MICROBIoLOGICAL REVIEWS, Mar. 1981, p. 1-80146-0749/81/010001/08$02.00/0

Transformation by Rat-Derived Oncogenic RetrovirusestEDWARD M. SCOLNICK

National Cancer Institute, Bethesda, Maryland 20205

Retroviruses are reverse transcriptase-con-taining infectious agents which replicate througha deoxyribonucleic acid (DNA) copy of theirgenomic ribonucleic acid (RNA) (44). Duringthe past decade, our understanding of the ge-netic structure and life cycle of retroviruses hasincreased rapidly due in large measure to thedevelopment of quantitative cell culture assaysfor retroviruses and the discovery of the enzy-matic machinery involved in the viral replicativecycle. The discovery of reverse transcriptase (1,45) removed a major intellectual barrier to our

understanding of RNA tumor viruses and pro-vided a rational approach for applying conven-

tional techniques of molecular biology to thestudy of how these agents transform specifictarget cells.The most intriguing biological aspect of retro-

virology has been the diverse array of oncogenicretroviruses that has been discovered. Most iso-lates of retroviruses replicate in cell cultures andanimals without causing any obvious pathogeniceffects. On the other hand, highly oncogenicretroviruses have been isolated from several ver-

tebrates. Approximately 10 to 20 of these highlyoncogenic variants have been isolated, and re-

views have been written recently categorizingthe viruses and discussing their biology and mo-lecular structure (3, 18, 35, 48). Each of theseviruses can cause abnormal growth of varioustarget cells in cell culture, and in some cases a

good correlation exists between the pathologicaleffects of the virus in cell culture and the onco-

genicity of the virus in its susceptible vertebratehosts.During the past 8 to 9 years, my laboratory

has been studying one particular group of thesehighly oncogenic viruses isolated from rats.Three different isolates of highly oncogenic ret-roviruses have been isolated from rats. The firstisolate was described in 1964 by Harvey, in thecourse of her studies with mouse-derived Molo-ney leukemia virus (Mo-MuLV) (7, 20). Inocula-tion of Mo-MuLV into mice results in overtthymic leukemia in infected animals. In earlystudies, investigators routinely maintained theirleukemogenic stocks by passage of these virusesfrom mouse to mouse. In the course of such

t Review based on the Eli Lilly & Co. Award Lecture givenat the 1980 American Society for Microbiology Annual Meet-ing.

animal passage, Harvey inoculated rats withMo-MuLV, and these animals also developedthymic leukemia. However, in the course of suchrat passage ofMo-MuLV, Harvey noted that thedisease syndrome changed when the rat-pas-saged virus was reinoculated into mice. Insteadof causing thymic leukemia after a relativelylong latent period, the virus induced the rapidonset of fibrosarcomas and an erythroid leuke-mia with splenomegaly. The etiological agentthat was responsible for the new disease syn-drome was named Harvey sarcoma virus (Ha-SV), and this agent was subsequently biologi-cally cloned. In 1967, a second isolate of a highlyoncogenic virus was isolated from rats. Kirsten(22, 23) inoculated another mouse leukemia-in-ducing virus into Wistar Furth rats and notedthat the disease syndrome induced in mice

changed after passage of.the virus in rats. Curi-ously, the disease syndrome observed in micewas similar to the pathology observed with Ha-SV. In 1978, Rasheed and her colleagues (26)isolated the first RNA sarcoma-inducing virusfrom cell culture. These investigators coculti-vated a rat tumor cell line with a rat embryo cellproducing rat type C viruses. After 2 to 3 weeksof cocultivation of these two cell lines, Rasheedwas able to isolate a focus-forming virus fromthe cell-free supernatant of her cultures. Thisvirus has been named the rat sarcoma virus (Ra-SV) since its isolation did not involve the use ofa mouse retrovirus, as did Ha-SV and Kirstensarcoma virus (Ki-SV) isolates. This paper willreview the studies of my laboratory of the ge-

netic structure of these three rat-derived onco-

genic viruses, Ha-SV, Ki-SV, and Ra-SV, and ofthe protein coded by each virus which is respon-sible for the oncogenicity of the viruses.Each of these rat-derived oncogenic viruses is

replication defective. Accordingly, propagationof infectious sarcoma virus requires coinfectionwith helper-independent type C retrovirus.Therefore, all infectious virus stocks containingany of these oncogenic viruses were a mixture ofat least two viruses: (i) the sarcoma virus, whichcould transform cells but which could not itselfreplicate, and (ii) the helper virus, which couldreplicate but not transform. In studies with avianretroviruses, certain unique strains of Rous sar-

coma virus (RSV) were found which could bothreplicate and transform (47). Because of this

