Endogenous type-C RNA viruses of mammalian cells

Biochimica et Biophysica Acta, 458 (1976) 323-354 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - Printed in T h e N e t h e r l a n d s BBA 87029

ENDOGENOUS

TYPE-C

RNA

VIRUSES

OF

MAMMALIAN

CELLS

S T U A R T A. A A R O N S O N a n d J O H N R. STEPHENSON

Laboratory of RNA Tumor Viruses, National Cancer Institute, Bethesda, Md. 20014 (U.S.A.) (Received February 12th, 1976)

CONTENTS I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

324

II.

General properties of m a m m a l i a n type-C R N A helper l e u k e m i a viruses . . . . . . . . A. M o r p h o l o g y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Viral g e n o m e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Virus-coded reverse transcriptase . . . . . . . . . . . . . . . . . . . . . . . D. O t h e r viral proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Properties in cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . .

324 324 325 325 326 327

II1. Distribution o f e n d o g e n o u s m a m m a l i a n type-C R N A viruses . . . . . . . . . . . . . A. Discovery o f genetic t r a n s m i s s i o n o f type-C viruses in the m o u s e . . . . . . . . . B. O t h e r e n d o g e n o u s viruses o f r o d e n t s . . . . . . . . . . . . . . . . . . . . . . C. E n d o g e n o u s viruses o f cats . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Feline leukemia virus . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. R D 114 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. E n d o g e n o u s type-C viruses o f pigs . . . . . . . . . . . . . . . . . . . . . . . E. E n d o g e n o u s type-C viruses o f primates . . . . . . . . . . . . . . . . . . . . .

328 328 330 331 331 331 332 332

IV. Biologic regulation of e n d o g e n o u s viruses . . . . . . . . . . . . . . . . . . . . . A. Differential regulation of three distinct e n d o g e n o u s m o u s e type-C viruses . . . . . . B. M e c h a n i s m s of type-C virus activation by h a l o g e n a t e d pyrimidines a n d inhibitors of protein synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. E n h a n c e m e n t of e n d o g e n o u s type-C virus release by steroid h o r m o n e s . . . . . . . D. Influence of t h e differentiated state of the cell on type-C virus regulation . . . . . . E. A gene affecting expression of class I virus . . . . . . . . . . . . . . . . . . . F. Genetic control of class III e n d o g e n o u s virus expression . . . . . . . . . . . . . G. A gene conferring susceptibility to e x o g e n o u s infection of m o u s e cells by xenotroptic virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Systematic regulation of e n d o g e n o u s type-C viruses of m o u s e cells . . . . . . . . 1. Biologic regulation of e n d o g e n o u s type-C viruses of other m a m m a l i a n species . . . .

334 334

V.

M a m m a l i a n s a r c o m a virus - a n o t h e r g r o u p o f type-C R N A virus c o n t a i n i n g e n d o g e n o u s viral genetic sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Biologic properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Defective n a t u r e o f m a m m a l i a n s a r c o m a viruses . . . . . . . . . . . . . . . . . C. Biochemical evidence o f the e n d o g e n o u s n a t u r e o f s a r c o m a viral genes . . . . . . . D. O t h e r type-C virus isolates o f m o u s e cells . . . . . . . . . . . . . . . . . . . .

336 339 339 340 341 341 342 343 343 343 344 344 345

VI. Biologic implications o f e n d o g e n o u s type-C viruses . . . . . . . . . . . . . . . . .

345

References

348

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

324 I. INTRODUCTION Since their initial discoveries more than 60 years ago [1,2], RNA-containing tumor viruses, designated type-C RNA viruses, have been isolated from a large and ever-increasing number of species. Only in recent years has it become evident that these viruses may interact with their host in a manner that appears to be unique for viruses of vertebrates. In many species, type-C RNA viruses, morphologically indistinguishable from leukemia viruses of the chicken and mouse, have been shown to be transmitted from one generation to next, and often in an unexpressed form, within the host genome. Under such conditions, these viruses appear to be subject to regulatory processes analogous to those affecting cellular genes. Endogenous type-C viruses can be defined as existing within the cellular genome of a species and containing the information necessary for production of viral-specific gene products. In many cases, complete type-C virus can be induced, but evidence has also been obtained that genetic information related to that of known endogenous viruses is present in many species from which complete virus has yet to be isolated. Another group of mammalian type-C viruses transforms cells in tissue culture but requires a type-C helper leukemia virus for its replication as an infectious virus. Increasing evidence indicates that these transforming viruses also originate at least in part from within the genome of their species of origin. Endogenous viruses exist in species as diverse as the chicken, mouse, and baboon, and there is evidence suggesting their presence within some species over a long period of evolution. These findings raise obvious questions concerning their natural functions. The present review contains a brief description of the known properties of mammalian type-C viruses and summarizes experimental evidence leading to the demonstration of their genetic transmission. The known distribution of endogenous viruses among mammalian species and evidence concerning the regulation of their expression by the host are presented. Finally, some of the possible biologic functions of endogenous viruses are discussed.

II. GENERAL PROPERTIES OF MAMMALIAN TYPE-C RNA HELPER LEUKEMIA VIRUSES

IIA. Morphology The type-C RNA virus consists of a roughly spherical, compact nucleoid (made of R N A and associated proteins) surrounded by an electron-lucent layer that gives electron micrographs of the virus a target-like appearance (Fig. 1). By electron microscopy, a crescent of electron-dense material is seen in association with a protrusion of the cell surface. This crescent becomes progressively circular, becoming the virus core as the plasma membrane develops into a microvillus-like structure and is eventually pinched-off from the membrane. The virion core subsequently becomes more electron-dense as the virus develops into a mature type-C virus particle [3,4].

325

Fig. I. Electromicrograph of a type-C RNA virus (120000). (Photograph courtesy of V. Zeve, NCI, NIH).

HB. Viral Genome The genome of the type-C virus consists of single-stranded R N A with an apparent molecular weight of around 107 as determined by its 60--70 S sedimentation coefficient in neutral sucrose gradienst [5]. Like many known messenger and viral RNAs, type-C viral RNA contains 150-200 nucleotide stretches of poly(A)-rich sequences [6-8]. Exposure of 70 S RNA to heat or mild denaturing conditions results in the formation of a 30-35 S structure [9]. Current evidence favors the concept that the viral genome is polyploid, containing two to three identical subunits, each of which possesses a molecular weight of approximately 2-3" 106 [10-13]. Thus, the total information content of the viral genome is sufficient to encode for around 300000 daltons of amino acid sequences. HC. Virus-Coded Reverse Transcriptase Mammalian type-C viruses contain a reverse transcriptase [14,15] with a molecular weight of around 70000 [16-18]. This enzyme can synthesize a D N A strand complementary to viral R N A and a second strand of D N A complementary to the first [19]. The reverse transcriptase also possesses RNAase H activity, a processive exonuclease which can degrade the R N A portion of D N A • R N A hybrids [20-23]. Analysis of temperature-sensitive mutants of avian [24-26] and mammalian [27] (Verma et al., in preparation) type-C viruses, whose reverse transcriptases and RNAase H activities are each temperature-labile, has established that both activities are viruscoded and physically located within the same molecule. The reverse transcriptase

326 provides a mechanism for integration of type-C viral RNA as a DNA provirus within the genome of the infected cell. Antisera which recognize the antigenic determinants of type-C viral reverse transcriptases have been used in the immunological identification of these enzymes. It has been possible to show, for example, that the type-C viral enzyme does not cross-react with normal cellular DNA polymerases [16]. However, there is partial immunological cross-reactivity between the reverse transcriptases of type-C viruses isolated from several mammalian species including the rat, mouse, cat, and hamster. These same antisera do not inhibit reverse transcriptases of avian type-C viruses or of other non-type-C viruses of mammalian origin [28,29]. liD. Other Viral Proteins

Initial attempts to identify and biochemically characterize type-C viral-coded proteins relied, to a large extent, on the use of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) [30,31]. However, these studies led to considerable disagreement concerning the numbers and properties of such proteins. A major advance resulted from the application of agarose gel filtration under denaturing conditions to the resolution and identification of type-C viral proteins [32,33]. By this approach, type-C viruses of mammalian origin were shown to contain a high molecular weight glycoprotein, a major virion structural protein, and several lower molecular weight viral proteins (Fig. 2). The designation of each protein is based upon the molecular weight (× 10-3) as determined by SDS-polyacrylamide gel electrophoresis for proteins over 30000 tool. wt. and by gel filtration in 6 M guanidine • HC 1 !

