endogenous c-type viruses in normal and â€œabnormalâ€šcell … · nomenon would be the...

[CANCER RESEARCH 37, 2957-2968, August 1977]

Summary

Endogenous C-type viruses were first discovered becauseof their association with cancer in birds. They have sincebeen visualized or recovered from cancers in many animalspecies. Their role in cancer of the human bladder hasrecently been suggested. They have been detected by electron microscopy in human tissues, but isolation of a confirmed human virus has not yet been achieved. Studies inmice form a basis for understanding these inherited virusesin mammalian species. Three classes of mouse C-type vimuseshave been identified by host range and serologicalstudies. One of these virus classes, exemplified by the xenotropic virus, is present in early embryos, in cells undergoing normal differentiation, and in cancer cells. The otherclasses, ecotmopic and amphotropic, are found primarily inanimals with pathological conditions. All these C-type vimusesare expressed spontaneously by cells at certain frequencies and titers during the lifetime of the mouse. Anonimmunogbobulin factor has been identified in normalmouse sera which specifically neutralizes the xenotropicvirus class of endogenous C-type viruses. Virus expressionthereforeappearstobe regulatedby intracellularaswellashumorab factors. These observations suggest that C-typeviruses play a role in natural life processes, particularlynormal maturation (embryogenesis and differentiation) andaging (autoimmunity and cancer). These viruses in micemay have counterparts in humans in whose tissues endogenous virus particles have been detected.

Introduction

Historically, viruses associated with many of the majorinfectious diseases of humans have been unwanted agents.Like bacteria, however, they can be latent in a host and notproduce disease. Moreover, just as certain bacteria may bepathogenic whereas others are vital for normal physiological processes (e.g. , digestion), viruses could play dualrobes. Some may be disease producing, whereas others mayprotect the species from invasion by certain virulent virusesor contribute to a necessary cellular function.

I Presented at the National Bladder Cancer Conference, November 28 to

December 1, 1976, Miami Beach, Fla. The research was supported by National Cancer Institute Grant CA13086, National Cancer Institute ContractCP43381, and a grant from the Council for Tobacco Research.

2 Recipient of Research Career Development Award 5K04 CA70990 fromthe National Cancer Institute.

In this century, viruses have been implicated in oncogenesis because of their recognized association with cancersin many animal species. Their involvement in human cancer, however, remains an important unanswered question.The factors involved in oncogenesis are complex, since theorganism has evolved several mechanisms to ward off thefinal event, i.e., cell transformation. Studying viruses as acause of cancer requires, then, the recognition that malignant transformation depends not only on a major change inthe physiological state of a cell, but also on other factorssuch as genetic makeup and degree of immunological competence of the host. Experiments conducted in animal systems have indicated that some inbred strains and immunologically depressed hosts are very susceptible to virus-induced cancer. Physical (X-ray, UV) and chemical factorsmay interact with viruses to produce the disease. Forexample, chemical or physical carcinogens could inducethe expression of viruses either inherited in the host genes(endogenous) or acquired (exogenous) by the host, and thisexpression may result in cell transformation. These samefactors might also depress the immunological response ofthe host. Alternatively, cells actively producing viruses maybe more susceptible to chemical transformation. Markedcellular alteration has been observed after methybchobanthrene treatment of cells chronically infected with mouseleukemia viruses (29, 81, 90). One could speculate thatbladder cancers result from chemical activation or potentiation of viruses present in the bladder epithelium. The recentobservation of virus-like particles in transitional cell carcinomas is therefore relevant (26, 28) (see â€œRNAVirusesâ€•).

Cancer is remarkably constant in its expression from yearto year (15, 47). If an exogenous source were the cause, onewould expect episodic fluctuations in the incidence of thisdisease. It appears, therefore, that cancer must occur primanly by an endogenous mechanism. C-type viruses, budding RNA viruses with lipoprotein coats, are inherited in thegenes of many species (i.e. , endogenous viruses) and havebeen implicated in cancers of several animals. They offer agood working model for studying the role of endogenousgenes in oncogenesis as well as other â€œpathologicalâ€•conditions. Moreover, since these C-type viruses have beendetected in developing embryos and normal tissues (61),they may also be involved in certain maturation processes.The incidence of cancer and autoantibodies increasessteadily as animals, including humans, age (59). Althoughthese conditions appear to be manifestations of â€œabnormalâ€•development, we believe they represent natural aging processes. Endogenous viruses, then, could be necessaryagents for the normal maturation and evolution of a species

AUGUST 1977 2957

Endogenous C-type Viruses in Normal and â€œAbnormalâ€•CellDevelopment1

Jay A. Levy2

Cancer Research Institute and the Department of Medicine, University of California Medical Center, San Francisco, California 94143

on July 25, 2021. © 1977 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

J. A. Levy

as well as for certain aging events that lead to its demise(Chart 1). Although indigenous to an animal species inwhich they may play an important role in early stages ofdevelopment, they might give rise to â€œabnormalâ€•conditionswhen passed to another species. These possibilities will beconsidered as we discuss the potential role of these endogenous C-type viruses in normal maturation (embryogenesisand differentiation) and aging processes (autoimmunity andcancer).

