Primate Origin of the Cell-Derived Sequences of Simian Sarcoma Virus KEITH C

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JOURNAL OF VIROLOGY, Feb. 1982, p. 721-725 Vol. 41, No. 2 0022-538X/82/020721-05$02.0O/0 Primate Origin of the Cell-Derived Sequences of Simian Sarcoma Virus KEITH C. ROBBINS,* ROBIN L. HILL, AND STUART A. AARONSON Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20205 Received 4 May 1981/Accepted 14 September 1981

We sought to identify the species of origin of the cell-derived (sis) sequences of simian sarcoma virus. A molecular clone comprised of sis DNA detected related nucleotide sequences at low copy numbers in normal cellular DNAs of species as diverse as humans and quail. The extent of hybridization and degree of base-pair matching with sis DNA were greatest with New World primate DNAs. The thermal denaturation curve midpoints of hybrids formed between sis and woolly monkey DNAs were indistinguishable from homologous sis DNA hybrids, establishing the woolly monkey (Lagothrix spp.) as the source ofsis sequences. In comparative studies, sis was shown to be more conserved among mammalian species than unique-sequence woolly monkey cellular DNA. There was no detectable homology between sis and the cell-derived sequences of other fibro- blast-transforming retroviruses. These findings indicate that sis is likely to be a unique onc gene among transforming retroviruses.

Retroviruses that transform cells in tissue Thus, the possibility exists that the SSV(SSAV) culture and induce solid tumors in vivo have complex was horizontally transmitted from been isolated from several vertebrate species. some other source to the woolly monkey in The only known sarcoma virus of primate origin which SSV was initially detected. In the present was initially obtained from a naturally occurring report, we have sought to identify the species of tumor of a woolly monkey (33). The application origin of SSV by examination of the sequence of recombinant DNA techniques to the investi- homology between the SSV cell-derived se- gation of RNA tumor viruses has recently made quences (designated sis) and DNAs of different it possible to clone in biologically active form species. In addition, we have examined the the integrated genome of this virus, designated relationship between sis and the cell-derived simian sarcoma virus (SSV) (26). The full-length sequences of other molecularly cloned trans- linear 5.1-kilobase-pair (kbp) SSV genome has forming retroviruses. been found to contain a 1.0-kbp segment of Previous studies have localized sis at 4.0 to helper virus-unrelated information localized to- 5.0 kbp on the physical map of X-SSV-11 Cl 1, a ward the 3' end with respect to SSV RNA. molecular clone containing the intact SSV Moreover, this genetic information has been genome (26). This region of SSV DNA was shown to be related to DNA sequences present subcloned in pBR322, and the sis insert was nick at low copy numbers within mammalian cellular translated for use as a probe. High-molecular- DNAs (26). Thus, SSV, similarly to other trans- weight cellular DNAs were prepared and treated forming retroviruses characterized to date, has with EcoRI, an endonuclease which did not cut arisen by recombination of a type C helper virus within the sis gene (26). After fractionation by with a host cellular gene. agarose gel electrophoresis, cellular DNA frag- The species from which SSV obtained its ments were blotted onto nitrocellulose filters cellular sequences has not as yet been identified. and hybridized with the sis DNA probe. The sis- The recombinational event that led to its genera- related DNA fragments were readily detected tion may have occurred in the woolly monkey within cellular DNAs of species as diverse as from which SSV was initially isolated. Howev- humans and quail (Fig. 1). Mouse cellular DNA er, simian sarcoma-associated virus (SSAV), the contained a sis-related fragment of around 18 type C helper virus isolated with SSV, is closely kbp, whereas a 16-kbp fragment was detected in related immunologically to several exogenous rat cellular DNA. One fragment of 10 kbp and type C viruses of gibbon apes (20, 28, 35) and two fragments of 8 and 4 kbp were detected in also possesses an unexpectedly high degree of mink and cat cellular DNAs, respectively. Of antigenic relatedness with endogenous viruses note, woolly monkey, gibbon ape, and human of certain Asian rodents (23). SSV is also known cellular DNAs each demonstrated a single sis- to contain some sequences of its helper virus (1). related fragment of around 21 kbp. As each of 721 722 NOTES J. VIROL.