30

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~5 4 p15 p12

40

60

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10Q NUMBER

plO

120

Fig. 2. Analysis of structural polypeptides of Rauscher murine leukemia virus by agarose gel filtration in the presence of 6 M guanidine • HCI. The individual polypeptides are designated according to their molecular weights relative to standards as follows: gp70 (70000); p30 (30000); p15 (15000); p12 (12000); p l 0 (10000).

327 for lower molecular weight proteins [34]. According to this nomenclature, mammalian type-C viruses contain glycoproteins of 700000 and 45000 daltons (gp70 and gp45) and several nonglycosylated proteins including p30, p15, p12, and pl0 [34]. Approaches toward characterizing type-C viral proteins have primarily been immunological. Early studies were based upon the use of complement fixation and immunodiffusion techniques [35-38]. However, the more recent application of radioimmunological methods to the detection and identification of viral proteins has greatly increased information concerning their properties [39-42]. Antigenic determinants possessed by viral structural proteins include (1) those unique to a particular virus isolate (type-specific); (2) those shared by corresponding proteins of different virus isolates of the same species (group-specific); and (3) those shared by corresponding proteins of virus isolates of different species (interspecies-specific). The demonstration that antigenic determinants of viral proteins, grown in cells of different species, are conserved, strongly indicates their virus-coded nature. Several mammalian type-C viral structural proteins including gp70, p30, p15, p12 and pl0 have been shown to be virus-coded by these criteria [43]. Two mammalian viral proteins, which do not as yet fulfill these criteria, have also been described. These include a 45000 mol. wt. glycoprotein, gp45 [34] which may represent a breakdown product of gp70 and a nonglycosylated 15000 mol. wt. protein, pl 5(E) [44], that is believed to be a constituent of the virion envelope [45]. pl5(E) appears to be a group-specific protein, immunologically distinguishable from p15 [44]. Further, p15 and pl5(E) exhibit slightly different molecular weights by SDS/polyacrylamide gel eletectrophoresis analysis [44]. liE. Properties in Cell Culture

Mammalian type-C helper or helper leukemia viruses, replicate in fibroblasts without causing morphologic alteration [46,47]. There are several reports indicating that infection of murine cells with these viruses can lead to a more rapid development of 'transformed loci' over many cell passages than would occur spontaneously [48, 49]. Hackett and Sylvester [50] have described a more rapid transformation assay involving focus-formation in cells of a clonal line, UCB-I, derived from BALB/3T3 mouse embryo fibroblasts. In vitro transformation assays for the Friend erytholeukemia virus [51] and Abelson leukemia viruses [52] have also recently been described. The most commonly used current methods for detecting virus infectivity include the measurement of virion-associated reverse transcriptase activity in tissue culture fluids [53-55] and the detection of viral antigens in infected cultures [56,57]. In addition, plaque-forming assays have been developed for the detection of a few helper leukemia viruses. In these assays, cells that form syncyctia in the presence of the particular replicating type-C virus, are plated on cultures infected several days earlier with virus. Plaques containing giant syncytial cells form in descrete areas of the culture where type-C virus is being released [58-61].

328 IlI. DISTRIBUTION OF ENDOGENOUS MAMMALIAN TYPE-C RNA VIRUSES

IliA. Discovery of Genetic Transmission of Type-C Viruses in the Mouse Studies by several laboratories led to the demonstration that mammalian type-C RNA viruses can be genetically transmitted within the germ line of a species. These studies originated with the development by Furth et al. [62] and McDowell and Richter [63] of inbred mouse strains, characterized by a very high natural incidence of leukemia. Their findings established the inherited basis for factors involved in leukemogenesis in mice and contributed to the discovery by Gross severa 1 years later that leukemia could be induced by inoculation of susceptible mice with filtrates of tumors of high leukemia incidence strains [64]. The agent responsible for these tumors was shown to be a type-C RNA virus [65]. A series of investigations originating with studies by Kaplan and co-workers subsequently demonstrated that in mouse strains with low natural incidences of cancer, leukemia developed at high incidence following fractionated total-body X-ray irradiation [66,67]. Filtered extracts prepared from tumors induced by X-ray irradiation were shown to cause lymphatic leukemia when inoculated into newborn mice of the same strain [68,69]. These findings further documented the intimate association of type-C viruses with the mouse and their association with tumors. In mouse strains that expressed high levels of infectious virus, the method of virus transmission was shown to occur by congenital infection within the reproductive tract or milk [70,71]. It was not initially recognized that type-C viruses were genetically transmitted, within the gametes themselves. Evidence leading to the demonstration of genetic transmission of the virus came from several independent discoveries. In immunologic investigations, Huebner and co-workers demonstrated by complement fixation, the expression of antigens of type-C viruses in embryonic and other tissues of mouse strains which did not yield infectious viruses [72,73]. These findings could most readily be explained on the basis of the inherited nature of the virus [72]. Evidence suggesting the genetic transmission of these viruses was also provided by the discovery that mouse cells, that remained virus-negative for many cell generations in tissue culture, could spontaneously begin to release type-C virus [74]. Subsequent findings that halogenated pyrimidines could activate virus from clonal lines derived from single cells of both high [75] and low [76] leukemia incidence strains demonstrated that endogenous viruses were present in an unexpressed form in all mouse cells. Independent evidence of the genetic transmission of type-C viral information in the mouse was obtained through the application of molecular hybridization techniques to this question. Viral D N A probes, prepared in reactions utilizing mouse type-C viral RNA and the viral reverse transcriptase, detected shared nucleotide sequences with cellular DNAs of different mouse strains [77-81]. By this method of analysis, type-C virus-specific information was found to be present in the form of multiple copies within the high molecular weight DNA of the mouse cell [78]. The kinetics of DNA hybridization are defined by the product of the DNA

329 concentration and time of reaction (Cot) at constant salt concentration and temperature. Thus, when cellular D N A shares nucleotide sequences with those of a doublestranded (ds) DNA probe synthesized using the type-C viral RNA as template for its reverse transcriptase, the increased concentration of virus-specific DNA in the reaction mixture causes the probe to reanneal at a faster rate. Fig. 3 illustrates the increased rate of reassociation of a mouse type-C viral ds D N A probe in the presence of mouse cellular DNA but not DNA of control chicken or monkey-cells. Singlestranded (ss) viral DNA probes, prepared as above but with the addition of actinomycin D [82], have also been utilized for the detection and quantitation of nucleotide sequence homology between type-C viral and cellular DNA. Here, 3H-labeled ss viral DNA hybridizes only if shared sequences are present in the unlabelled cellular DNA. The Cot at which the probe anneals, relative to the Cot of unique cellular DNA sequences defines the number of viral copies per cell. The maximum hybridization of the probe achieved is a measure of the extent of sequence homology of the virus genome with the cellular DNA. Fig. 4 illustrates a DNA reassociation experiment in which a large fraction (over 80 ~ ) of the ss-[JH]DNA probe is annealed by BALB/c or C57BL/6 liver DNA, but not by calf thymus DNA. Hybridization using ss-DNA probes is the more sensitive technique for detecting small differences between virus-related sequences in cellular DNAs of different sources. The availability of inbred strains and the relative ease with which genetic analyses can be performed with mice has made even more rigorous proof of the chromosomal localization of mouse type-C viral genes possible. Findings that endogenous viruses are inducible from cells of some strains, while cells of other i

