Proc. Nati. Acad. Sci. USA Vol. 75, No. 2, pp. 923-927, February 1978 Genetics Genetic recombination between mouse type C RNA viruses: A mechanism for endogenous viral gene amplification in mammalian cells (type-specific radioimmunoassay/molecular hybridization/genetic mapping) MARIANO BARBACID, KEITH C. ROBBINS, SHIGEO HINO, AND STUART A. AARONSON Laboratory of RNA Tumor Viruses, National Cancer Institute, Bethesda, Maryland 20014 Communicated by Henry S. Kaplan, October 28, 1977

ABSTRACT A strategy based on the identification of kemic BALB/c mice infected with Rauscher MuLV at birth type-specific antigenic determinants in the transitional products or from leukemic C58 animals. After propagation of these vi- of gag(pl5, p12, and p3O proteins), pol (reverse transcriptase), and env(gp7O glycoproteins) genes of mammalian type C viruses ruses in canine fetal thymus cells, they were cloned at limiting has been used to study genetic recombination between these dilution by the microtiter procedure. Clonal virus isolates, tested RNA viruses. By this approach, recombinants involving exog- and shown to retain their xenotropic host range, were grown enous and endogenous mouse type C viruses have been identi- in canine fetal thymus cells to mass culture and concentrated, fied and genetically mapped. Analogous techniques have been from 250 ml of tissue culture fluids by centrifugation, for im- upplied to investigate the genetic relationships between dif- munologic analysis. ferent classes of endogenous virus that exist within the same mouse cells. Proteins of the inducible class of xenotropic virus Radioimmunoassays. The p30 and p15 structural proteins were shown to exhibit extensive antigenic homology with the of BALB:virus-1, BALB:virus-2, and NIH-MuLV were purified gag but not the env gene products of the ecotropic virus class. to homogeneity and radiolabeled as described (10, 11). Double Instead, the env gene-coded glycoproteins of the inducible and antibody competition radioimmunoassays were performed as noninducible xenotropic virus classes possessed striking anti- described (8) and included those for the above proteins as well genic relatedness. These results, as well as supporting findings as for the p12 (7) and gp7O (8) structural proteins of from molecular hybridization, favor the concept that the in- endogenous ducible xenotropic virus of mouse cells arose by a recombina- type C viruses and for the p30 (12), p15 (13), p12 (14), reverse tional mechanism involving the progenitors of the other two transcriptase (15), and gp7O (16) proteins of Rauscher- endogenous virus classes. MuLV. DNA-RNA Hybridization. BALB:virus-1 and BALB:virus-2 A large number of mammalian species have been shown to [3H]cDNAs were prepared, purified, and hybridized as de- contain information coding for type C RNA viruses (for review, scribed by Cabradilla et al. (17). At equal DNA/RNA molar see ref. 1). Among available systems for the study of endogenous ratios, each probe protected around 70% of its homologous 70S type C viruses, the mouse model has been one of the best [32PJRNA from pancreatic RNase (10 Ag/ml) digestion. The characterized. Investigations by several laboratories have re- extent of cDNA hybridization was assayed with SI nuclease (18, vealed the existence of at least three partially related endoge- 19). nous mouse type C viruses (2-6), each of which is independently regulated by the same host cell (1). These viruses are also dis- RESULTS tinguishable by their reapctivities in radioimmunoassays for two structural proteins, p12 and gp7O (7, 8). In the present report, Immunologic Analysis of Recombinants between