1 2 3 4 5 6 7 8 for hybrids formed between the sis probe and these rodent cellular DNAs were around 12°C kbp lower than the Tm of hybrids formed between the sis probe and X-SSV-11 Cl 1 DNA. Mink and cat cellular DNAs hybridized around 40o of the 2 0 0- No _ w m probe and demonstrated ATm values of 9.2 and 8.7°C, respectively. Primate cellular DNAs ex- 963- hibited greater homology with the sis probe (Table 1). Human, gibbon ape, and rhesus monkey DNAs hybridized the sis DNA probe to a 6.5 II maximum extent of 50% with ATm values ranging from 5.5 to 6.0°C. The highest extents of annealing were exhibited by DNAs of New World primates with values ranging from 56 to 65%. Thus, sis was most closely related to sequences present in New World primates. The 2.2 ATm values of the hybrids formed helped to FIG. 1. Detection of sis-related sequences in unequivocally identify the source of sis se- DNAs of different vertebrate species. High-molecular- quences. These values were 3.5 and 1.5°C for weight cellular DNA, prepared by the method de- squirrel and spider monkey DNAs, respectively. scribed by Canaani and Aaronson (10), was treated Findings that the Tm of woolly monkey cellular with EcoRI, fractionated by agarose gel electrophore- DNA-sis DNA hybrids was identical to that of sis, and subjected to Southern blotting analysis by homologous sis DNA hybrids (Table 1) demon- using conditions previously reported (26). The sis strated that sis arose from within the woolly DNA fragment located from 4.0 to 5.0 kbp on the monkey genome. restriction map of X-SSV-11 Cl 1 DNA (26), was nick To compare evolutionary conservation of sis translated for use as a probe. EcoRI-treated DNAs extracted from QT-6 (24), NIH/3T3 (19), NRK (12), DNA relative to unique-sequence woolly mon- MvlLu (ATCC 64), and FEF (27) cells and woolly key cellular DNA, we examined the ability of monkey fibroblasts (26), gibbon ape spleen, and hu- various mammalian cellular DNAs to hybridize man embryo lung fibroblasts were electrophoresed in a probe comprised of unique woolly monkey lanes 1 to 8, respectively. Reference markers of cellular DNA sequences. The highest extents of HindIII-digested k phage DNA were electrophoresed annealing were exhibited by woolly and spider in parallel. monkey cellular DNAs with values of 100 and 97%, respectively (Table 1). The ATm values the fragments detected in normal cellular DNA indicated the well-matched nature of the hybrids was larger than the 1.0-kbp SSV sis gene, each formed. These results are consistent with the must contain cellular flanking sequences, inter- classification of both species in the same sub- vening sequences, or both. family Atelinae. Squirrel monkey cellular DNA To identify the species from which sis origi- annealed 82% of the probe and demonstrated a nated, we examined the extent of annealing and ATm value of 3.3°C. Cellular DNAs ofOld World thermal denaturation (Tm) of hybrids formed primates hybridized the probe to a lesser extent between different cellular DNAs and labeled sis (around 50%) with ATm values ranging from 6.2 DNA. To prepare a single-stranded probe neces- to 7.1°C. There was no significant homology sary for these experiments, 0.25 jig of sis DNA between nonprimate cellular DNAs and woolly was 32P labeled by nick translation (25). The monkey unique-sequence DNA. Since these probe was then heat denatured and hybridized same nonprimate cellular DNAs hybridized as with 300 Fg of SSV(SSAV) RNA. This amount much as 40% of the sis DNA probe, sis appears of RNA was determined to contain a 10-fold to be more conserved among mammalian spe- excess of plus-strand sis sequences. DNA-RNA cies than unique-sequence cellular DNA. hybrids, which contained minus-strand sis Recent findings have indicated that onc genes DNA, were separated from double-stranded sis present in sarcoma viruses independently isolat- DNA hybrids by CsCl gradient centrifugation ed from the same (11, 13, 15, 16, 32) and even (17). RNA was removed from the DNA-RNA from different (2, 4, 5, 30) vertebrate species can hybrid molecules by alkali treatment, and the be genetically related. Thus, the number of remaining single-stranded sis DNA was ethanol cellular genes which have been transduced by precipitated and suspended in water before use. type C viruses in the formation of sarcoma Table 1 shows the extents of annealing of viruses appears to be limited. The availability of different normal cellular DNAs with the single- molecular clones of several avian and mammali- stranded sis DNA probe. Rat and mouse DNAs an sarcoma viruses made it possible to test hybridized to a maximum extent of 23%. The Tm whether their cell-derived genes shared nucleo- VOL. 41, 1982 NOTES 723 TABLE 1. Comparison of the relatedness of sis DNA with DNAs of different mammalian species' Hybridization with:

sis DNA Woolly monkeyDNAunique-sequence DNA source Maximum Maximum hybridization ATm (CC) hybridization ATm (°C) (%) (%) New World primate Woolly monkey (Lagothrix spp.) 65 0 100 0 Spider monkey (Ateles spp.) 63 1.5 97 1.1 Squirrel monkey (Saimiri spp.) 56 3.5 82 3.8 Old World primate Gibbon ape (Hylobates lar) 47 5.8 47 6.5 Rhesus (Macaca mulatta) 48 6.0 48 6.2 Human (Homo sapiens) 50 5.5 47 7.1 Carnivore Cat (Felis catus) 40 8.7 5 ND Mink (Mustela vison) 39 9.2 5 ND Rodent Mouse (Mus musculus) 23 11.7 3 ND Rat (Rattus rattus) 23 12.2 3 ND a The sis probe was prepared from a DNA fragment located 4.0 to 5.0 kbp on the restriction map of X-SSV-11 Cl 1 DNA (26). This DNA fragment was 32P labeled by the nick translation method, and selected for single- stranded DNA complementary to SSV RNA as described in the text. Unique-sequence cellular DNA was prepared from woolly monkey cells grown for 24 h in medium containing 10 ,uCi of [3H]thymidine per ml. Labeled cellular DNA was extracted and sheared as previously described (9). Reiterated DNA sequences annealing at a Cot of 100 mol - s * liter-' were removed by fractionation on hydroxyapatite as previously described (9). After heat dissociation, 0.5-ml hybridization reaction mixtures containing 2 mg of total cellular DNA, 12,000 cpm of radioactively labeled DNA probe, 0.01 M Tris-hydrochloride (pH 7.4), 0.6 M NaCl, and 1 mM EDTA were incubated at 62°C. At various times, 0.025-ml samples were quick-frozen and stored at -70°. Hybridization was determined by the S1 nuclease method (6, 22). Results are expressed as the final extent of hybridization at a Cot of 10' normalized to the extent of hybridization of sis or unique-sequence DNA probes by X-SSV-11 Cl 1 and woolly monkey total cellular DNA, respectively. The thermal stability of hybrids formed was determined by heating samples of the reaction mixture at 5°C increments from 65 to 95C for 3 min, followed by digestion with Si nuclease. The Tm's were 89 and 90°C for the homologous sis probe hybrid and self-associating unique-sequence DNA, respectively. The ATm is the difference in Tm between the other DNA-DNA hybrids and the Tm of the homologous hybrid. ND, Not determined. tide sequence homology with SSV sis. Molecu- probes of these viral onc genes also failed to lar clones of murine, feline, and avian sarcoma hybridize with unlabeled sis DNA. Thus, SSV viruses were excised from their respective vec- sis appears to be a distinct onc gene. tors and purified by preparative agarose gel Molecular cloning of the integrated SSV electrophoresis. Approximately 500 ng of each genome has made it possible to characterize its insert and 5 ng of the SSV insert were electro- molecular structure. We have identified within phoresed in agarose and visualized by ethidium the viral genome a 1.0-kbp DNA segment bromide staining (Fig. 2A). DNA was blotted which is unrelated to its helper virus but is onto nitrocellulose by the Southern technique related to cellular DNA (26). In the present and hybridized with sis probe by relaxed hybrid- report, we have investigated the species of ori- ization conditions. The sis probe readily detect- gin of the SSV sis gene. Although homology ed the SSV insert, but none of the onc genes with sis DNA was demonstrated with cellular present in Moloney (34), BALB (3), and Harvey DNAs of a wide variety of species, none an- (14) murine sarcoma viruses, Abelson murine nealed to an extent or with the high degree of leukemia virus (31), Snyder-Theilen feline sarco- base-pair matching comparable to that observed ma virus (K. C. Robbins, M. Barbacid, and with woolly monkey DNA. Thus, our results S. A. Aaronson, unpublished data), Rous sarco- establish SSV as the first transforming retro- ma virus (29), or avian myelocytomatosis virus virus, whose cell-derived sequences are of pri- (21) (Fig. 2B). In reciprocal studies not shown, mate origin. 724 NOTES J. VIROL. A B