Virusesand Cancer

Viruses were first suggested as oncogenic agents in 1773when Peynilhe attempted experiments to prove that cancerwas causedby contagiousagents(38). Sanarelli,in 1888,considered that an infectious virus was responsible formyxomatosis, a highly contagious disease of rabbits (38).The 1st isolation of oncogenic viruses, however, did notoccur until the early part of this century when filterableagents were recognized as the cause of certain avian tumoms.These experiments in chickens, conducted by Ellenman and Bang (25) and by Rous (93), laid the foundation forthe study of cancer viruses. It took nearly 50 years, however,before the majority ofthe scientific community accepted thefact that cancer could be caused by a virus. Part of thestimulus for this acceptance came from Gross' isolation in1950 of an infectious cancer agent from mice (37, 38). Thisdiscovery gave credence to the idea of a mammalian oncogenic virus. In his experiments Gross used a highly inbredstrain of laboratory house mouse, the AKR, which spontaneously develops thymic lymphomas by 8 to 9 months ofage. He showed that filtered extracts from the spontaneouslymphomas gave rise to a similar disease in susceptiblemice. Among the important facts learned from these studieswerethat:(a)thegeneticmakeup ofthehostdetermineditssusceptibility to cancer, and (b) the virus responsible wasinherited vertically by the species (endogenous virus) andwas not acquired by horizontal (exogenous) means.

MATURATION PROCESS AGING PROCESS

LIPOPROTEINENVELOPECOAT

(type specificneutralizing

antigen)

Chart 2. Model of C-type virus. The locations of certain group- and typespecific antigens are shown. Reprinted with permission from the AmericanSociety of Clinical Pathologists (59).

EndogenousC-typeViruses

The viruses isolated from tumors in AKR mice and inchickens have been identified as RNA-contamning particles,and their integration as endogenous viruses in host DNA hasbeen well established (19, 38, 49, 94). They are classified asC-type by morphological criteria, on the basis of electronmicroscopic examination (Fig. 1) (11). These viruses have adensity in sucrose of 1.16 g/mI and a central RNAcontaining core that is surrounded by nucleopmoteins withgroup- and type-specific antigens (Chart 2) (104). Withinthis comeis located an enzyme, reverse transcniptase, whichafter infection makes a DNA copy from the viral RNA (6,106). By this means the viruses can integrate into the chro

mosome of the host and be passed to subsequent cellgenerations. The outer envelope of the C-type virus is composed of lipoproteins and glycoproteins on which resideboth group- and type-specific antigens (105). The latterantigens distinguish the individual C-type virus isolates ofvarious species. The virus replicates by budding from thecell surface. Lysis does not generally occur, so viable cellscan produce large quantities of virus after an establishedinfection.

Since the 1950's, C-type viruses have been identified byelectron microscopy or isolated directly from a wide varietyof vertebrate species (Table 1) (62). Although observed byelectron microscopy in several different human tissues, aconfirmed human C-type virus has not yet been isolated(see â€œRNAVirusesâ€•).

These viruses are generally passed to the species in thegerm cell (i.e. , endogenous virus), but horizontal (onexogenous) infection can also occur. As endogenous agents, theC-type viruses are usually found in multiple copies in the

RNA

(group specificantigen(s) gs)

EMBRYOGENEI IMUNITY

DIFFERENTIAT

Chart 1. Endogenous C-type viruses as double agents in normal lifeprocesses. Endogenous C-type viruses may be involved in life processesrepresenting 2 sides to development: maturation and aging. The host responseand the extent of virus or viral antigen expressionmay determinethe degree of normal maturation in the early Stages of development as wellas the type of condition occurring during the late stages, or processes ofaging.

2958 CANCER RESEARCH VOL. 37



C-type Viruses in Development

rus to pass in a latent form to subsequent cell generationsand maintain transformation. The oncogenic potential ofviruses is therefore enhanced when they can integrate easily into cellular DNA and when they do not destroy theinfected cell. For the latter reason, the RNA tumor viruses,which bud from the cell without lysing it, have better transforming capacities than do the DNA viruses which on replication usually kill the cells that they infect (Table 5). DNAvirus transformation is most efficient in cells that are nonpermissive for their replication or when the virus itself hasreduced meplicative ability. Integration can take place without cell death. Certain DNA viruses, such as members of theherpes group, have greater transforming potential after UVirradiation or inactivation by neutral red dye treatment (86).This fact has alarmed dermatologists who use neutral reddye to treat human herpesvirus infections. The applicationof these laboratory observations to clinical situations, however, requires further study.