Exog- typing immunoassays for gag, pol, and env gene products of enous and Endogenous Mouse Type C Viruses. Previous prototype endogenous viruses have been developed and applied studies have shown that both in vtvo and in vitro passage of to the analysis of potential type C virus recombinants. The mouse ecotropic viruses can generate viruses with xenotropic usefulness of these approaches in genetic mapping is demon- host range (20). Moreover, there is evidence that such viruses strated. may arise by a mechanism involving recombination between the infecting virus and endogenous viral information (21). METHODS Therefore, we propagated clonal Rauscher-MuLV in newborn Cells and Viruses. Embryo cell cultures were established BALB/c mice. To screen for recombinants between the eco- from C58, DBA, C3H/He, A/He, and C57BL/6 mice (7). In- tropic Rauscher-MuLV and either of the xenotropic endogenous duction of ecotropic and xenotropic endogenous viruses from viruses of BALB/c mouse cells, animal-passaged Rauscher- these cells were performed as described (4). Prototype endog- MuLV was first selected for xenotropic host range. Individual enous type C viruses representing three distinguishable classes virus clones, isolated by the microtiter procedure, were purified included inducible ecotropic BALB:virus-1 (4), inducible xe- by sucrose gradient centrifugation and subjected to immuno- notropic BALB:virus-2 (4), and noninducible xenotropic NIH- logic analysis. As shown in Fig. 1, a representative virus clone, murine leukemia virus (MuLV) (5). NZB- (6) and Rauscher- D17-3B3, was assayed in homologous radioimmunoassays for (9)- MuLV were also utilized. All viruses were cloned prior to the type-specific gag gene-coded proteins, p15, p12, and p30, use. of the potential parental viruses, Rauscher-MuLV, and the Recombinant Virus Selection. Potential recombinant viruses endogenous xenotropic viruses BALB:virus-2 and NIH-MuLV. with xenotropic host range were isolated from plasma of leu- The last virus was used as prototype for the noninducible en- dogenous virus, whose antigens have been shown to be ex- The costs of publication of this article were defrayed in part by the pressed by BALB/c cells and found to be indistinguishable from payment of page charges. This article must therefore be hereby marked those of NIH-MuLV (22). The gag gene products of D17-3B3 "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: MuLV, murine leukemia virus. 923 Downloaded by guest on October 1, 2021 924 Genetics: Barbacid et al. Proc. Natl. Acad. Sci. USA 75 (1978)

gag pol env mGppp-t i poly A

I REVERSE I (NlSIpl2lp30 I TRANSCRIPTASE ilL

ii

t00 v. D z 80fa 8K) do Rauscher-MuLV D ) Rauscher-MuLV Immunoassays 0 0 40 *0 100 Immunoassays kwco .2345 60 '0I

BALB : virus-2 ) 0 BALB : virus-2 Immunoassays 4C) 0 Immunoassays <_8 Xlo w 1 3 NIH-MuLV NIH-MuLV LU Immunoassays ) - v Immunoassys v , . I 1 23 4 COMPETING VIRUS (loge ng) FIG. 1. Immunologic analysis of D17-3B3 virus, a recombinant between exogenous and endogenous mouse type C viruses. Detergent-disrupted D17-3B3 virus (0), Rauscher-MuLV (0), BALB:virus-2 (a), and NIH-MuLV (v) were tested as competing antigens in type-specific radioim- munoassays for Rauscher-MuLV pl5, p12, p30, reverse transcriptase, and gp70 (A-E, respectively) as well as for the p15, p12, p30, and gp7O proteins of BALB:virus-2 (F-I) and NIH-MuLV (J-M), as described (8). The gene order depicted above for D17-3B3 viral translational products, exhibiting antigenic determinants specific for parental Rauscher-MuLV (Eo) or BALB:virus-2 (tn), conforms to that described for known murine (29) and avian (31-33) type C viruses.

