing both species-specific and interspecies antibodies. Thus, whcn MuLV interspecies reactivity was purified by a heterologous interspccies reactive serum, MuLV species-specific reactivity was co-purified. To indicate the efficacy of the immunoabsorbent SDSpolyacrylamide gel electrophoresis was made on the eluted fraction. As shown (Fig. 5), a single viral component corresponding to a mol. wt of - 30.000 was specifically bound to and eluted from the column. Additionally, some physically adsorbed gamma-globulin co-eluted from the columns as evidenced by protein bands corresponding to heavy and light chains.
DISCUSSION Previous studies have demonstrated that antisera with species-specific properties and heterologous antisera with interspecific properties were reactive with the same viral protcin, p30 (Gilden et al., 1971: Oroszlan et al., 1971h). While one could argue that differential reactivity was a function of potency rather than reactions with distinct determinants, the data presented here strongly support the latter hypothesis. Thus: ability of heterologous p30s to remove all reactivity of heterologous antisera without effect on species-specific antisera effectively defines two qualitatively distinct
Fig. 5. SDS-polyacrylamide gel electrophorcsis analysis of acetic acid elution peak 2 (A) from the interspecies antibody-Sepharose immunoadsorbent. The main band has an estimated mol. wt of 30,000. The other two bands represcnt heavy and light chains of gamma-globulin which occasionally co-eluted with the viral protein. These bands were identified on the lower gel (Bt in which 0.1 M acetic acid eluate was electrophoresed which was obtained from a Sepharosc column to which only goat gamma-globulin was coupled. This is similar to previous cxperienccs reported by others (Pihko et al., 1973) and the amount of contaminating gamma-globulin could be reduced or almost completely eliminated by more drastic washing of the adsorbent before coupling the antigen. Migration is from right to left.
66
S. OROSZLAN, D. BOVA and R. V. GILDEN
Groves W, E., Davis F. O. C., Jr. and Sells B. H. (1968) Anacategories of reaction. Identity reactions in gel diffulyt. Biochem. 22, 195. sion must then result from occurrence of both deterHaase A. T. and Pcreira H. G. (1972) J. Imm,n. 108, 633. minants on a single molecule. The use of immunoadHartley J. W., Rowe W. P., Capps W. and Huebncr R..I. sorbent techniques further emphasizes this conclusion. (1969) J. Virol. 3, 126. Interspecies reactive antibodies, purified by this proHuebner R. J. (1967) Prec. natn. Acad. Sci. U.S.A. 58, 835. cedure and attached to the adsorbent, detected a single Huebner R. J., Armstrong D., Okuyan M., Sarma P. S. and molecular species identified as p30 by acrylamide gel Turner H. C. (1964) Prec. hath. Acad. Sci. U.S.A. 51,742. electrophoresis. This purified protein demonstrated Huebner R. J., Kelloff G. J., Sarma P. S., Lane W., Turner both interspecies and species-specific determinants. H. C., Gilden R. V., Oroszlan S, Mcicr H., Mycrs D. D. Immunoadsorption techniques similar to those deand Peters R. L. (1970) Prec. hath. 4cad. Sci. ~ .S..,I. 67, 366. scribed here have been successfully used in combinaHuebner R. and Todaro G. (1969) Prec. mat,..toad. Sci. tion with chemically modified p30s to isolate different U.S.A. 64, 1087. antibodies belonging to species-specific class (see KelloffG., Huebner R. J., Lee Y. K., Toni R. and Gilden R. accompanying paper). Experiments are in progress to V. (1970) Prec. natn. Acad. Sci. U,S.A. 65, 310. determine whether similar procedures could be used to Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. purify type-specific antibody populations from antiJ. (1951) J. biol. Chem, 193. 