Many scientists who link viruses to cancer believe theseagents act by carrying oncogenic information into a cell(exogenous infection) or by activating the cancer-producing gene(s) (the oncogene) already present in the cell (38,49, 62). Alternatively, the causative agent might be an integrated endogenous virus containing an oncogene that wasinherited through the germ cell (49, 62). This virus could beactivated by a variety of environmental factors includingexogenous virus infection. The prototype of this latter phenomenon would be the release of endogenous RNA C-typeviruses from mouse cells infected with the DNA-containingherpesviruses (39). The suggested requirement of a herpesvirus and avian C-type viruses for induction of Marek'sdisease in chickens could be another example of a concerted robeof exogenous and endogenous viruses in cancer(79). DNA and RNA viruses could then be either directly

Table 1Animal vertebrate species in which C-type viruses have been

detectedAnimals listed below are those in which viruses with C-type

morphology have been described. In some cases, the isolation ofthe virus has not beenachieved; in others, the classification of thevirusesasC-typeis not conclusive (seeRefs.62 and 78 for specificreferences).

MammalianCatCowDeerDogGuineapigChinesehamsterSyrian golden hamsterHorseMousePigRabbitRatSheep

Primates:BaboonChimpanzeeGibbonHumanMarmosetMonkey

Fig. 1. Gross AKR murine C-type viruses budding from cultured mousefibroblasts. x 40,000. Cells prepared and photographed by Dr. Ellen R.Dirksen, UCLA School of Medicine, Los Angeles, Calif.

Fig. 2. C-type particle budding from the trophoblast layer of a humanplacenta. x 75,000. Tissue prepared and photographed by Dr. Ellen R.Dirksen, UCLA School of Medicine, Los Angeles, Calif.

PiscesFish

ReptiliaViper

AyesChickenDuckPheasantQuailTurkey

AUGUST 1977 2959

.cD

genes of the host species (5 to 20 copies per haploid genome) (10). Their presence in some species probably datesback many million years ago (108). Some of these virusesmay have entered their host within the past 3 million yearsby exogenous infection (transspecies infection) (9). Endogenous virus production occurs spontaneously at differentfrequencies by various tissues and cells of the host (61, 68).Since these virus genes have been tightly conservedthrough evolutionary development and their expression appears well regulated by host genes, we believe that C-typeviruses, besides being oncogenic agents, may also havefunctions beneficial to the survival of the species (62).

OncogenicViruses

The concept of cancer viruses has now been widelyadopted, primarily because of the substantial experimentalwork with both DNA and ANA viruses in animal modelsystems. Tables 2 to 4 list those viruses that have beenimplicated in cancers in animals. Readers interested in details on each of them can refer to excellent reviews on thesubject (3, 38, 84, 85, 109). Biological and biochemicalstudies have indicated that cell transformation takes placeafter integration of the virus into the host chromosomes.Virus replication does not necessarily occur, but the transforming protein(s) is produced. Integration enables the vi



VirusHostTumorPapovaPapillomaCow,

human,rabbitWarts,

laryngealpapilbomas,

carcinomasPolyomaMouseTumors(rodents)Simian,

vacuolatingMonkeyLymphomas,virus(SV4O)sarcomas(rodents)BK,

JCHumanSarcoma (hamster)?Progressive multi

focal leukoencephalopathy (human)

Tumors(human)AdenovirusesBird

DogMonkeyCowHumanSarcomas

(rodents)PoxShopeRabbitFibromas,

fibrosarcomasMolluscumHumanSkinpapillomascontagiosumYabaMonkeySkin

nodulesOrfSheepSkinnodules

Herpesviruses and cancerTissue

culturecellVirusHostClinicalsyndromeTumorinductiontransformationLuckOFrogRenal

adenocarcinomaFrogMarekChickenNeumolymphomatosisChickenChickenHerpesvirus

sylvilagusCotton tailrabbitLymphomaRabbitâ€”HerpesvirussaimiriSquirrel monkey(New

World)Lymphomaand acute

lymphocytic leukemiaMonkeys,rabbitsMonkeyleukocytesHerpesvirus

ate/esSpider monkey (NewWorld)LymphomaMarmoset,

monkeyMonkeyIeukocytesGuinea

pig herpes-likevi Guinea pigLeukemiaâ€”Hamster, guineapigvirusleukocytesHerpes-like

virusCowLymphosarcomaâ€”â€”Herpes-likevirusSheepPulmonaryadenomatosisâ€”â€”Herpes-likevirusHorseâ€”?Hamstemlymphomaâ€”Epstein-BarrHumanBurkitt's

lymphomaOwl monkey,marmosetHuman

leukocytesHerpesvirus

type 2Human?Cervical carcinomaHamsterHamster, mouse,humanHerpesvirustype 1Humanâ€”â€”Hamster,mouseCytomegalovirusHumanâ€”â€”Hamster

J. A. Levy

musesinteract with endogenous C-type viral genes in thesehosts and thereby induce cancer must be considered.

HumanCancerViruses

In the past 20 years the association of specific viruseswith human cancer (Table 6) has been frequently discussed.