virus were identical to those of Rauscher-MuLV as determined nucleotide sequence homology of no more than 85% between by their complete competition in the homologus Rauscher- the two viral genomes (data not shown). The above data indi- MuLV p15, p12, and p3O assays (Fig. 1 A-C). Moreover, cate that D17-3B3 arose as a result of recombination between D17-3B3 and Rauscher-MuLV viruses showed similar, and only Rauscher-MuLV and BALB:virus-2, most likely somewhere partial, crossreactivity in the homologous immunoassays for the within the env gene region of the viral RNA. p15, p12, and p30 proteins of BALB:virus-2 (Fig. F-H) and Demonstration of Recombination between Endogenous NIH-MuLV (Fig. 1 J-L). Mouse Type C Viruses. An analogous experimental approach Further identity between Rauscher-MuLV and D17-3B3 was utilized to determine whether genetic recombination could virus was found when each virus was analyzed for the type- be demonstrated between viruses integrated within the same specific antigenic determinants of its pol gene product. Both cellular genome. For this purpose, the C58 mouse strain, which viruses competed to a full extent in the homologous radioim- contains information for several endogenous viruses, was uti- munoassay for Rauscher-MuLV reverse transcriptase (Fig. 1D). lized. C58 mouse cells are permissive for replication of the In contrast, saturating amounts of BALB:virus-2 and NIH- chemically inducible ecotropic virus C58-MuLV which has MuLV only displaced 50% of the input l25I-labeled viral en- been implicated in leukemias of this strain (23). C58 cells also zyme. These results provide evidence for the involvement of contain a chemically inducible xenotropic virus (7, 24). These both gag and pol genes of Rauscher-MuLV in the generation viruses were shown to be identical to prototype BALB:virus-1 of D17-3B3 virus. and BALB:virus-2, respectively, by both biological and im- The immunologic reactivity of the major glycoprotein, gp70, munological criteria (7). Morever, C58 cells also express antigens of D17-3B3 virus was next analyzed. Although this viral protein of a third endogenous virus, immunologically indistinguishable was not identical to the env gene-coded proteins of Rauscher- from those of NIH-MuLV (unpublished data). To assay for MuLV, BALB:virus-2 gp70, or NIH-MuLV, its reactivity possible recombinants involving endogenous viruses of the C58 closely resembled that of BALB:virus-2 gp7O. Moreover, strain, extracts of leukemic spleen tissue were inoculated onto D17-3B3 viral gp70 also exhibited a set of antigenic determi- canine fetal thymus cells to select for viruses possessing xeno- nants related to those of Rauscher-MuLV gp70 but not found tropic host range. Viruses grown in this manner were then in either BALB:virus-2 or NIH-MuLV gp7O (Fig. 1E). In con- cloned by infection of canine fetal thymus cells at limiting trol experiments, the partial crossreactivity of D17-3B3 virus dilution in microtiter. in immunoassays for BALB:virus-I structural proteins was like Five separate clones demonstrating xenotropic host range that observed with the parental viruses (data not shown). These were assayed in competition radioimmunoassays for the results exclude participation of the ecotropic endogenous structural proteins of prototype BALB:virus-1, BALB:virus-2, BALB:virus-1 in the formation of D17-3B3 virus. To establish and NIH-MuLV. One clonal virus designated V702, like the further that the immunological differences between the ecotropic endogenous virus of C58 cells, competed fully in D17-3B3 virus and Rauscher-MuLV were not the result of a BALB:virus-1 p15, p12, and p30 immunoassays (Fig. 2 A-C), mutation, reciprocal molecular hybridization studies were demonstrating the identical nature of the gag gene products performed with cDNA probes and RNAs prepared from of these two viruses. As further evidence for this conclusion, Rauscher-MuLV and D17-3B3 virus. The results indicated V702 virus, like C58 MuLV, only partially competed in ho- Downloaded by guest on October 1, 2021 Genetics: Barbacid et al. Proc. Natl. Acad. Sci. USA 75 (1978) 925

gag pol env mGppp I -H--- poly A jP15p12I P30 P10l

-r~~~~~~'\ c 100 D 80 a z 60 : 0 40 .00 m a 20 0 z LLJ - = K - 100 H L 80 , n z c 60 3-0 < a 40- 91 PO 20- mtu K 100 Yv30L J~

GN 60 . 40- v + f V -L Iw 'vvvv IV 20r 0 1 2 3 0 1 2 3 0 1 2 3 V1234 COMPETING VIRUS (Ilog10 ng) FIG. 2. Immunologic analysis of V702 virus, a recombinant between endogenous mouse type C viruses. Detergent-disrupted V702 virus (-) and prototype endogenous viruses including ecotropic C58-MuLV or BALB:virus-1 (a), inducible xenotropic viruses from C58 or BALB/c mice (a), and NIH-MuLV (v) were tested as competing antigens in type-specific radioimmunoassays for the p15, p12, p30, and gp7O structural proteins, respectively, of BALB:virus-1 (A-D), BALB:virus-2 (E-H), and NIH-MuLV (I-L). The gene order of the V702 virus translational products, exhibiting antigenic determinants specific for the parental ecotropic (D) or inducible xenotropic (Ca) endogenous viruses of the C58 mouse, is as shown in Fig. 1.