265. serums made against the various murine Type C virus McAllister R. M., Nicolson M., Gardner M. B., Rongcy R. p30s. In addition to the immunological data (Strand W., Rasheed S., Sarma P. S., Huebner R. J., Hatanaka M., and August, 1974; Gilden et al., 1974), we have recently Oroszlan S., Gilden R. V,, Kabigtin A. and Vernon L. obtained biochemical evidence for the existence of . (1972) Nature N e w Biol. 235, 3. type-specific differences in the primary structure of Meier H., Taylor B. A., Cherry M. and Huebner R. J. (1973) Prec. natn. Acad. Sci. U.S.A. 70, 1450. these viral proteins (Oroszlan et al., 1974). Okabe H., Gilden R. J. and Hatanaka M. (1973) Prec. ~latn. Acad. Sci. U.S.A. 70, 3923. Acknowledgement,s~This work was supported by Contract Oroszlan S., Bova D., Huebner R. J. and Gilden R. V. NOI-CP-3-3247 from the Virus Cancer Program of the (1972a) J. I/irol. 10, 746. National Cancer Institute, National Institutes of Health, Oroszlan S., Bova D., Toni R. and Gilden R. V. (1972ht Bethesda, Maryland 20014. The authors wish to thank Mrs. Science 176, 420. Carol Foreman and Mr. Larry Masters for excellent techni- Oroszlan S., Bova D.. White M. H. M., Toni R., Foreman cal assistance. C. and Gilden R. V. (1972c) Prec. natn. Acad. Sci. U.S.A. 69, 121 I. Oroszlan S., Fisher C, F., Stanley T. B. and Gilden R. V. (1970) J. gen. Firol. 8, 1. REFERENCES August T.. Bolognesi D. P., Fleissncr E., Gilden R. V. and Oroszlan S., Foreman C., Kelloff G. and Gilden R. V. ( 197 la) Virology 43, 665. Nowinski R. (1974) Virology, 60, 595. Davis J., Charman H.. Oroszlan S. and Gilden R. V. (1974) Oroszlan S., Huebner R. J. and Gilden R. V. (1971h) Prec. hath. Acad. Sci. U.S.A. 68, 901. lntervirol. (in press). Oroszlan S., Summers M., Foreman C. and Gilden R. V. Davis J., Gilden R. V. and Oroszlan S. (1973) Virology 56, (1974) J. Virol. (in press). 411. Geering G., Aoki T. and Old L. J. (1970) Nature, Lend. 226, Parks W. P. and Scolnick E. M. (1972) Prec. natn. Acad. Sci. U.S.A. 69, 1766. 265. Pihko H., Lindgren J. and Ruoslahti E. (1973) hmmmocheGeering G.. Old L. J. and Boyse E. A. (1966) J. exp. Med. mistry 10, 381. 124, 753. Gilden R. V. and Oroszlan S. (1972) Prec. hath. Acad. Sci. Porath J., Axen R. and Ernback S. (1967) Nature, Lend. 215, 1491. U.S.A. 69, 1021. Gilden R. V., Oroszlan S. and Hatanaka M. (1974) Virus, seh/ifer W., Pister L., Hunamann G. and Moinnig V. (1973) Nature New Biol. 245, 75. E~,olution and Cancer (Edited Maramorosch K. and Kurstak E.) from Second Int. Congress of Comparative Viro- Strand M. and August J. T. (1974) J. Virol. 13, 171. logy, 1973, Montreal: Academic Press, New York. (in Theilen G. H., Kawakami T. G., Rush J. D. and Mann K. J. (1969) Nature, Lend. 222, 589. press). Ting R. C. (1968) J. |irol. 2, 865. Gilden R. V., Oroszlafl S. and Hucbncr R. J. (1971} Nature Weber K. and Osborn M. (1969) J. hiol. Chem. 244, 4406. New Biol. 231, 107. Gildcn R. V., Toni R., Hanson M., Bova D., Charman H. Wright B. S., O'Brien D. A., Shibley G. P,, Mayyasi S. A. and Las Fargues J. C. (1967) Cancer Res. 27, 1672. P. and Oroszlan S. (1974) J. hnmun. 112, 1250.