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dual property of clonal isolates of RSV, thegenetics and genome mapping of viral codedfunctions could proceed in ways not possiblewith replication-defective oncogenic retrovi-ruses. The RSV-transforming (src) gene wasdefined, using conditional and deletion mutantsof helper-independent, replication-competentstrains of this virus (12-14, 49). The src gene isthat gene which codes for a protein required forthe maintenance of the transformed phenotype.Although the genetics of mammalian transform-ing viruses were much more rudimentary, wewere able to isolate a conditional mutant of Ki-SV which allowed us to show that a src gene(s)existed in Ki-SV (5). However, the absence of anextensive collection of genetically altered Ki-SV's precluded the identification of the src genewith the same genetic precision achieved byavian retrovirologists.

Despite this limitation, we chose to study thestructure of Ki-SV by molecular hybridization.By using complementary DNA probes synthe-sized from the genomic RNAs of Ki-MuLV andMo-MuLV or Ki-SV and Ha-SV, we learnedthat Ki-SV contained two distinct nucleic acidsequences. Ki-SV genomic RNA was shown tobe comprised of portions homologous to Ki-MuLV and rat genetic information (31). A simi-lar conclusion was reached about the structureof the Ha-SV genome (30). Because of the his-torical method of isolation of Ki-SV, we postu-lated that Ki-SV had arisen by recombinationbetween Ki-MuLV and rat genetic informationand that the src gene had been acquired by aprocess analogous to transduction in bacterialsystems (31). The lack of genetics precluded amore precise analysis of the origin of the srcgene of Ki-SV. Subsequent elegant experimentson the src gene of RSV by Stehelin et al. (43)and Hanafusa et al. (19) more precisely definedthe origin of src genes. Their work clearly estab-lished that src genes did derive by a processanalogous to transduction. In addition, an im-portant property of src genes was established,namely, that they are highly conserved in avariety ofvertebrate species (41, 42). I will returnto a comparison of the structure ofRSV and thestructure of Ha-SV later in this paper.

In subsequent experiments, we proceeded tomap the genomic RNA of Ki-SV and Ha-SV,using oligonucleotide fingerprinting and molec-ular hybridization (38). We were extremely for-tunate in this work to have the help of JohnCoffin, who taught us the techniques of oligo-nucleotide fingerprinting with polyacrylamidegels. By protecting oligonucleotides with com-plementary DNAs representing either mouse orrat sequences of Ha-SV and Ki-SV, we could

identify which oligonucleotides of the viruseswere of mouse origin and which were of ratorigin. We then mapped the order of these oli-gonucleotides with respect to the 3' polyadenylicacid-containing end of each virus. In addition, apicture of the structure of Ha-SV and Ki-SVbegan to emerge from subsequent mapping stud-ies and heteroduplex analyses performed byChien and co-workers (8). These studies re-vealed the structure of Ha-SV and Ki-SV (Fig.1). The structure of the parental prototypemouse type C helper virus is shown at the top ofFig. 1. In Ki-SV and Ha-SV, only the very 5' andthe 3' ends of the parental mouse virus areretained. All of the genes coding for the viralstructural proteinsgag,pol, and env (2) ofmousetype C helper-independent viruses have beendeleted, thus explaining why these two onco-genic viruses are replication defective. Ha-SVand Ki-SV were shown to contain 5,500 and6,500 bases of rat-derived genes, respectively,between the 5' and 3' ends of their parentalmouse viruses. The crosshatched areas of Ha-SV and Ki-SV indicate portions of incompletegenetic homology. The blackened areas of themap of each virus were found to be derived froman unusual rat retrovirus-like molecule calledthe rat 30S subunit (16, 28, 45, 46). The rat 30Sgene(s) is present in multiple copies in rat DNA,and heterogeneity between the multiple copiesexists. RNA transcripts of these unusual ratgenes can be efficiently packaged by type Chelper-independent retroviruses growing in ratcells (16). The exact nature of this rat 30S geneis poorly understood. Recently, similar 30Sgenes have been described in mice (11, 32), andrecombinant DNA phages containing mouse 30Sgenes have been found to have repeat sequencesat the termini which are similar to the longterminal repeat of other retroviruses (E. Keshet,personal communication). However, precise in-terpretation of the physical map and the defini-tion of the src gene remained elusive since thecomplexity of the rat-derived genes was so muchgreater (5,500 bases) than that of the src gene ofRSV (1,800 bases). Furthermore, no genetic def-inition of the src gene existed within the 5,500-base sequence of acquired rat information.With the change in the guidelines that gov-

erned the application ofrecombinantDNA tech-nology to the study ofanimal virology, we turnedto the task of cloning the DNA proviral form ofHa-SV into bacterial virus vectors. In this phaseof the project, we are indebted to Wallace Roweand Malcolm Martin of the National Institute ofAllergy and Infectious Diseases. Our own labo-ratory in particular and animal virology in gen-eral are indebted to the efforts of Rowe and