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Fig. 3. Reassociation of all-labeled KiMuLV D N A in the presence of unlabelled cellular D N A . Reaction mixtures contained 1.5 • 10-2 #g/ml double-stranded 3H-labeled KiMuLV DNA, 2.5 " 10 -3 M EDTA, 0.6 M phosphate buffer, pH 6.8, and 2.5 mg/ml BALB/c ([~); C57BL/6 ( I ) ; chicken (O); or African Green monkey ( ~ ) DNA in a volume of 1.7 ml. The reaction mixture was heat-denatured at 96 °C and allowed to reassociate at 68 °C. Aliquots were removed at various times and renaturation of viral D N A analyzed by hydroxyapatite chromatography [77].

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Fig. 4. Analysis of type-C viral genetic sequences in normal mouse cell DNA by molecular hybridization utilizing single-stranded all-labeled KiMuLV DNA. Reaction mixtures contained 3 • 10-~ /~g/ml labelled viral DNA, 0.6 M NaCI, 10 mM Tris" HCI, pH 7.5, 1 mM EDTA and 5 mg/ml BALB/c ( ~ ) ; C57BL/6 (11) or calf thymus (Q) DNA in a volume of 0.4 ml. Reaction mixtures were heat-denatured at 96 °C and allowed to reassociate at 62 °C. Aliquots were removed at various times and analyzed for double-stranded DNA by the $1 nuclease method [79].

strains lack these same inducible viruses, led to genetic investigations in which virus inducibility was shown to be a dominant genetic characteristic [83,84]. BALB/c cells were found to have one [85], AKR, two [84,86] and C58, three or more loci [87] for activation of viruses with very similar biologic properties. The locus for induction of one of these viruses, AKv-I, of A K R cells has been genetically mapped in linkage group 1, about 12 map units from Gpi-1 [88]*. Evidence that loci for virus induction represent viral structural rather than regulatory genes remained indirect until the demonstration by molecular hybridization that cellular DNAs of mouse strains containing one class of inducible virus, also possess quantitatively more virus-specific DNA related to this virus than do noninducible strains [79-81]. Moreover, these viral sequences can be found only in those genetic crosses, inducible for that particular virus [89]. The question of whether the viral structural information at this locus represents the entire or only a portion of the viral genome is not as yet resolved.

IIIB. Other Endogenous Viruses of Rodents At the same time or soon after the discovery of the induction of mouse endogenous viruses by halogenated pyrimidines, it was found that clonal rat cell lines could be activated to release type-C virus by these same chemicals [90,91 ]. The host range and serologic characteristics of induced rat type-C viruses differ from those of known mouse type-C viruses [91,92]. Subsequent studies have demonstrated extensive nucleotide sequence homology between the rat endogenous virus and rat * See Note Added in Proof, p. 354.

331 cellular DNA, but little if any, with D N A of mouse cells [93]. Syrian and Chinese hamster cells, have also been shown to contain inducible type-C viruses, that differ markedly immunologically and biochemically from each other and from mouse or rat type-C viruses [94]. Endogenous viruses of each of these species can be propagated in tissue culture, although they tend to be poorly infectious for cells of the species in which they reside. Recently, the search for endogenous viruses of mouse species other than Mus museulus has led to the discovery of a virus endogenous to M. earoli, an Asian feral mouse [95]. This virus is of particular biologic interest because of its striking immunologic similarities to a very closely-related group of infectious type-C viruses that have been isolated in the past few years from certain colonies of gibbon apes [96,97] and, in one instance, from a woolly monkey [98]. These latter viruses appear to be horizontally transmitted as infectious agents and causative of naturally-occuring tumors in gibbon apes [96]. Since they lack genetic homology with the cellular DNA of gibbon ape or woolly monkey and cannot be induced from cells of these animals, they are clearly not endogenous primate viruses [80,99]. Thus, current evidence is consistent with the possibility that an endogenous virus of a species of feral mouse related to M. earoli has crossed species to become an infectious cancer-producing type-C virus of certain primates.

lIIC. Endogenous Viruses of Cats 1. Feline leukemia virus. Jarrett and co-workers first showed that typical type-C viral particles could be identified in lymphoid tumors of cats and that feline lymphosarcoma could be transmitted by inoculation of cell-free filtrates [100-I02]. Type-C virus particles were later found at high frequency in tissues of cats with spontaneous lymphosarcoma [103-106]. Extensive seroepidemiological studies have indicated that feline leukemia virus (FeLV) is horizontally transmitted as an infectious virus [107-109] and is naturally causative of lymphosarcomas in the cat. Although FeLV is horizontally transmitted among domestic cats, genes partially related to those of FeLV are found, not only in the DNA of the domestic cat Felis eatus [110,111 ], but also in other species of closely-related cats, including the European wildcat (F. sylvestris), the sand cat (F. margarita), and the jungle cat (F. chaus) [111,112]. Thus, information related to FeLV appears to have been maintained within cats for a considerable period of time. The partial rather than complete homology of FeLV with cat cell DNA may reflect the more rapid rate of evolution as an infectious virus, of what was at one time an endogenous cat virus. This may be analogous to the situation with certain laboratory strains of mouse type-C virus, such as Moloney and Rauscher leukemia virus. These viruses originated in the mouse but have been passaged extensively as infections viruses for several years and no longer show complete sequence homology with mouse cell DNA. 2. RD 114. McAllister and co-workers (1971) reported the development of disseminated tumors of human karyotype in cats following fetal inoculation with a human rhabdomyosarcoma line (RD) [113]. When the cells were found to be releasing type-C

332 virus, attempts were made to determine whether the virus was of feline or human origin [114]. These studies demonstrated that p30 and reverse transcriptase of the new virus isolate, designated RD114, were immunologically distinct from the corresponding FeLV polypeptides [114,115]. Since there had been no previous example of a single species with two such different endogenous type-C viruses, the findings raised the possibility that the RD114 virus might be of human origin. In subsequent studies, viruses indistinguishable from RDI14 were isolated from cultured cells of two species of cats, F. catus [116-118] and F. sylvestris [119]. The fact that the virus could be activated from virus-negative clonal cell lines indicated that it was endogenous to feline cells. This was further supported by the demonstration of nucleic acid homology between the RD114 viral genome and cellular DNA of several species of cats but not DNA of human cell origin [112,120-124]. RD114 is not known to be causative of any diseases in the cat.