DNAViruses.Oneofthe 1Stassociationsofanyviruswithneoplasia came with the early experiments on human warts(38, 96). The wart virus, now considered part of the papovagroup of DNA viruses, was recognized as a filterable etiological agent in 1890 (96). Papillary tumors in humans mayalso be caused by papovaviruses as are similar neoplasias inother animal systems (85). A connection of wart virus withmalignant transformation, however, has not been shown.Recently, the BK virus, another human papovavirus, hasbeen implicated in human cancers of various origins including rhabdomyosarcomas and transitional cell carcinomas ofthe bladder (G. Mayorca, personal communication; Ref.27).Itsrobeinthisdiseaserequiresfurtherstudy.

The relationship of the herpesviruses, particularly Epstein-Barr virus and herpesvirus type 2, to human cancer isalso not yet clear. As a class of viruses, herpes has beenimplicated in cancer in several different animals (Table 3)(84). Some herpesviruses including cytomegaboviruses cantransform human cells in tissue culture (34, 35). The Epstein-Barr virus, associated with infectious mononucleosis,Bumkitt's lymphoma, and nasopharyngeal tumors, can induce human lymphocytes to divide indefinitely (35, 44).Moreover, it has given rise to conditions ranging from lymphocytosis to frank lymphoma in New World monkeys (99).These observations suggest that the Epstein-Barr viruscould produce human cancers under certain conditions.The relationship of herpesvirus type 2 to cervical cancer,however, is less convincing since these data are based onlyon serological (75) and limited biochemical information(92). A definitive robe for herpesvirus type 2 in any cancer,therefore, has not been established.

RNA Viruses.The endogenousRNAviruseshave beenimplicated in human cancer chiefly through electron microscopic studies and biochemical assays on a wide variety of

Table3

Table 2

DNA tumor viruses

oncogenic or could act as cocarcinogens in cell transformation. These viruses might also act as mutagens by integrating into parts of the cellular genome and disruptingnormal cell function. In this case they need not be transcribed, but their presence could initiate the transformedstate.

Some DNA viruses, nonpathogenic for their own species,cause cancer when transmitted to other animals. Humanadenoviruses, for instance, produce cancers in rodents buthave not been implicated in any cancer in man. Pobyomaand SV4Oviruses, papovavimusesof mice and monkeys, arerarely involved in tumors in these species but can transformhamster, mat,and human cells. The significance of theselaboratory observations to natural events remains to bedetermined. Moreover, the possibility that these DNA vi

(1 _ , not known.




VirusHostTumorTypeBMammaryMouse,

monkeyBreasttumorstumorType

CLeukosisChicken, mouse,Leukemia,viruseshamster,

cat, rat,cow, primate, viper,fishlymphoma,

Wilms'tumor (chicken

C-type)SarcomaChicken,mouse,cat,SarcomasvirusesprimatesType

DMason-PfizerRhesusmonkey (Old

World)?BreasttumorsM432MouseSMRVSquirrel

monkey (NewWorld)â€”

The viruses listed below havebeen implicated in humancancer.However,no conclusiveevidencethat any virus causescancersinman

has beenfound.VirusesCancerDNAPapillomaWart

CarcinomasBKCarcinomasHerpesEpstein-Barr

Bumkitt'slymphomaNasopharyngealcarcinomaHerpes

type 2 CervicalcarcinomaRNAB-type

MammarytumorC-typeLymphomasLeukemiasSarcomasCarcinomas

Characteristics of the oncogenic DNA and RNA tumorvirusesDNARNAProductive

infection with celllysis.Productive

infection leads totransformation. No celllysis.Abortive

infection beads to celltransformation with little orno virus production.Abortive

infection leads totransformation with no virus

production.Transformation

is inefficient(10@to 1O@virus particles/transformation event).Transformation

is fairly efficient(102 to 10@ virus pamticbes/transformationevent).Some

induce cellular DNAsynthesis.Induces

cellular DNAsynthesis.


Table 4RNA tumor viruses

budding particles were seen. Ubtrafibtrates of tumor homogenates and fluids from tumor tissues established inculture caused cytopathic changes in chicken and humancells. Chicken embryo cells (CEF) appeared to be mostsusceptible to the virus. Two-thirds of the patients had antibodies to the virus as assayed in CEF, whereas only 20% ofsera from control patients showed reactivity. The identityof the virus isolates has not yet been reported, but it ispossible that they represent endogenous C-type viruses ofchickens which were activated by the bladder tumor cellpreparations. Studies of human bladder carcinoma by otherworkers have not bed to the isolation of viruses (88). Thesignificance of the virus-like particles observed in thetumors remains, therefore, to be determined.