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TRANSFORMATION BY ONCOGENIC RETROVIRUSES

5, -gag. -

M-MuLV 10 9 8 7 6 5 4 3 2 1 OkMW

K-uSV o _ _ ___---- 3

HMuSV 0_---------------

FIG. 1. Structure ofHa-SV, and Ki-SV (Ki-MuSV) genomes, deduced from oligonucleotide fingerprints andheteroduplex maps. The experiments which support these maps are described in references 8 and 40. Cross-hatching indicates areas ofHa-SV and Ki-SV of incomplete homology. Poly(A), Polyadenylic acid. Symbols:q, helper; -, host; -, homology between Ki-SV and Ha-SV.

Martin in helping the National Institutes ofHealth, under Donald Frederickson, to revisethe National Institutes of Health guidelines gov-erning recombinant DNA experiments. The suc-cessful cloning of infectious Ha-SV DNA intolambda bacteriophage by Hager and Lowy (17)in collaboration with Martin's laboratory en-abled us to define precisely the src gene of Ha-SV and to reveal the complexity of the structureof Ha-SV (6, 15). Heteroduplex studies on mo-lecularly cloned Ha-SV and biological studieswith the clonedDNA revealed that the structureof Ha-SV was tripartite (Fig. 2): (i) mouse typeC sequences at the 5' and 3' ends of the virus,including the long terminal repeat; (ii) 4,400bases representing the rat 30S genes; and (iii)toward the 5' end of Ha-SV, 1,000 bases repre-senting an evolutionarily conserved rat gene re-sponsible for the transforming ability of the vi-rus. The conserved rat gene codes for the trans-forming protein of Ha-SV, p21 (see below). Thetripartite structure of Ha-SV seems at this pointto be unique among oncogenic RNA viruses.The important features of the structure are thedifferent properties of the 30S and p21 genesfound in normal rat DNA and in other verte-brate DNA. (i) The 30S gene(s) is detected in 40to 80 copies in rat DNA, and the p21 gene isdetected in just a few copies (?2 to 4); and (ii)there are marked evolutionary differences be-tween the rat 30S genes of Ha-SV and the p21src gene of the virus. The p21 src gene is wellconserved from one vertebrate species to an-other, and the 30S-related genes are poorly con-served; yet in Ha-SV the 30S and the src genesare juxtaposed and transmitted together in astable form. This unusual structure is somewhatreminiscent of the structure of the transposableelement of yeast, his4-917, and raises importantquestions for us as to the mechanism by whichHa-SV arose (27). Are the 30S genes linked tothe transforming gene in the normal rat genome,

5' 3'

K P

FIG. 2. Structure of Ha-SV and Ki-SV, deducedfrom studies on molecularly cloned Ha-SV. The ex-periments which support this map are described inreferences 6 and 15. K and P indicate the restrictionendonuclease sites Kpn and Pst, respectively (6, 15).Symbols: _, mouse; %, transforming andp21-codingregion; E, rat.

and were both the 30S gene and the src geneacquired in a single recombination event? Orwere the 30S and the src genes acquired as twoindependent events, suggesting that the forma-tion of transmissible oncogenic viruses requiresmultiple recombinational steps? Are the 30Sgenes analogous to the Ty family of repeatedsequences found in yeasts (27)? I will discussthese questions further at the end of the paper.The other aspect of the study of Ha-SV and

Ki-SV has been the search for the src geneproduct. This protein, p21, was identified firstby in vitro translation of the RNA of Ha-SV.However, characterization of the p21 proteinrequired the preparation of antisera to p21 (37).For the RSV src protein, rabbits were found tobe excellent sources of antiserum, and that dis-covery provided a stimulus to our own ongoingsearches for antisera to p21 (4). For Ha-SV,antisera were finally prepared by transplantingHa-SV-transformed cells in rats (37). Nonpro-ducer rat kidney cells transformed by Ha-SVwere inoculated into 10-day-old syngeneic rats.Sera were obtained from the tumor-bearing rats4 to 5 weeks later. By methionine labeling andimmunoprecipitation of Ha-SV-infected cells,we demonstrated that Ha-SV-transformed cellscontained the 21,000-dalton protein p21. A sim-ilar p21 protein was detected in Ki-SV-trans-formed cells, and this protein could also be trans-lated from Ki-SV genomic RNA. In a series of