llID. Endogenous Type-C Viruses of Pigs Several continuous cell lines derived from tissues of domestic pigs have been shown by electron microscopy to spontaneously release morphologically typical type-C viral particles [124,125]. Biochemical and immunological analyses of these particles have indicated that they represent a group of type-C viruses distinct from previously described endogenous murine, rat, feline, hamster, and primate viruses [126,127]. Evidence that this virus is endogenous to pig cells derives from the demonstration of virus activation from a permanent cell line of pig origin following treatment with 5-bromodeoxyuridine and dimethylsulfoxide [128,129]. The detection of multiple copies of gene sequences related to this virus within the DNAs of both domestic pigs (Sus serofa) and other members of the family Suidae suggests that this type-C virus has been associated with the pig cellular genome for a long period of evolution (millions of years) [130]. HIE. Endogenous Type-C Viruses of Primates Electron microscopic analysis of placentas of primate species, including rhesus monkeys, baboons, and humans have demonstrated the appearance of particles morphologically resembling type-C viruses [131-133]. By cocultivation of baboon placental tissues with susceptible assay cells, it was possible to isolate a type-C virus [134]. A closely-related virus has also been isolated from tumor cells of a second baboon species [135]. Nucleic acid hybridization studies have shown that the genetic sequences of the baboon virus are present within baboon cellular DNA, indicating that this virus is art endogenous primate virus [136]. Moreover, partial homology of baboon type-C viral specific sequences with DNAs of other Old World monkeys has been reported, the degree of relatedness correlating with the presumed evolutionary diversity of the species [136,137]. This has been taken as evidence for the presence of endogenous viruses, evolutionarily related to the baboon virus, in other Old World monkeys and higher apes [136,137].

333 The baboon virus has rather marked biochemical [138] and immunologic [139] similarities to RD114 virus of cats. Analysis of the distribution of baboon-RD114 viral sequences within the DNAs of various primate and feline species has indicated their much wider distribution among primates. This has suggested that virus of this class has been associated with primate cellular genome for a longer period of evolution than with the feline genome [138]. Thus, Benveniste and Todaro have speculated that an endogenous primate virus may have crossed species several million years ago to become secondarily established as an endogenous virus of the cat [138]. The known endogenous viruses of mammalian cells and the species in which they have been obtained are summarized in Table I. It should be noted that in many species from which virus has not yet been isolated, molecular hybridization studies have indicated the presence of viral genetic sequences (Table I). There is as yet no endogenous type-C virus isolate of human origin. However, there have been reports indicating low levels of sequence homology between human cellular DNA and that TABLE I M A M M A L I A N SPECIES K N O W N TO CONTAIN G E N E T I C I N F O R M A T I O N O F E N D O G E N O U S TYPE-C VIRUSES Mammalian species

Rodents mouse (Mus musculus, Mas caroli) rat (Rattus norvegicus) hamster ( Cricetulus griseus,

Mesocricetus auratur) Carnivores cat ( Felis catus, Fells sylvestris)

( Felis margarita, Felis chaus) Artiodactyls pig (Sus scroJa) bush pig (Potamochoerus porcus) wart pig (Phacochoerus aethiopicus)

Isolation of endogenous virus

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Primates baboon (Papio cynocephalus, Papio papio,

Papio hamadryas) mandrill ( Mandrillus sphinx) mangabey (Cercocebus atyls) patas ( Erythrocebus patas) African green (Cercopithecus sabaeus) Macaque stumptailed ( M. arctoides) pigtailed ( M. nemistrina) crab eating ( M. fascicularis) rhesus ( M. mulatta) celebes ( M. maura)

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334 of the baboon endogenous virus [140]. These reports provide suggestive, but p.ot as yet convincing evidence for the presence of endogenous type-C viruses of humans. However, the large number of species already shown to contain endogenous viruses makes the continued search for analogous viruses of humans an active area of investigation.

IV. BIOLOG1C REGULATION OF ENDOGENOUS VIRUSES There is much evidence indicating that conditions favoring high levels of type-C virus production are detrimental to the survival of the host. When type-C viruses exogenously infect an animal, permissive for virus release and spread, there is an associated high incidence of lymphoid tumors [64,65]. Moreover, in certain inbred mouse strains, where highly infectious endogenous viruses are spontaneously activated during embryonic development, there is also an associated high incidence of neoplasia [62,63]. Thus, if there were any advantage to the host conferred by the evolutionary persistence of type-C viral genes, there must be strong selective pressure for the development of regulatory mechanisms that control active virus replication. The mouse provides an excellent experimental model for studying endogenous virus regulation due to the availability of inbred strains and the comparative ease with which genetic analysis can be undertaken. Further, biochemical and immunological probes required for quantitation of the transcriptional and translational products of endogeneous viruses have been developed in the mouse system.

IVA" Differential Regulation of Three Distinct Endogenous Mouse Type-C Viruses Mouse cells have been shown to contain information for at least three biologically-distinguishable endogenous viruses [141-143]. These viruses can be partially differentiated on the basis of standard host range and serologic tests. The development of radioimmunologic techniques for the detection of certain of their structural polypeptides has further helped to distinguish these close-related viruses. The most useful immunoassays are those that detect the type-specific antigenic determinants of the p12 [141,143] and gp7Q [42,144] viral proteins. Each mouse endogenous virus isolate so far examined can be placed into one of three classes according to the immunologic properties of its p12 and gp70 in radioimmunoassays for the type-specific antigenic determinant of each protein. The different patterns of immunological reactivity of viruses of each class in type-specific immunoassays for viral p12 are illustrated in Fig. 5. Analogous differences have been demonstrated between the antigenic characteristics of the gp70s of endogenous mouse type-C viruses of each class [144]. While the type-specific antigenic determinants may represent only small differences in amino acid sequence, the different immunologic reactivities of the pl2s and gp70s of each virus demonstrate that these proteins are specific for each virus class. It is now known that p12 is synthesized as a component of a high molecular weight precursor that also contains p30, p15 and pl0 [145,146].

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COMPETING VIRUS (ng/ml) Fig. 5. Comparison of immunologic reactivities of endogenous type-C viruses of B A L B / c and

N1H Swiss mouse cells in type-specific immunoassays for the p12 virion structural polypeptide. Detergent-disrupted viruses were assayed at 2-fold serial dilutions by measuring their capacity to compete with a251-1abelled p l 2 polypeptide for limiting antibody as previously described [143]. Results are expressed as the percent of the total t2~I-labelled p l 2 precipitated, standardized to 1 0 0 ~ in the absence of competing antigen. The reactivity of each virus was tested in homologous typespecific competition immunoassays for prototype Class 1 (/5); Class II ( I ) ; and Class llI ( O ) endogenous mouse type-C viruses. Viruses tested included: (A) Class I; and (B) Class I1 isolates of the BALB/c strain; and (C) a Class III isolate of the N I H Swiss strain.

Information for these proteins (including the precursor of which p12 is a part) would thus, account for one-half to two-thirds of a viral genome of 2-3 × 106 daltons. The three prototype viruses can be further differentiated by the manner in which the cell regulates their expression (Fig. 6). One class of endogenous virus, inducible from cells of the inbred BALB/c mouse strain, preferentially replicates in N I H Swiss mouse cells. This virus can be spontaneously activated [74] or induced by chemicals such as IdUrd [75,76]. Many strains have been shown to contain one or more loci for the induction of this virus [84-87]. An endogenous mouse virus representative of a second class was initially shown to be inducible from BALB/c cells [147]. This virus is noninfectious for cells of most inbred strains, but replicates well in cells of several other species [147]. Viruses with this host range have been termed xenotropic [148]. While the endogenous Class II virus of BALB/c mouse cells is spontaneously activated at very low level [149], its frequency of induction can be markedly increased by exposure of cells to halogenated pyrimidines [147,150] or inhibitors of protein synthesis [151] (Fig. 6). Viruses of this class have also been shown to be present in embryo cells of many strains [146]. In BALB/c cells, the locus for induction of Class II endogenous virus segregates independently from that of Class I virus [147]. Information for a third endogenous virus is also present in each of many inbred strains tested to date. While this third virus cannot be activated by known inducers

336

L

Fig. 6. Differential regulation of three classes of endogenous mouse type-C RNA virus. from embryo cells in culture [83], it has been isolated from NIH Swiss mice [152], and from N I H Swiss spleen cells in culture [148]. Like Class II virus, the third class of endogenous virus is xenotropic in host range. Viral polypeptides of this class are present at relatively high levels in embryo cells of the N I H Swiss and many other strains in the absence of detectable virus release [144, 146, 153] (Fig. 6). Embryo cells of one strain, NZB, have been shown to spontaneously generate this virus at readily detectable levels [143,155]. Table 1I summarizes some of the inbred strains that have been studied, the origin of these strains and their known endogenous type- C viruses.