mologous assays for the gag gene-coded proteins of BALB: whereas NIH-MuLV gp7O possessed most of the antigenic de- virus-2 (Fig. 2 E-G) and NIH-MuLV (Fig. 2 I-K). In immu- terminants of BALB:virus-2 gp70 (Fig. 2 D, H, and L) (8). Thus, noassays for the env gene-coded gp70s of the prototype en- the genetic homologies among the three prototype endogenous dogenous viruses, V702 virus showed only limited homology type C viruses, as detected by immunologic techniques, ap- with BALB:virus-1 or NIH-MuLV gp7O (Fig. 2 D and L). In- peared to be localized to specific genes. stead, the reactivity of the V702 virus gp7O was identical to that In attempt to obtain additional evidence for this possibility, of the inducible xenotropic virus of C58 cells, each showing the relatedness of the genomes of the three endogenous viruses complete homology with BALB:virus-2 gp7O (Fig. 2H). These were also studied by molecular hybridization techniques. results demonstrate that V702 virus is a recombinant containing BALB:virus-1 and BALB:virus-2 cDNAs were hybridized ex- the gag gene of C58-MuLV and the env gene of the inducible tensively by their homologus viral RNAs but only partially xenotropic virus of C58 cells. The other virus clones showed (40-60%) by the heterologous viral RNAs (Table 1). If BALB: immunologic reactivities indistinguishable from those of virus-2 arose by a mechanism involving recombination of the V702. two other endogenous viruses, it was reasoned that hybridization Origin of the Chemically Inducible Xenotropic Endoge- of BALB:virus-2 cDNA by the combination of BALB:virus-1 nous Mouse Type C Virus by a Mechanism Involving Ge- and NIH-MuLV RNAs should be greater than that by either netic Recombination. Immunologic analysis of prototype alone. The addition of NIH-MuLV RNA to BALB:virus-1 RNA endogenous viruses not only provided the necessary controls increased the final extent of hybridization to 84% of that ob- for characterization of in vivo generated recombinants but also served with the homologus RNA. These findings further support helped to define genetic relationships between these endoge- the concept that BALB:virus-2 was generated by recombina- nous viruses. Fig. 2 revealed that the gag genes of BALB:virus-l tion. and BALB:virus-2 were most closely related. In immunoassays Previous studies have indicated that many inbred strains for the highly type-specific p12 protein, the homology between contain endogenous viruses analogous in their biologic prop- BALB:virus-1 and BALB:virus-2 was as high as 85% whereas erties to the chemically inducible ecotropic and xenotrotropic NIH-MuLV only exhibited 30% crossreactivity (Fig. 2 B, F, and BALB/c viruses (7). It has been shown that these mouse strains J). Similar although less pronounced differences were observed also contain a noninducible endogenous virus whose immu- in radioimmunoassays for p15 and p30 proteins. In marked nologic properties so far has been indistinguishable from those contrast were results obtained by analysis of the env gene of NIH-MuLV (7). Comparisons were made of the immuno- products of these viruses. Here, the gp70s of BALB:virus-1 and logic reactivities of a number of such virus isolates in homolo- balb;virus-2 were found to be almost completely unrelated, gous radioimmunoassays for the gag gene- and env gene-coded Downloaded by guest on October 1, 2021 926 Genetics: Barbacid et al. Proc. Natl. Acad. Sci. USA 75 (1978)