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studies of the thermolability of the p21 proteinofa conditional transformation mutant of Ki-SVand of the presence of p21 in cells transformedby various molecular clones of Ha-SV, we une-quivocally demonstrated that p21 was the srcgene product of Ha-SV and Ki-SV (6, 15, 29, 36)and that it was required for both the initiationand the maintenance of the transformed phe-notype.A surprising result was the observation that

Ra-SV coded for a related protein, p29 (52). Inthis case, the src protein was a p21-like proteinfused to a rat type C viral structural proteinwhich appears to be a part of the gag precursorof rat type C viruses (H. Young, personal com-munication). Thus, the p29 protein of Ra-SVappears to be a fusion protein analogous to thelarger fusion proteins detected in a variety ofother oncogenic retroviruses. The molecularstructure of the genomic RNA of Ra-SV has notbeen elucidated, but rat 30S genes do not seemto be present in Ra-SV (52). This observation isconsistent with our recent studies of Ha-SV,which showed that the p21-coding gene in Ha-SV is not comprised of rat 30S genetic sequences(15). Thus, each of these three independent rat-derived viruses, Ha-SV, Ki-SV, and Ra-SV,codes for a p21 protein which is the src proteinof each virus. It is curious that a gene coding fora related protein was acquired in three separateisolates. The coincidence is somewhat surprsingsince rat cells, like other vertebrate cells, containother endogenous sarc genes as well as p21-coding sarc genes. These results suggest that theendogenous p21-coding sarc gene(s) has somespecial structural features which make it espe-cially susceptible to recombination with repli-cating retroviruses. Future studies on molecularclones ofendogenous sarc p21-coding genes mayelucidate what these special features are.

In the past year we have begun to investigatethe biochemistry of the p21 protein. As with thesrc protein of RSV, there is a phosphoproteinform of p21, and both a phosphorylated and anon-phosphorylated form of p21 can be detectedin Ha-SV-infected cells (37). A binding assay forthe detection of p21 was devised which involvesthe use of antiserum to p21 and labeled guaninenucleotides (29). The [3H]guanosine 5'-diphos-phate (GDP)-binding assay is shown in Fig. 3.Extracts of Ha-SV-infected cells are incubatedwith [3H]GDP, and antisera containing anti-bodies to p21 are added. The resulting immunecomplex is captured with the aid of Staphylo-coccus containing protein A on its surface, andthe amount of guanine nucleotide bound in theimmune complex can be quantitated. The nu-cleotide-binding assay for p21 seems to be spe-

p21 + PIHJGDP * [3HJGDP p21

+ nti-p21 Ab

[3H]GDP p21

IAnti-p21 Ab

+ Staph. A

then Wash and Centrifugation

FIG. 3. Guanine nucleotide-binding assay for p21.Experiments with this assay are described in refer-ences 29 and 34. Ab, Antibody; Staph., staphylococ-cus.

cific for GDP or guanosine 5'-triphosphate(GTP), and the assay detects the p21 protein ofHa-SV and Ki-SV and the p29 protein of Ra-SV. Recently, we have been able to purify theHa-SV p21 protein approximately 265-fold withthis assay, and incubation of p21 with GTPlabeled with 32P in the terminal phosphate re-sults in phosphorylation of this purified p21protein (34). The phosphorylated amino acid isphosphothreonine, and the same phosphopep-tide(s) and threonine residue are identified bothin vitro, in p21 phosphorylated with GTP, andin vivo, when the phosphorylated form of p21 isisolated from Ha-SV-transformed cells.The enzymology of GTP and p21 is of obvious

importance in the study of the biochemicalmechanisms involved in the etiology of cancer.The comparative biochemistry of p21, the RSVp60 src protein, and the enzymologically relatedproteins Abelson virus p120 and Snyder-Theilenfeline sarcoma virus p85 should provide novelinsights into the mechanisms of the etiology ofcancer in the coming decade (9, 21, 24, 50). Thestudy of the transforming genes and gene prod-ucts of oncogenic RNA tumor viruses, I believe,has initiated a new era in the study of cancerbiology. For the first time in history, biochemical