IVB. Mechanisms of Type-C Virus Activation by Halogenated Pyrimidines and Inhibitors of Protein Synthesis The mechanisms by which chemical agents such as ldUrd and cycloheximide cause endogenous virus release are subjects of investigation in several laboratories. It is known that IdUrd must be incorporated into cellular DNA in order for it to induce virus. Drugs that inhibit D N A synthesis are able to inhibit virus induction by IdUrd [156]. Further, with synchronized cell populations, the period of inducibility corresponds to the S phase of the cell cycle [157-159]. The ability to undergo cell division dose not seem to be a requirement for virus activation, since pre-treat-

337 TABLE 11 DISTRIBUTION OF E N D O G E N O U S TYPE-C VIRUSES A M O N G INBRED STRAINS OF MICE The origins of inbred strains were derived from the following source: Bielschowsky and Goodall [250]. The designation ( + ) indicates that complete virus of the particular class has been isolated or that gene products specific for that class have been identified. Geographic origin

Boston, Mass., 1907

Inbred strain

Endogenous virus class 1

lI

II!

DBA

+

+

+

CBA

+

+

+

C3H/He

+

+

+

BALB/c

+

+

+

A/He

+

+

+

Cold Spring Harbor, NY

C58

+

+

N.T.*

Granly, Mass., 1921

C57BL/6

--

+

+

C57BL/10

+

+

+

Philadelphia, 1928

AKR

+

+

N.T.

Ohio, 1913

Switzerland, 1920

NIH Swiss

--

--

+

Rockefeller Institute, 1926

SWR

--

--

+

Imperial Cancer Laboratories, England

NZB NZW

-+

-+

+ N.T.

* N.T., not tested.

ment with mitomycin C under conditions which prevent further cell division does not markedly reduce the frequency of virus induction in response to IdUrd [157]. It has been reported that IdUrd treatment increases both the nuclear and cytoplasmic level of viral RNA, leading to the suggestion that this drug increases viral-specific RNA at a transcriptional level [158]. Once incorporated into DNA, halogenated pyrimidines may cause several biologic effects among which are mutation [160], DNA strand-breaks [161], and alteration of protein binding to DNA [162]. The high frequency of virus activation achieved with halogenated pyrimidines tends to argue against a mechanism involving mutation, especially since many other chemicals that induce mutations, do not cause type-C virus activation (ref. 156, and Aaronson, et al., unpublished observations). Teich et al. [156], reported that by increasing the incidence of DNA strand-breaks by exposure of IdUrd-treated cells to high intensity white light, they observed increased levels of virus activation. One explanation for these findings is that type-C virus induction by IdUrd may involve an excision mechanism. However, the fact that physical agents such as ultraviolet and X-ray irradiation which cause DNA strand-breaks either do not induce type-C virus or induce at only very low frequency

338 [156] indicates the need for further investigation. Whether I d U r d incorporation into D N A alters the binding of a control protein, analogous to repressor proteins in bacterial model systems [163], is as yet experimentally untested. However, knowledge gained f r o m studies of the mechanism o f action of inhibit0rs of protein synthesis as virus inducers (see below) makes this possibility an attractive one for further investigation. Several chemicals including cycloheximide, puromycin, and pactamycin, that inhibit different steps in protein synthesis, have been shown to be efficient inducers o f a type-C virus. These drugs are only active at concentrations that markedly inhibit cellular amino acid incorporation. Moreover, many metabolic inhibitors whose primary action is to inhibit cellular R N A or D N A synthesis do not cause virus induction. These findings argue for inhibition of protein synthesis rather than nonspecific disruption of cellular metabolism as the primary event in virus induction by these agents [151]. Unlike IdUrd, inhibitors of protein synthesis do not require cellular D N A synthesis for their action [157]. Further, these drugs do not become

I

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Fig. 7. Kinetics of cycloheximide induction of Class 11 endogenous virus and virus-specific RNA from BALB/c mouse cells. (A) Exponentially growing cultures containing 5 10~ Kirsten murine sarcoma virus-transformed BALB/c mouse cells were exposed to cycloheximide (10/~g/ml) (O); or cycloheximide plus actinomycin D (1 pg/ml) (11) for 12 h at 37 °C. At subsequent times, as indicated, cells were transferred for infectious center assay on NRK cells and the frequency of virus induction determined as previously reported [164]. (B) Exponentially growing BALB/3T12 cultures were exposed to cycloheximide (10/~g/ml) (O) or cycloheximide plus actinomycin D (1 /~g/ml) (11) as in Fig. 4A. At the times indicated, the concentration of type-C virus-specific RNA in cells was determined by hybridization of total cellular RNA to BALB: virus-2 [3H]DNA product as previously described [164].

339 incorporated into the cell DNA. This may be the reason for their much more transient disruption of the cellular control of endogenous viruses than that observed with IdUrd [151]. To study the mechanism of virus activation by inhibitors of protein synthesis, the levels of type-C virus-specific RNA in BALB/c cells following exposure to cycloheximide have been investigated [164]. As shown in Fig. 7A, transient virus release follows drug treatment. During drug exposure, there is an increase in the concentration of virus-specific RNA (Fig. 7B). This can be prevented by simultaneous exposure of the cells to actinomycin D. Since the total amount of RNA per cell does not change significantly during the course of drug treatment, this increase in virusspecific RNA reflects an absolute increase in viral RNA per cell. Simultaneous, but not subsequent, exposure of cycloheximide-treated cells to actinomycin D blocks type-C virus activation [164]. Thus, there is a de novo requirement for RNA synthesis during, but not after, exposure to cycloheximide and the evidence suggests that the required RNA species is that of the virus, itself. These findings are consistent with a mechanism, whereby inhibitors of protein synthesis block synthesis of a labile control protein that either inhibits viral RNA transcription or acts at a post-transcriptional level to degrade viral RNA. The rapid return of cells to the nonvirus-expressing state would presumably be due to the reaccumulation of the control protein following resumption of protein synthesis. There have also been reports that 2-deoxyglucose induces type-C virus from rat embryo cells [I 65] and that cyclic AMP can activate an endogenous virus of Chinese Hamster cells [166]. There is yet no information concerning how general are the inducing activities of these latter two chemicals nor their mechanisms of action.