In summary, no virus isolate has fulfilled the necessarycriteria for a human oncogenic agent, although severallisted in Table 6 are under intense investigation. Mostadvances in this field have come from studies of animalmodel systems.@1_ , not known.

human cancers ranging from skin cancers to tumors of thebladder (5, 20, 23, 26, 28, 32, 42, 55, 71, 97, 100). Spiegelman et al. (100) maintain that the oncogenic genes in manyhuman tumors are acquired and not inherited. They havenoted, for instance, a new ribonucleoprotein particle inhuman tumors (5, 20, 42, 55, 100). Similar to endogenous C-type viruses, this particle has a density of 1.16 g/mI,contains reverse transcriptase, and has 70 S RNA, which insome cancers is partially homologous to the RNA of murineleukemia viruses (42, 55). Isolation of an infectious virus,however, has not been achieved. The recent identificationin acute myebogenous leukemia cells (31) and in humanembryonic kidney cells (77) of viruses resembling the primate and baboon C-type viruses requires further confirmation and characterization before one can conclude that theyare human tumor viruses. Particles similar to the mousemammary tumor B-type viruses have been detected in human milk, but also no connection of these particles tohuman disease has been shown (74, 97).

Virus-like particles resembling the endogenous C-typeRNA viruses have been described in transitional-cell carcinomas of the human renal pelvis, ureter, and bladder(26, 28). In 14 of 23 tumors, these virus-like particles were

observed by electron microscopy in the cytoplasm; no

Table 5

Table 6Viruses associated with human cancer

AUGUST 1977 2961

The Mouse Model System

For the understanding of various factors involved in human disease and other life processes, animal model systems have been very helpful, since inbred strains can beused and experimental procedures in vivo can be performed. The mouse has been particularly useful for examining the role of endogenous viruses in nature, since thisanimal develops diseases similar to those in humans andcontains at least 3 types of endogenous C-type RNA viruses.Classified descriptively by their host range properties (Table7), these C-type viruses share some proteins and nucleicacid sequences in common but can be distinguished fromone another by certain serological and molecular properties(16, 19, 40, 58, 102). These mouse viruses are probablyprototypes of similar endogenous agents in other animalspecies.

The mouse ecotropic C-type virus infects and spreadsreadily in cells of the species of origin (e.g. , the Gross AKRvirus in the mouse) (38). The name of this virus class wasderived from the Greek word for home, oikos, since this



J. A. Levy

virus has a tropism for cells of its â€˜â€˜homeâ€•species (59).ecotropic viruses from several other animal species havebeen identified (Tables 1 and 4). They differ among themselves in many ways, but they all productively infect cellsof their own species.

The endogenous mouse xenotropic (Greek: xenos, foreign) virus can productively infect cells from many foreignspecies including avian cells, but not mouse cells (Table 8)(58-61). Ecotropic C-type viruses of animals such as mice,rats, hamsters, and, recently, fish (78) were discovered because of their association with cancer. In contrast, thexenotmopic (X-tropic) C-type viruses were first recognized byelectron microscopic examination of nonmalignant mousetissue (24) and were first isolated from mouse embryo andkidney cells (69). Similar agents have now been recoveredfrom normal tissues from cats and baboons (7, 72) and maybe present in most animal species. These other X-tropicviruses differ from the mouse virus and each other, but theyhave the same restricted host range. The role of X-tropicendogenous viruses in nature is still unknown.

The amphotropic (Greek: amphos, both) virus, recentlydiscovered in wild house mice from the canyons of LosAngeles, has a dual host range (40, 87). It can infect not onlycells of its own species (e.g. , mouse), but also cells fromother mammals and even birds. Amphotropic viruses havethus far been described only in wild house mice. They mayrepresent recombinants or progenitors of the ecotropic andxenotropic viruses and may cause the leukemia, neurobogicabdisorders, or both observed in wild mice.

The variety of endogenous C-type viruses in mice atteststo their potential as normal physiological factors in manyphases of development including cancer. The ecotropicviruses have been considered the oncogenic agents. Exemplified by the isolate Gross obtained from lymphomas ofinbred AKR mice, these viruses spread easily through themouse and are associated with the bymphomas and leukemias which develop at 8 to 9 months of age. Ecotropicviruses can be recovered from many but not all strains ofhouse mice. Their spontaneous production by most othermice, moreover, is less than it is in the inbred AKR strain.Their robe in cancer in all mice, therefore, has been questioned (62, 65). The xenotropic viruses, although isolatedfrom some bymphomas (59), are often found associated withnormal mouse tissues including embryos and may representanother side to the function of C-type viruses in development; that is, agents of normal maturation (62). Yet, adefinite distinction in the robes of these different endogenous viruses cannot yet be made (62) (see â€œDiscussionâ€•).

Table 7Classesof endogenousC-typevirus

1. Ecotmopic (Greek: oikos, home, one's environment; Greek:tropos, turning).Viruses that infect and replicate efficiently in cells from theirown host species.

2. Xenotropic (Greek:xenos, foreigner).Viruses that infect and replicate efficiently only in cellsfrom an animal species foreign to the host.

3. Amphotropic (Greek:amphos, both).Viruses that infect and replicate efficiently both in cellsfrom their own host speciesand in cells from heterologousspecies.

Table8Animal cell lines susceptible to infection by mouse xenotropic

virusesSensitivity of the cells to xenotropic viruses was measured by

extent of focus formation and progeny production after injectionwith a standardquantity of the NZBxenotropic virus pseudotypeofmurine sarcomavirus (68).