Table 1. Molecular hybridization analysis of genetic relationships Table 2. Immunologic relationships of endogenous mouse type C between endogenous type C viruses of BALB/c cells viruses with the prototype inducible xenotropic virus class, BALB:virus-2 BALB:virus-1 BALB:virus-2 [bklt3HlcDNA [3H]cDNA Maximum competition* in BALB:virus-2 immunoassays, % Hybridiza- Hybridiza- env gene- 70S Viral RNA tion Additivity tion Additivity gag gene-coded coded BALB:virus-1 100 60 Virus source p15 p12 p30 gp7O BALB:virus-2 56 100 Inducible N-tropic NIH-MuLV 39 62 BALB/c (BALB:virus-1) 85-90 75-80 90-95 35-40 BALB:virus-1 C58 (C58-MuLV) 85-90 75-80 90-95 35-40 + NIH-MuLV 100 84 22 DBA 85-90 75-80 90-95 35-40 BALB:virus-2 C3H/He 85-90 75-80 90-95 35-40 + NIH-MuLV 55 0 100 - A/He 85-90 75-80 90-95 35-40 Saturating amounts of 70S viral RNA (as determined in separate C57BL/6 85-90 75-80 9095 35-40 experiments) were hybridized to either BALB:virus-1 or BALB: Inducible xenotropic virus-2 [3H]cDNA; 83% of the BALB:virus-1 [3H]cDNA and 81% of BALB/c (BALB:virus-2) 100 100 100 100 the BALB:virus-2 [3H]cDNA were found to anneal with their re- C58 100 100 100 100 spective homologous viral RNAs. These values were defined as 100% DBA 100 100 100 100 hybridization for each [3H]cDNA probe. C3H/He 100 100 100 100 A/He 100 100 100 100 structural proteins of BALB:virus-2. In each case the proteins C47BL/6 100 100 100 100 of inducible xenotropic viruses of different strains demonstrated Noninducible xenotropic complete homology with those of BALB:virus-2 (Table 2). Al- NIH Swiss (NIH-MuLV) 60-65 40-45 70-75 90-95 though inducible ecotropic viruses could be readily distin- NZB (NZB-MuLV) 60-65 40-45 70-75 jo9J guished in each of these assays, they consistently exhibited a Competing viruses were tested at 2-fold serial dilutions in homol- much higher degree of homology with BALB:virus-2 in their ogous radioimmunoassays for the structural proteins ofBALB:virus-2. gag gene products than in their env gene products. In contrast, In each case, 2 .g of protein from Triton X-100-disrupted BALB: representative isolates of the noninducible class of xenotropic virus-2 completely displaced the 2-5I-labeled probe (10,000 cpm) for virus, although showing only a limited degree of relatedness binding limiting amounts of goat anti-BALB:virus-2 serum. to BALB:virus-2 in their gag gene products, possessed env * Maximum competition values indicate the degree of displacement gene-coded gp7Os that were immunologically identical to achieved by saturating amounts of protein (5-20 jg) from Triton BALB:virus-2 gp7O. These findings further indicate that the X-100-disrupted heterologous viruses and represent the mean of immunologic relatedness between different endogenous mouse at least three separate experiments. type C viruses can be localized to specific genes and strengthen The gag and po! gene translational products of one of the the concept that the class of inducible xenotropic virus arose viruses, D17-3B3, as well as part of its gp7O molecule, were by a mechanism involving genetic recombination. shown to be specific for Rauscher-MuLV. The other parental virus, BALB:virus-2, provided the remaining env gene-coded DISCUSSION antigenic determinants. These findings suggest that D17-3B3 Genetic approaches have yielded important