IVC. Enhancement of Endogenous Type-C Virus Release by Steroid Hormones Glucocorticoids have been shown to enhance type-C virus release in response to both halogenated pyrimidines [167,168], and inhibitors of protein synthesis [168]. Unlike virus inducers that impair chronic virus release at concentrations at which they are potent activators of endogenous virus [168], steroid hormones also enhance type-C virus release by chronically producing cells. This suggests that steroids act to enchance rather than to initiate virus synthesis [168,169]. Recently Wu et al., [170] reported that these drugs affect type-C virus production post-transcriptionally since no increase was detected in the level of virusspecific RNA in steroid-treated cells. In contrast, with mouse mammary tumor virus (MMTV), another RNA containing virus whose release can be enhanced by steroids [171], treatment with dexamethasone is associated with an increase in the cellular level of MMTV-specific RNA [171,172]. These findings suggest an effect at the level of transcription of MMTV RNA. Thus, the mechanism of enhancement of type-C virus release by steroids remains to be further clarified. 1VD. Influence of the Differentiated State of the Cell on Type-C Virus Regulation Immune stimulation as in the graft-versus-host reaction and in mixed lympho-

340 cyte cultures can lead to release of either mouse cell tropic [173] or xenotropic [174] virus. In studies utilizing more defined antigenic stimuli, exposure of BALB/c mouse spleen cells to a B-ceU mitogen, lipopolysaccharide, has been shown to be associated with a specific and very marked stimulation of xenotropic virus production [175-177]. The biologic and immunologic properties of this virus are indistinguishable from those of the Class II virus of BALB/c cells [175]. A number of other mitogens, including concanavalin A and phytohemagglutinin, that are at least as active in causing blast0genesis, are much less effective or completely inactive in causing virus release [175,176]. By use of purified subpopulations of spleen cells, the major target of lipopolysaccharide-stimulated virus release has been shown to be the B-cell. T-cells, macrophages, and BALB/c embryo fibroblasts are poorly, if at all, inducible by lipopolysaccharide [176], suggesting that the differentiated-state of the cell is an important determinant of endogenous virus regulation. IVE. A Gene Affecting Expression of Class I Virus Fv-1 is a gene known to affect cell susceptibility to exogenous ,infection by many strains of MuLV [178,179] and, thus, is an important determinant of host susceptibility to leukemogenesis. The Fv-1 locus has been reported to be on chromosome 4 [180] and to possess two alleles, Fv-l" and Fv-1 b. Different mouse strains can be classified as N-type or B-type, respectively, depending on their Fv-I genotype [181]. Mouse type-C viruses which replicate preferentially in N-type mouse cells are said to be N-tropic, while viruses which replicate in B-type cells are B-tropic. This genetic restriction to virus was originally investigated by in vivo titration of the spleen focus forming activity of Friend leukemia virus [178,182] and more recently by the in vitro XC plaque assay [183]. In both assay systems similar dose-response relationships were initially reported. High titer, one-hit curves were obtained in Fv-1 susceptible hosts, and low-titer, two-hit curves were observed with Fv-1 restrictive hosts [183.184]. However, the two-hit nature of virus in nonpermissive cells has recently been called into question [185,186]. The mechanism of action of Fv-1 is not yet known. Genetic studies have indicated that restriction at this locus is dominant since in F1 crosses between N- and B-type mice, both N-tropic and B-tropic virus replication is restricted [178,181]. While the Fv-1 gene appears to act at step in virus infection beyond adsorption or penetration [187-189], it is not clear whether the restriction precedes or follows integration of the viral genome. The recent report of a soluble cell extract of nonpermissive cells, which can confer Fv-1 resistance to permissive cells [190], may provide a molecular approach toward understanding the mechanism of Fv-I restriction. Induction of embryo ceils of genetic crosses containing class I (N-tropic) virus and permissive for N-tropic viruses at Fv-1 results in the reproducible establishment of chronic virus production (Fig. 8A). In contrast, there is a reduced ability of induced class I virus to persist in Fv-1 nonpermissive cells [83,150,191]. Whether this restriction pertains only to virus spread or also involves the activation process remains

341

104 A

I

i

I

I

I

I

I

12

16

B

o/ /

~LL 103 o

> 102

(o Z

I

10'

4

8

12 16 4 8 TIME FOLLOWING TREATMENT (days)

Fig. 8. The effect of Fv-1 genotype on activation and persistence of different classes of endogenous virus. NIH Swiss × (NIH Swiss x BALB/cF1 clonal cell lines nonproductively transformed by Kirsten murine sarcoma virus and containing either (A) Class 1 endogenous virus; or (B) Class II endogenous virus were treated for 24 h with 20 #g/ml IdUrd. Culture fluids were then assayed at the times indicated for focus formation on NIH/3T3 (Class I virus) or NRK (Class II virus) as described previously [150]. The Fv-1 genotypes were Fv-l"" (©) or Fv-1"b (CD.

to be resolved. There does not appear to be any effect of the known alleles at Fv-1 on the activation and persistence of class lI virus. As shown in Fig. 8B, the magnitude of the initial burst of class II virus release is similar, but this virus fails to persist in embryo cells permissive for replication of N-tropic of B-tropic viruses [150]. " IVF. Genetic Control o f Class III Endogenous Virus Expression The genetic basis for the high spontaneous levels of class III endogenous virus release by cells of the NZB strain has been investigated. Embryo cells of F1 hybrids between NZB and strains such as N I H Swiss, that express high levels of class III viral antigens in the absence of detectable virus, release class III virus at an intermediate level, 5-20-fold lower than that of NZB parental cells. Analysis of virus expression in embryo cells of backcross and F2 generations involving NZB and N1H Swiss strains, has indicated a pattern of virus release consistent with that expected for segregation of a partially dominant gene restricting spontaneous release of class III virus by the N I H Swiss strain [155]. The mechanism of action of this gene is not as yet resolved. IVG. .4 Gene Conferring Susceptibility to Exogenous Infection o f Mouse Cells by Xenotropic Virus An effective restriction to virus infection can occur at the cell surface through a block to virus adsorption and]or penetration. Findings that murine sarcoma virus pseudotypes of xenotropic virus, which possess the xenotropic virus envelope, are absolutely restricted in their ability to induce focus formation in ceils of most inbred mouse strains [ 147,150], strongly suggested that restriction to xenotropic virus infection

342 occurs at an early step such as virus adsorption/penetration or virus integration within the cell genome. Unlike cells of known inbred strains, embryo cells of wild mice from several geographical regions have been found to be susceptible to focus formation by MSV pseudotypes of xenotropic virus [192,193]. Further, embryo cells derived from genetic crosses between mouse strains such as NIH Swiss or BALB/c and the wild mouse are also susceptible. These results suggested that the wild mouse possesses a dominant gene for xenotropic virus susceptibility. As a test of this hypothesis, the pattern of susceptibility to xenotropic virus infection has been examined in embryo cells of backcross and F2 generations involving the wild mouse and the NIH Swiss strain, The evidence obtained from these studies argues for segregation of a dominant gene of the wild mouse that confers susceptibility at an early step in xenotropic virus infection [193]. The absence of this allele in most, if not all, inbred strains appears to be a major factor responsible for the "xenotropic" host range of Class II and III endogenous viruses. IVH" Systemic Regulation o f Endogenous Type-C Viruses o f Mouse Cells

There is accumulating evidence that leukemogenesis associated with Class 1 type-C virus is influenced by one or more genes within the H-2 region of chromosome 17 of the mouse. Strains of mice possessing the H-2 k haplotype exhibit high incidences of lymphoid leukemia, spontaneously (AKR, C58) or following inoculation of exogenous Class I virus (C3Hf/Bi, C57BR). In contrast, strains with other H-2 haplotypes are generally resistant to leukemogenesis. In genetic crosses between H-2 k and H-2 b mice, resistance to Class I virus leukemogenesis is dominant and segragates with H-2 b [194,195]. Further, studies with recombinant H-2 haplotypes by Lilly have indicated that the portion of H-2 associated with susceptibility and resistance to leukemogenesis, designated Rgv-1, is toward the K end of the H-2 region [195]. One possibility is that Rgv-I is a specific case of an Ir-1 region effect. This is supported by the fact that genes of the H-2 ~ region, which are closely linked to H-2k, are known to be involved in determining the level of immune responsiveness to a variety of other antigens. However, other more complex explanations could also be invoked to explain these findings. Additional evidence has been recently obtained for host immunologic responses to their endogenous type-C viruses. By measurement of immunoprecipitation of radioactively labelled intact virus, antibodies to several type-C viral structural polypeptides have been detected in sera of many inbred strains of mice [196-198]. Further, mice of several strains have been shown to develop antibody to the major 70 000 tool. wt. envelope glycoprotein of class I virus following spontaneous activation of this virus in older animals [199]. Sera of many mouse strains have also been shown to possess high-titered neutralizing activities directed against xenotropic class II and III endogenous viruses [200]. However, recent evidence indicates that this activity differs from known immunoglobulins [201,202].