XenotropicViruses

Mammalian

Avian

HumanLionMarmosetMinkMiopithicusMongoose(African water)Mongoose(black-footed)MuntjacOrangutanRabbitRacoonRatRhesusmonkey

The NZB mouse provided the 1st isolate of this class ofendogenous C-type viruses (69). The NZB mouse strain wasderived by Dr. Marianne Bielschowsky in the 1950's whenshe arrived in New Zealand and was encouraged by herhusband to obtain an inbred strain of mice. Taking agouticolored animals from the random-bred mouse colonies acquired some years before from England, she selected forblack coat color by brother-sister matings and derived theNZB strain (12). She and her colleagues subsequently demonstrated that this mouse had characteristics resemblingautoimmune disease in man, particularly lupus erythematosus and Sjogren's syndrome (12). Some animals developedimmunoblastic lymphomas (73). East et al. (24) and Melborsand Huang (73) were among the first to demonstrate byelectron microscopy that the cells from this mouse spontaneously produced barge numbers of C-type particles. Theysuggested that the virus was responsible for the diseasesyndrome in this strain. Although, initially, the NZB viruscould not be isolated as an infectious agent, it had themorphological characteristics of the ecotropic Gross AKRviruses. We demonstrated that this virus was infectious butthat it had an unusual host range, since it infected cellsfrom many foreign species but not from the mouse (59, 61,69).

This xenotmopic (X-tropic) virus was subsequently foundin several different laboratory mouse strains, includingnude mice, as well as wild house mice captured in Japanand California (1, 8, 61). Presence of the virus in nude micewas an important observation, since this immunologicallycompromised animal is used extensively for transplantationof heterologous cells. Each of the mouse X-tropic virusisolates shared similar antigenic properties, although somedifferences between them have been reported (16, 102). TheX-tropic virus was detected in tissues at various times during the life of a mouse. Cells of the NZB mouse had unu

AnteaterArmadilloBatBearCatChimpanzeeCowDeer(black-footed)DogGazelleGorillaGuinea pigHorse

DuckQuailParakeet

PheasantPigeonTurkey

CANCER RESEARCH VOL. 372962




sually high expression of this endogenous virus (61, 68). Allof its cells, both from embryo and adult tissues, spontaneously produced infectious X-tropic virus. Cells from otherstrains spontaneously released less virus than did NZB, andsome tissues did not express infectious virus at all (61).Strains, such as 129/J and SWR, produced little if anydetectable virus. Titers of virus produced by cells in cultureranged between 100 and 10,000 infectious particles/mb depending on the mouse and tissue tested. Individual cellsfrom a single NZB embryo, moreover, also differed 100-foldin the amount of X-tropic virus spontaneously produced(68). These observations have suggested an intracellularregulation of these endogenous viruses which operates atdifferent levels in NZB mouse cells and in cells from othermouse strains (68). It is dominant, since crosses of NZBmice with 129/J or SWR/J mice have given rise to hybridsthat are all producing substantial titers of virus (21).@Webelieve that this intracellular modification in X-tropic virusexpression is involved in the control of cell function (seeâ€œDiscussionâ€•),

C-type Virusesin Embryogenesisand Differentiation

Since high titers of X-tropic virus were recovered fromNZB embryos (68), we considered the possibility that theseendogenous viruses played a role in certain normal maturation processes (62). We therefore looked for C-type virusesin developing embryos of other mouse strains. Infectious Xtropic virus was detected in 10- to 15-day embryos andplacentas from NIH Swiss, C57L, and SJL/J mice (61). Noecotropic virus was detected. Like our observation withother tissues of mice, the bevelof X-tropic virus productionand the frequency of virus recovery were much less inembryos from other strains than in those from NZB mice.The identification of infectious virus in mouse embryos confirmed earlier reports of C-type virus antigen and virus partides in these tissues (48, 110). We have also looked atcultured mouse eggs at the 2-, 4-, and 8-cell stages and inmorulae and blastocysts. Our data thus far indicate that infectious X-tropic virus can be detected at the late morulaand blastocyst stage.4 This detection of X-tropic virus inblastocysts confirmed the electron microscopic observation by Chase and Piko (18) of C-type viruses in mouse eggsat this stage of embryonic development.

Hellman and Fowler (43) demonstrated the induction of C-type virus antigens in uteri of ovariectomized mice aftertreatment with estradiob. In collaborative studies with theseinvestigators, we isolated infectious X-tropic virus from theuteri of NIH Swiss mice during estrus and after stimulationwith estrogens. Uteri from ovariectomized mice did notyield detectable virus. These observations further suggestan importance of endogenous C-type viruses in the earlystages of development. Perhaps they stimulate rapid growthof the embryo. C-type viruses do enhance cell replication intissue culture (30). Perhaps these viruses enhance the cellto-cell contact necessary for implantation of an embryo.

3 J. Levy, J. Joyner, K. Nayar, and R. Kouri, Proc. Am. Soc. Microbiol.

(Abstract), p. 294, 1977.4J. Levy,P. Kazan,and M.Golbus,manuscriptin preparation.