information con- virus was generated by a single recombination event, assuming cerning the organization of avian and murine type-C viral ge- that the murine viral gene order is 5'-gag-pol-env-3' as has been nomes. Recombinant viruses, essential to such studies, have been described for avian type C viruses (31-33). The V702 virus, a isolated by various selection systems utilizing parental viruses recombinant containing the gag gene region of C58-MuLV, with conditional lethal or nonconditional defects (for review, was shown to contain antigenic determinants of a xenotropic see ref. 25). Analysis of recombinant viruses has, for the most viral gp7O. Moreover, the high degree of specificity of the part, involved the oligonucleotide fingerprint technique (26-28) typing immunoassays made it possible to establish that this gp7O or the use of radioimmunoassays for detection of different was derived from the inducible, as opposed to noninducible, virus-coded proteins in high molecular weight precursors and xenotropic virus of C58 cells (8). their cleavage products (29). Previous studies have shown that ecotropic viruses selected The present report describes an approach to type C virus for xenotropic host range in human cells retained their parental genetic analysis that takes advantage of the fact that proteins virus p12, implying their origin as recombinants between represented within different regions of the viral genome possess ecotropic virus and xenotropic viruses (21). More recently, vi- highly type-specific antigenic determinants (30). This technique ruses with amphotropic host range have been isolated from makes it possible to identify most type C viral proteins as be- tissue culture-passaged ecotropic viruses (34) or from thymic longing to a specific parent, because the range of type-specific tissues of preleukemic or leukemic AKR mice (35). The latter antigenic determinants associated with a particular viral protein have been implicated in the pathogenesis of naturally occurring is unlikely to be altered sufficiently by a mutation or deletion leukemias of this strain. Interference and neutralization studies to cause that protein to become immunologically identical to have suggested that the amphotropic nature of these viruses a type-specific protein of some other virus. The validity of this may result from a recombinational event involving ecotropic approach was demonstrated in the genetic analysis of two and xenotropic viral glycoproteins (34, 35). The present findings naturally occurring recombinant viruses. One was shown to indicate that recombination within the env region of ecotropic involve genetic sequences of Rauscher-MuLV and one of the and xenotropic viruses does not necessarily lead to a broadened two known xenotropic endogenous viruses of BALB/c mouse virus host range. Whether recombination in this region of the cells. The second was identified as containing genetic infor- D17-3B3 viral genome directly results in a more pathogenic mation of an ecotropic and xenotropic endogenous virus of the leukemia virus remains to be determined. C58 mouse strain. Endogenous type C viruses are known to exist within the Downloaded by guest on October 1, 2021 Genetics: Barbacid et al. Proc. Natl. Acad. Sci. USA 75 (1978) 927