343

IVI. Biologic Regulation of Endogenous Type-C Viruses of Other Mammalian Species While knowledge of cellular controls affecting endogenous viruses of other mammalian species is less advanced, there is evidence to suggest that analogous restrictions may exist. For example, endogenous type-C virus of the rat cell is only transiently induced by halogenated pyrimidines; its kinetics of activation resemble those observed with I d U r d induction of mouse type-C virus [90,91]. Moreover, rat cells are relatively nonpermissive to exogenous infection by the induced rat type-C virus, further suggesting cellular restrictions to its replication. Similar to the Fv-1 restriction to Class I virus in the mouse cell, the rat cellular restriction can be partially overcome by infection at high virus multiplicity and by continued active cell growth [203]. The restriction to exogenous infection of their cell of origin for viruses activated from hamster, cat, and baboon species also appear to be partial rather than absolute, since low levels of virus replication can be induced at high virus multiplicity. That these same cells are fully permissive for growth of type-C viruses of other species argues for the specific nature of the restriction imposed upon these endogenous viruses.

MAMMALIAN SARCOMA VIRUSES - ANOTHER GROUP OF TYPE-C RNA VIRUS CONTAINING ENDOGENOUS VIRAL GENETIC SEQUENCES V.

VA. Biologic Properties The endogenous type-C viruses described in the preceding sections belong to a group, designated type-C helper viruses or helper-leukemia viruses. As summarized in Table III, sarcoma viruses constitute the second major group of mammalian type-C viruses. In contrast to helper viruses which do not cause visible alteration of cells in culture, sarcoma viruses induce foci of morphologically-altered cells in fibroblasts in vitro and the formation of solid tumors in vivo. While endogenous type-C helper viruses have been isolated from many mammalian species, natural TABLE III PROPERTIES OF MAMMALIAN TYPE-C RNA SARCOMA VIRUSES

Natural occurrence Isolates from mouse, rat, cat, and woolly monkey

Structure A. Single-stranded RNA B. Structural proteins conferred by helper leukemia virus

Functions A. In vivo - cause solid tumors B. In tissue culture - transform cells; mammalian sarcoma viruses require leukemia virus for their replication. Cells transformed by sarcoma virus in the absence of exogenous leukemia virus are designated "nonproducer cells"

344 isolates of sarcoma viruses are relatively rare. Available evidence indicates that at least a portion, and possibly the entire genome of the mammalian sarcoma virus, shares nucleotide sequences with cellular D N A of its species of origin. Hence, these viruses may be broadly classified as endogenous type-C viruses. Most sarcoma virus isolates have been obtained from mice or rats. They were initially discovered when solid tumors were observed in a rare type-C helper virusinoculated animal [204-206]. Such tumors were shown to release both a helper virus and a focus-forming virus. Subsequently, there have been spontaneous sarcoma virus isolates from the mouse [207,208], and cat [209,210], and one from a New World primate, the woolly monkey [211,212]. All mammalian sarcoma virus isolates to date have been shown to be associated with a type-C helper virus.

VB. Defective Nature of Mammalian Sarcoma Viruses Early studies indicated that murine sarcoma virus required the coinfection of a helper leukemia virus in order to induce focus formation [213,214]. This was in contrast to the pattern obtained with most tumor viruses, which exhibited a linear or "one-hit" relationship between the number of transformed colonies and the multiplicity of virus infection. It was later shown that murine sarcoma virus was not defective for transformation, but instead was defective in functions required for its replication as a competent infectious virus. This was initially demonstrated in tissue culture by the isolation of transformed cells, designated nonproducer cells, that were morphologically indistinguishable from sarcoma virus and helper leukemia virusproducing cells. In contrast to producer cells, these transformants lacked any detectable viral antigens and released no infectious virus. The sarcoma viral genome could, however, be rescued by the addition of exogenous type-C helper virus [215,216]. Findings that the host range and neutralization properties of the rescued sarcoma virus were identical to those of the helper leukemia virus added, indicated that at least the envelope properties of the sarcoma viral genome were conferred by its helper virus [216,217]. Recently, a temperature-sensitive mutant of murine leukemia virus, possessing a temperature-labile reverse transcriptase, has been isolated [218]. Evidence that the sarcoma virus pseudotype of this ts mutant is restricted at the nonpermissive temperature in functions required for transformation strongly argue that the helper viral reverse transcriptase is an additional function provided the defective sarcoma virus genome [27]. VC. Biochemical Evidence of the Endogenous Nature of Sarcoma Viral Genes In the initial biochemical analysis of the genome of a mammalian sarcoma virus, it was shown that a large part of the genome of the Kirsten strain of murine sarcoma virus lacked homology with its helper virus [219]. Subsequent studies demonstrated that another portion of this sarcoma virus shared genetic sequences with those of its helper virus [220]. Those sequences not shared with the helper virus, were found to be closely related to D N A sequences of the rat cell from which the sarcoma virus was isolated [220]. The Harvey strain of murine sarcoma virus, another

345 sarcoma virus isolate of the rat has been reported to have a similar genetic composition [221]. Investigation of the genetic composition of the Moloney strain of murine sarcoma virus, which originated in a mouse inoculated with mouse leukemia virus, has indicated that this sarcoma virus contains genetic sequences homologous to those of its helper virus as well as unrelated sequences shared with mouse cellular D N A (Frankel, personal communication). Sarcoma viruses of the woolly monkey and cat are not yet biochemically characterized, although it is known that the woolly monkey sarcoma virus contains information at least partially related to that of its helper virus [222,223]. Evidence obtained to date is consistent with the hypothesis that mammlian sarcoma viruses have originated as a result of recombination between a type-C helper virus and genetic sequences of cells, replicating that type-C helper virus. Whether the, as yet undefined, sarcoma virus-specific sequences represent those of another class of type-C RNA virus [221] or some other cellular information, coding for transforming function(s), remains to be resolved. In this regard, recent studies have indicated that the transforming sequences of avian RNA tumor viruses are of cellular origin [224]. Knowledge concerning the regulation of endogenous sarcoma viralspecific genes awaits further information concerning those situations where sarcoma virus-specific information is "normally" transcribed.

VD. Other Type-C Virus Isolates of Mouse Cells Several other mouse type-C viruses, which appear to be defective for their own replication, have biologic activities that differ from those of typical sarcoma viruses. Rauscher and Friend viruses induce progressive enlargement of the spleen and liver of inoculated animals after a relatively short latent period [225,226]. In contrast to conventional mouse leukemia viruses, which induce thymic tumors, Friend and Rauscher viruses induce a disease pathologically classified as erythroleukemia. Another mouse type-C virus, Abelson virus, induces lymphosarcoma in vivo [227, 228] and transforms cells of lymphoid origin in vitro [52,229]. The genetic relationship of the "transforming" sequences of the Rauscher, Friend, and Abelson viruses with those of known sarcoma viruses is currently under investigation in several laboratories.