Certain C-type viruses induce syncytial formation amongcells (82, 83, 95).

Studies of the mouse pancreas as it develops in vivo havealso suggested a robeof endogenous viruses in maturation.X-tropic virus was first detected in the pancreas of ICR miceat a stage when pancreatic differentiation began, not at thesomite stage.5

The observations of C-type viruses in placentas and embryos are not limited to mice. The viruses have been detected in the same tissues from other animal species including humans (Table 9) (7, 17, 51, 53). We have confirmed thepresence of virus-bike particles in the trophobbast layerof placentas from normal individuals as well as frompatients with lupus erythematosus (22, 52) (Fig. 2). Theseparticles occur more readily in placentas from women whoare multiparous (Ref. 22; M. L. Vernon, personal communication). The presence of these virus particles at the trophoblast layer, where maternal and fetal cells meet, brings tomind the enhanced C-type virus production noted duringgraft-versus-host reactions (46, 65, 98). Perhaps activatedby antigenic differences between mother and fetus, theseviruses are important in embryogenesis. An increasedchance of survival for embryos that are more dissimilar tothemotherhas been reported(54).

C-typeVirusesin AutoimmuneDiseaseand Cancer

We have discussed the recovery of infectious X-tropicC-type viruses from mouse tissues during embryogenesisand differentiation. Their isolation from mice undergoingautoimmune disorders and cancer has also been described.Evidence for any function of these endogenous viruses inthese maturation and aging processes, however, is nowbased only on their expression in the mouse during thesestages of development. In attempts at understanding themole they might play in nature, the consequences of aninteraction of C-type viruses with host cells should be considered.

Virus-Cell Interaction. As illustratedby Chart 3, bothvirus and cell can be modified by a virus infection. If a cellcontaining an endogenous C-type virus is infected by another C-type virus, phenotypic mixing often occurs. In thiscase, the genome of the viruses remains unaltered, but theiroutside envelopes are exchanged. Since this outer coatdetermines the host range of the virus, virus genes canenter cells previously resistant to them. Phenotypic mixingamong endogenous C-type viruses of mice (63) leads toecotropic virus genomes in the envelope of xenotropic vimusesand vice versa; xenotropic viruses carry ecotropicvirus coats. By this phenotypic mixing, oncogenic messages from ecotropic viruses can enter heterobogous cellsand give rise to cancers. We have, for instance, inducedsarcomas in ducks with a mouse sarcoma virus in an Xtropic virus coat.6

Phenotypic mixing can also occur between C-type virusesof different taxonomic classes. Under the proper conditionsin tissue culture, a mouse virus can acquire a chicken coat,

5 J. Levy, P. Kazan, R. Pictet, and W. Rutter, manuscript in preparation.

SJ, Levy,manuscriptin preparation.

AUGUST 1977 2963



C-type viruses andembryogenesisC-typeviruses have been visualized by electron microscopyorisolated

directly from the placenta and/or embryos of theanimalspecieslisted.Placenta

EmbryoMouseMouseRatRatRabbitBaboonRhesus

monkeyHumanMarmosetBaboonChimpanzeeHuman

J. A. Levy

Table 9 (61, 89). Perhaps they are responsible for some hemolyticsyndromes.

NeutralizingFactor

We have recently described in mouse serum a factorassociated with mouse lipoproteins which specifically neutralizes xenotropic and not ecotropic viruses (56, 67). Thisneutralizing factor is found at various levels in normal seraof nearly all house mice but is found at highest titers(1:10,000) in sera from NZB mice (61). The discovery of thisnonimmunoglobulin antiviral factor was exciting because itsuggested to us a mechanism by which the host couldmediate cell function. We have discussed above that intracellular factors probably control the extent of X-tropic virusexpression by mouse cells. Infectious virus production isnot required, in fact, for the appearance of virus envelopeglycoprotein on the cell surface (45, 57, 76). The neutralizing factor could bring about changes in cells by binding tothese virus antigens serving as receptors on the cell membrane (see â€œDiscussionâ€•).This system is analogous to interaction of protein hormones at the cell surface (91)

Discussionand Conclusions

Endogenous C-type viruses, originally discovered in cancers of chickens and mice, have now been isolated fromnormal tissues, developing embryos, and placentas. Theirantigens are readily found in early embryos as well as innormal adult tissues. These endogenous viruses appearconserved in nature for some benefit to the species, sincenormal evolutionary pressure should have led to their extinction. The original concept of them as strictly oncogenicagents should be modified to include their possible roles in

VIRUS@

and a chicken virus can acquire a mouse coat (64). Theoccurrence of this event in nature, however, needs to beevaluated.