genomes of a large number of vertebrate species (1). The an- 13. Strand, M., Wilsnack, R. & August, J. T. (1974) J. Virol. 14, tigenic relatedness of several of the respective proteins of all 1575-1583. known mammalian type C viruses argues strongly that they 14. Tronick, S. R., Stephenson, J. R. & Aaronson, S. A. (1973) Virology 54, 199-206. originate from a common progenitor (36). Within any given 15. Krakower, J. M., Barbacid, M. & Aaronson, S. A. (1977) J. Virol. species, multiple copies of the viral genome may exist (37-40). 22,331-39. If, as the evidence favors, these viruses initially became ge- 16. Strand, M. & August, J. T. (1974) J. Virol. 13, 171-180. netically associated with the cell as long ago as millions of years 17. Cabradilla, C. D., Robbins, K. C. & Aaronson, S. A. (1976) Proc. (41, 42), mechanisms can be considered for the presence of Natl. Acad. Sci. USA 73,4541-4545. multiple, partially related viral copies within the same cell. It 18. Leong, J., Garapin, A. C., Jackson, N., Fanshier, L., Levinson, is known that productive virus infection in tissue culture can W. E. & Bishop, J. M. (1972) J. Virol. 9,891-902. lead to integration of more than one viral copy (43). Whether 19. Benveniste, R. E. & Scolnick, E. M. (1970) Virology 51, 370- duplication of endogenous viral genes has taken place during 382. A. Nature the course of evolution is not known. Current theories on mo- 20. Aaronson, S. (1971) 230,445-447. 21. Stephenson, J. R., Anderson, G. R., Tronick, S. R. & Aaronson, lecular evolution postulate that amino acid substitutions in a S. A. (1974) Cell 2,87-94. given protein accumulate as a linear function of the generation 22. Stephenson, J. R., Tronick, S. R., Reynolds, R. K. & Aaronson, S. time of a given species (generation-time theory) (44, 45) or as A. (1974) J. Exp. Med. 139,427-438. a linear function of absolute biologic time (evolutionary-clock 23. Stephenson, J. R., Greenberger, J. S. & Aaronson, S. A. (1974) J. theory) (46, 47). Either hypothesis would argue for relatively Virol. 13, 237-240. constant antigenic drift of proteins of diverging endogenous 24. Callahan, R., Lieber, M. M. & Todaro, G. J. (1975) J. Virol. 15, viruses within the same cellular genome. This prediction is 1378-1384. borne out by the equal divergence of analogous proteins of the 25. Vogt, P. K. (1977) in Comprehensive Virology, eds. Fraenkel- ecotropic and noninducible xenotropic endogenous mouse type Conrat, H. & Wagner, R. R. (Plenum Press, New York), Vol. 9, viruses. is to account pp. 341-355. C Evolutionary divergence unlikely for 26. Lai, M. M. C., Duesberg, P. H., Horst, J. & Vogt, P. K. (1973) Proc. the present demonstration that gene products of the inducible Natl. Acad. Sci. USA 70,2266-2270. class of mouse xenotropic virus demonstrate striking antigenic 27. Wang, L. H., Duesberg, P. H., Beemon, K. & Vogt, P. K. (1975) homology to proteins coded for by different regions of two other J. Virol. 16, 1051-1070. endogenous mouse type C viral genomes. Instead, the present 28. Coffin, J. M. & Billeter, M. A. (1976) J. Mol. Biol. 100, 293- immunological evidence, as well as supporting findings from 318. molecular hybridization, favor the hypothesis that the inducible 29. Barbacid, M., Stephenson, J. R. & Aaronson, S. A. (1976) Nature xenotropic virus arose by recombination between progenitors 262,554-559. of inducible ecotropic and noninducible xenotropic endogenous 30. Stephenson, J. R., Barbacid, M., Tronick, S. R., Hino, S. & Aar- A. in Cancer Research: Cell Molecular viruses. If so, genetic recombination provides a mechanism by onson, S. (1977) Biology, Biology and Tumor Virology, ed. Gallo, R. (CRC Press, Cleve- which endogenous viral genes can be amplified in mammalian land, OH), Vol. II, pp. 37-50. cells. 