VI. BIOLOGIC IMPLICATIONS OF ENDOGENOUS TYPE-C VIRUSES Investigations summarized in the preceding sections have established the genetic transmission of type-C viral information in a wide variety of mammalian species. Although not within the scope of this review, evidence of a similar nature has been obtained from studies with avian cells, representing a separate class of vertebrates (for review, see ref. 230). The methodology used to date to demonstrate the existence of endogenous viruses, will, with time, undoubtedly lead to the discovery of additional species in which these viruses naturally reside. The diversity of their

346 known hosts as well as accumulating evidence of virus persistence within a particular species over a long period of evolution pose obvious questions concerning the natural biologic role of these viruses. Two theories have been proposed concerning the origin and possible functions of type-C viruses. The protovirus theory [231] postulates that a reverse transcriptase allows cellular R N A to serve as a template for new DNA, that can become integrated into D N A of the same or adjacent cells. This process of R N A - D N A information transfer is thought to be involved in "inducing an orderly conversion of the other cells into the same or related differentiated state" [231]. In addition, it is postulated to have a role in other biologic processes such as memory and antigen recognition. Cancer, according to the protovirus theory, results from variation of the normal physiologic evolution of protovirus D N A either through its mutation or integration at an incorrect site in the cellular genome. A major prediction of the protovirus theory is that the creation of type-C virus occurs randomly and as a result of this information transfer. Thus, the protovirus theory argues against the existence of complete type-C virus within all cells. Evidence that the germ lines of many species contain information for complete type-C virus, that these viruses can be induced at high frequency from clonal lines, and that different isolates of endogenous virus of the same species are strikingly similar, all argue against this last idea. The oncogene theory [232] proposes that genetic information for type-C R N A virus (virogene) is present within all cells. Evidence summarized above, has established, in many species, the validity of this postulate. Further, in the original formulation of this theory [232]. Huebner and associates suggested a possible role of the endogenous virus in such processes as cellular differentiation. Another major prediction of this theory is that a portion of the virogene, the oncogene, contains information coding for transformation. The oncogene is seen as the universal mediator of the oncogenic effects of known environmental carcinogens, which are thought to act by disrupting cellular mechanisms that normally suppress the oncogene. The possibility exists that the oncogene may be expressed in the absence of the complete virogene. However, implicit in this theory is the prediction that the oncogene is a component of the complete virus [232]. Questions have been raised concerning this last postulate, especially among scientists working on environmental carcinogens that are known causes of cancer. The need to invoke a single common pathway (the type-C virus) for the actions of these agents appears to many investigators philosophically and scientifically unnecessary. If the oncogene were a part of the complete virogene, infection of normal cells with type-C virus or even transient activation of an endogenous virus might be expected to cause transformation. This, as yet, has not been found to occur. Another argument derives from the striking morphologic diversity of transformants that occur within the same clonal line spontaneously or following exposure to different oncogenic viruses and chemical carcinogens. It is difficult to ascribe such different transformed phenotypes to the action of only a single oncogene. An early criticism of the oncogene theory was that it was untestable. However, the development and

347 refinement of molecular hybridization techniques over the past few years now makes it possible to determine whether or not particular sequences of the endogenous virus are uniformly expressed in all tumors. In any further consideration of the biologic implications of endogenous viruses, it is important to emphasize the known detrimental effects to the host of uncontrolled type-C virus proliferation. As reviewed above, naturally and experimentally transmitted infectious type-C viruses, as well as the few endogenous viruses known to be infectious for the species in which they reside, are all causative of neoplasia. In the mouse, for example, chemically inducible viruses that replicate at high titer, have been demonstrated to induce tumors of the hematopoietic system [233,234]. Moreover, the magnitude of early virus replication is a good prognostic indicator of the likelihood of subsequent tumor development [235]. In view of the known detrimental effects of endogenous viruses, their elimination as a result of evolutionary selection pressures might be expected. However, as the weight of experimental evidence now indicates, these viruses have instead been conserved, and a variety of specific controls have evolved to regulate their expression. It is worth considering benefits that might be conferred to the host by retention of endogenous viral genes. As Temin postulated, it is not difficult to imagine a role of the reverse transcriptase in a number of physiologic processes. However, it must be recognized that the whole virus, and not just the reverse transcriptase, has been conserved within many species. Thus, there may be physiologic functions, in which the whole virus is involved. One such role could be in the transduction of genetic information. Each time an endogenous virus becomes integrated within the genome of another cell, it transfers its own genetic information. That the virus can transduce other genetic information also has some experimental support [219,220, 221,224]. A random process of information transfer mediated by the virus might be beneficial to the species if the target of this process were the germ cell. Here, the transduction of cellular information would provide additional genetic sequences upon which evolutionary selection pressures could be applied. Thus, the type-C virus might provide a mechanism for acceleration of evolutionary change. Type-C virus-mediated transduction of cellular information between somatic cells could be involved in well-organized processes like somatic cell differentiation. However, if information transfer in somatic cells occurred on a random basis, it would seem more likely to lead to disorganization of the cell and possibly even to loss of growth control and neoplasia. Evolutionary persistence of the endogenous virus might also be explained on the basis of a paradoxically beneficial role in the prevention of tumors. Experimental evidence indicates that the expression of type-C viral antigens by tumor cells can cause those cells to be much less malignant [221,236,237]. This appears to be due to increased immunogenicity conferred by the presence of the viral antigens. It is known that normal tissues of many species demonstrate subviral expression of their endogenous viruses [72,153,238,239]. Moreover, in some cases, the ability of the host to immunologically respond to endogenous viral gene products has been demonstrated

348 [196-199,240]. Thus, if in the course o f n e o p l a s t i c t r a n s f o r m a t i o n , the t u m o r cell expressed higher levels o f t y p e - C viral antigens, the virus might indirectly p a r t i c i p a t e in the i m m u n o s u r v e i l l a n c e mechanism leading to t u m o r rejection. Such a m e c h a n i s m m i g h t allow g r o w t h o f only those t u m o r cells expressing low level o f viral antigens. The b i o l o g i c significance o f e n d o g e n o u s viruses for h u m a n s awaits conclusive evidence as to whether such viruses exist in man. There have been several reports o f t y p e - C viruses isolated f r o m h u m a n cells [241-247]. However, each virus, t h a t has been characterized, has been f o u n d to be indistinguishable f r o m some k n o w n virus o f a n o t h e r species [248,249]. The present lack o f a t y p e - C virus o f h u m a n origin does n o t exclude the possibility o f its covert existence. I n fact, the d o c u m e n t e d presence of e n d o g e n o u s viruses in such a wide variety o f species inducing several higher primates, m a k e s it seem likely t h a t such viruses exist in h u m a n cells as well. In the event that an e n d o g e n o u s h u m a n virus is found, efforts to elucidate the cellular processes regulating e n d o g e n o u s virus expression a n d d e t e r m i n e the biologic functions o f these viruses in m o d e l systems s h o u l d be directly a p p l i c a b l e to a n a l o g o u s studies in man.

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NOTE ADDED 1N PROOF (see p. 330) Mouse cells also contain genetic information for endogenous viruses, that differ biologically from one another (see Section IV). Genetic studies have demonstrated that loci for induction of endogenous viruses representing two different classes segregate independently.