f.; 4 f,8 9 k bp

10) so

.3--

0) 3 --

FIG. 2. Lack of homology between the cell-derived nucleotide sequences of SSV and those of other transforming retroviruses. Approximately 500 ng of the inserts of molecular clones of Moloney murine sarcoma virus (34), BALB murine sarcoma virus pP-7 (3), Harvey murine sarcoma virus BS-9 (14), Abelson leukemia virus X-AM-1 (31), integrated Snyder-Theilen feline sarcoma virus (Robbins, Barbacid, and Aaronson, unpublished data), Rous sarcoma virus X-SR-RSV-D-4 (29), and avian myelocytomatosis virus, a BamHI subclone of X-MC29-1 (21), were electrophoresed in lanes 3 to 9, respectively, and visualized by ethidium bromide straining (A). Reference markers of HindIll-digested A phage and HaeIII-digested (X-174RF DNAs (lane 1) and 5 ng of X-SSV-11 Cl 1 insert (lane 2) were electrophoresed in parallel. DNAs were blotted from the gel onto nitrocellulose filters as previously described (26), hybridized with sis probe by using relaxed hybridization conditions (18), and exposed to X-ray film (B).

The detailed mechanisms involved in the gen- more conserved among mammalian species than eration of transforming retroviruses are not yet unique-sequence DNA. By contrast, type C known. However, the evidence indicates that helper virus genes appear to exhibit much great- they have arisen rarely as a result of recombina- er divergence than unique-sequence DNA in tional events that have occurred in the labora- species in which these viruses have presumably tory after continuous passage of leukemia virus- evolved as part of the host cell genome (7, 8). es in vivo or in animal populations infected at It has previously been shown that certain high frequency with type C viruses. SSV is retroviruses independently isolated from the known to contain some sequences of its helper same species contain related cell-derived onc virus SSAV (1). Although viruses closely related genes (11, 13, 15, 16, 32). Recent reports have to SSAV have been isolated from gibbon apes, also indicated that certain onc genes of viruses such viruses have not been found in woolly isolated from different species also are genetical- monkey populations. Thus, SSV was isolated ly related (2, 4, 5, 30). Thus, only a limited from the only woolly monkey known to have number of cellular genes have been transduced been infected by SSAV. These results suggest by type C viruses in the generation of sarcoma that there may be an unusual propensity for viruses. The lack of homology between the SSV SSAV infection of the woolly monkey to in- sis gene and cell-derived genes of several other crease the probability of sarcoma virus genera- sarcoma-inducing retroviruses indicates that sis tion. is likely to be a unique onc gene. In view ofthese The sis-related cellular sequences exhibited findings and findings indicating that this primate- those characteristics expected of a well-con- derived SSV sis gene readily detects homolo- served cellular gene. One or at most a few DNA gous sequences in normal human DNA, sis fragments were detected in cellular DNAs by DNA should be useful as a probe to search for using labeled sis DNA as a probe. Moreover, expression of related sequences in different sis-related sequences were found in DNAs of forms of human cancer. species as diverse as humans and quail. Quanti- tative analysis of the divergence of sis-related We thank G. Cooper, T. Papas, and E. Scolnick for gifts of sequences compared with that of unique-se- molecular clones of Rous sarcoma virus, avian myelocytoma- quence cellular DNA revealed that sis DNA was tosis virus, and Harvey sarcoma virus, respectively. VOL. 41, 1982 NOTES 725 LITERATURE CITED 19. Jainchill, J. L., S. A. Aaronson, and G. J. Todaro. 1969. 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