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studies are being carried out on proteins whichare directly responsible for the induction andmaintenance of the transformed phenotype. Ibelieve that cancer cells, studied with the exper-imental tools fashioned by investigators of on-cogenic retroviruses, will finally yield to cancerbiologists major insights into the metabolicpathways involved in their cause.The final topic that I would like to discuss

relates to the origin of the src genes of highlyoncogenic retroviruses, such as Ha-SV and RSV.Studies on both of these viruses indicated thattheir src genes were acquired by recombinationbetween helper-independent type C viruses andgenes present in uninfected vertebrate cells. Thesrc gene of RSV was shown to be derived froma highly conserved gene present in all vertebratespecies. The acquired genetic sequences of Ha-SV have been shown to be of dual evolutionaryorigin, with the p21-coding gene being conservedlike the pp6O-coding gene of RSV. Since thegenes from which src genes are derived are sohighly conserved, Bishop and colleagues havehypothesized that such genes have an importantnormal function in all vertebrate species (41, 42).

Recently, we have been studying the expres-sion of endogenous p21 in a variety of vertebratecells. As in the case of the pp6O ofRSV, we havefound that all cells that we have examined ex-press low levels of p21. However, a unique he-mopoietic cell line which expresses markedlyelevated levels of endogenous p21 has beenfound (32). The cell line, 416B, was originallydescribed by Mike Dexter and colleagues andarose from a long-tern culture of bone marrowfrom BDF1 mice. The 416B cell line has manyproperties of hemopoietic stem cells. A varietyof more committed hemopoietic precursor cellsdo not express such markedly elevated p21 levels(32). The markedly elevated levels of endoge-nous p21 are shown in Fig. 4. These recentresults have led us to speculate that the expres-sion of endogenous p21 may play a role in deter-mining whether a stem cell undergoes self-re-newal or commitment, and study of the expres-sion of p21 and p21 biochemistry may providethe first molecular marker for hemopoietic stemcells. Expression of a normal protein related toAbelson virus p120 has been detected in hemo-poietic cells but not in fibroblasts (51). It ispossible that certain endogenous src genes playan important role in nonnal hemopoiesis andthat studies of endogenous src gene expressionand src gene products will be important tools forinvestigating the developmental biology of he-mopoietic tissues, especially the biochemicalevents concerning self-renewal versus commit-ment to differentiation of such cells.

FIG. 4. Expression ofp21 in a hemopoietic precur-sor ceU line. An autoradiogram of I'SJmethionine-labeled cells processed by immunoprecipitation andpolyacrylamidegel electrophoresis is shown. Detailedstudies are described in reference 32. Lanes 7 and 8,416B cells labeled and immune precipitated withcontrol serum (lane 7) and antiserum (lae 8). Thearrow indicates p21. Lanes 1 through 6, Other he-mopoietic cels processed sinilarly (see reference 32).

UNRESOLVED QUESTIONSAfter a decade of rapid growth of our under-

standing of the origin of the transforming genesof retroviruses, investigators are still left withmany vexing questions which need to be ad-dressed in the next decade. (i) By what mecha-nism do the recombinational events occur whichgenerate these oncogenic retroviruses that nowcarry cellular genes? Recent data on the struc-ture of the termini of retroviruses have led tothe speculation that retroviruses are like procar-yotic transposition elements (10, 39, 40). It ispossible that mechaniss simiar to transposi-tion in bactenra are a necessary step in the gen-eration of oncogenic retroviruses. As I noted

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earlier, the structure of Ha-SV is striking in itssimilarity to the transposable his9l7Ty (27). (ii)How many different endogenous src genes arethere? Is each proto-src gene truly unique, or iseach a member of a small family of genes? (iii)Are any naturally occurring cancers the result ofenhanced expression or gene rearrangements ofnormal endogenous src genes in the cell? (iv)What are the biochemical pathways involved inthe action of src gene products? Recently, evi-dence has been presented to suggest that phos-phorylation of tyrosine residues is an importantfunction of the RSV src product and Abelsonvirus p210 (50) and that at least one target ofthe RSV p60 kinase, a 34,000-dalton protein, hasbeen found (21, 25).With the aid of the two new powerful tools of

modem biology, hybridoma cell lines and recom-binant DNA technology, RNA tumor virologyshould continue to lead the intellectual attackon the mysteries of cancer cells in the nextdecade and, perhaps, begin to unravel for thefirst time the basic mystery of what controlstheir growth.

ACKNOWLEDGMENTSThis work was supported by the Virus Cancer Pro-

gram of the National Cancer Institute.I thank Wade Parks, Thomas Shih, and Howard

Young for their collaborative efforts over the courseof this project.

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