31. Joho, R. H., Billeter, M. A. & Weissmann, C. (1975) Proc. Natl. Acad. Sci. USA 72,4772-4776. We thank J. M. Krakower for helpful discussions and for providing 32. Wang, L. H., Duesberg, P. H., Kawai, S. & Hanafusa, H. (1976) the reverse transcriptase radioimmunoassay data and C. Y. Dunn, C. Proc. Natl. Acad. Sci. USA 73,447-451. A. Deren, and C. Winters for excellent technical assistance. This work 33. Wang, L. H., Galehouse, D., Mellon, P., Duesberg, P., Mason, was supported in part by Contract NOl 6-1024 from the Virus Cancer W. & Vogt, P. K. (1976) Proc. Natl. Aced. Sci. USA 73,3952- Program of the National Cancer Institute. M.B. is a Visiting Fellow of 3956. the National Institutes of Health, on leave of absence from the Instituto 34. Fischinger, P., Nomura, S. & Bolognesi, D. (1975) Proc. Natl. de Bioquimica de Macromoleculas, Madrid, Spain. Acad. Sci. USA 72,5150-5155. 35. Hartley, J. W., Wolford, N. K., Old, L. J. & Rowe, W. P. (1977) Proc. Natl. Acad. Sci. USA 74, 789-792. 1. Aaronson, S. A. & Stephenson, J. R. (1976) Biochim. Biophys. 36. Barbacid, M., Stephenson, J. R. & Aaronson, S. A. (1977) Cell 10, Acta 458,323-354. 641-648. 2. Lowy, D. R., Rowe, W. P., Teich, N. & Hartley, J. W. (1971) 37. Gelb, L. D., Milstein, J. B., Martin, M. A. & Aaronson, S. A. (1973) Science 174, 155-156. Nature New Biol. 244, 76-79. 3. Aaronson, S. A., Todaro, G. J. & Scolnick, E. M. (1971) Science 38. Chattapadhaya, S. K., Lowy, D. R., Teich, N. M., Levine, A. S. 174, 157-159. & Rowe, W. P. (1974) Proc. Natl. Acad. Sci. USA 71, 167- 4. Aaronson, S. A. & Stephenson, J. R. (1973) Proc. Natl. Acad. Sci. 171. USA 70, 2055-2058. 39. Benveniste, R. E. & Todaro, G. J. (1974) Nature 252,170-173. 5. Todaro, G. J., Arnstein, P., Parks, W. P., Lennette, E. H. & 40. Tronick, S. R., Golub, M. M., Stephenson, J. R. & Aaronson, S. A. Huebner, R. J. (1973) Proc. Natl. Acad. Sci. USA 70,859-862. (1977) J. Virol., 23, 1-9. 6. Levy, J. A. (1973) Science 182, 1151-1153. 41. Benveniste, R. E. & Todaro, G. J. (1976) Nature 261, 101-108. 7. Stephenson, J. R., Reynolds, R. K., Tronick, S. R. & Aaronson, S. 42. Benveniste, R. E., Callahan, R., Sherr, C. J., Chapman, V. & A. (1975) Virology 67,404-414. Todaro, G. J. (1977) J. Virol. 21, 849-862. 8. Hino, S., Stephenson, J. R. & Aaronson, S. A. (1976) J. Virol. 18, 43. Khoury, A. T. & Hanafusa, H. (1976) J. Virol. 18,383-400. 933-941. 44. Laird, C. D., McDonaughy, B. L. & McCarthy, B. J. (1969) Na- 9. Rauscher, F. J. (1962) J. Natl. Cancer Inst., 29, 515-543. ture 224, 149-154. 10. Barbacid, M., Stephenson, J. R. & Aaronson, S. A. (1976) J. Biol. 45. Kohne, D. E. (1970) Q. Rev. Biophys. 33, 1-48. Chem. 251, 4859-4866. 46. Zuckerkandl, E. & Pauling, L. (1962) in Horizons in Biochem- 11. Greenwood, F. C., Hunter, W. M. & Glover, J. S. (1963) Biochem. istry, eds. Kasha, M. & Pullman, B. (Academic Press, New York), J. 89, 114-123. pp. 189-225. 12. Parks, W. P. & Scolnick, E. M. (1972) Proc. Natl. Acad. Sd. USA 47. Wilson, A. C., Carlson, S. S. & White. T. J. (1977) Annu. Rev. 69, 1766-1770. Biochem. 46,573-639. Downloaded by guest on October 1, 2021