Coinfection of a cell by 2 C-type viruses might also resultin mecombination in which a virus is produced sharing partof the genome of both viruses. Recently, a new type of virus,MCF, was recovered from bymphomas of AKR mice (41). Ithas antigenic properties of both xenotropic and ecotropicviruses and probably represents a recombinant. The delayin cancer in the AKR may be related to the time intervalneeded to generate this recombinant virus. MCF could berepresentative of other viruses which form from the interaction of the different classes of endogenous C-type viruses.The function of these new viruses would depend on theextent of recombination. Such modifications in virusescould also be responsible for somatic changes in cells otherthan transformation. Recombination among the chickenviruses (111) and among the mouse viruses (103) has beenreported. It is uncertain, however, whether genetic recombination could occur between 2 distinct taxonomicclasses of C-type viruses.

The cell may also be altered by C-type virus infection. Itmay transform and/or express viral antigens on its outsidesurface. The autoimmune syndrome of NZB mice could beexplained by a hypemproduction of C-type virus antigens onthe cell membrane (59, 61). If the mouse reacts against theviruses, as suggested by binding antibodies (50) and neutralizing factor(s) (56, 67), it might also affect the cell onwhose surface the virus proteins are located. A carrierhapten effect illustrated by the studies of Lindenmann (70)with influenza virus infection of Ehmlich ascites cells couldresult. Lindenmann demonstrated that tumor cells becameantigenic only after infection by the influenza virus. Thevirus obtained from the tumor was also capable of immunizing mice against the tumor, whereas the tumor alone wasnot immunogenic (70). Since the influenza virus, similar toC-type viruses, buds from the cell surface, this phenomenon of tumor rejection was explained by the presence onthe virus envelope of tumor cell antigens derived from thecell membrane. The host recognized the virus as foreignand, in mounting an immune reaction against the virus,responded as well against the tumor antigens previouslyunrecognized. In an analogous way, a hyperproduction ofthe endogenous C-type viruses could lead to an anti-selfreaction by the host and result in autoimmunity. C-typeviruses have been observed budding from ABC membranes

MODIFICATION

Chart 3. Virus-cell interaction. C-type virus infection of a cell may lead toalteration in the virus and/or the cell. The incoming virus may exchange Itsoutside envelope with that of a virus already present in the cell. The cell mayacquire antigens coded for by the incoming virus (presumably the virusenvelope glycoproteins) and/or it may become transformed.

IDENTIFICATION





ii)

Chart 4. Possible role of endogenous C-typeviruses in early and late stages of development.Endogenous C-type viruses were first discovered associated with malignant tissues (a). Recently, their presence has been noted in embryos, differentiating organs, and normal adulttissues (b) A more extensive role of these viruses in natural life processes should be considered.

other life processes. Certain speculations on their functionin nature can be made. Some C-type viruses, exemplified bythe ecotropic virus, might cause cancer. Others, exemplified by the xenotropic virus, might function in early development. One virus may give rise to the other, they may acttogether, or each virus may have dual roles. Non pathogenicecotropic viruses exist (66), and X-tropic viruses may beresponsible for the immunobbastic lymphomas associatedwith NZB, NIH Swiss, and C57L mice, strains that lackendogenous ecotropic virus expression (61, 62). Moreover,a virus that serves a beneficial function in its host of originmay be oncogenic in certain heterobogous hosts. The Xtropic virus may be present in cells as a virogene lackingtransforming potential, but with time (i.e., aging) it maypick up a â€œresidentâ€•oncogene and emerge as a canceragent. Likewise, virus spread which presents more opportunities for virus integration at certain sites in specific cellsand for virus: virus interactions may be necessary for transformation. Ecotropic viruses, such as the AKA virus, require8 to 9 months after their spontaneous production in themouse to induce lymphomas. The recent description of theMCF virus in AKR bymphomas (41), in fact, suggests that

recombination between endogenous viruses may be theprerequisite for cancer. The same process may lead toother kinds of somatic changes.

Our experiments suggest that viral proteins on the cellsurface may be receptors for the transfer of information tothe cell from its outside environment. Virus production appears controlled by intracellular mechanisms that act atdifferent levels even in the same mouse. The reason for thisvariability in virus expression remains to be determined. Insome situations, these viruses or their antigens may act asindicators of unbalanced growth and induce an immuneresponse against the altered cell (107). In others, externalcontrol of the cell may be mediated by a substance such asthe neutralizing factor, which could alter the cell by interacting with viral antigens when present on the cell membrane. Neutralizing factor may be a prototype of other factors which bind to cell receptors and influence cell function.Embryogenesis, differentiation, and other early stages ofdevelopment may occur with normal expression of the virus. Low or no virus expression may lead to poor development of the species. Hyperproduction of endogenous vimusesor their antigens by cells could lead to autoimmunityand/or cancer (e.g. , the NZB strain). Autoimmunity, in fact,may be an attempt of the host to eliminate transformed cells

expressing the virus antigen. Normal fetal development,therefore, could be mediated through the interaction ofneutralizing factor with viral antigens on the surface of theembryo (62). This kind of action is reminiscent of tumorenhancement (80). Moreover, 1 of the maturation antigenson the surface of thymocytes (13) could be of xenotropicvirus origin. Reaction of neutralizing factor with these thymocytes may be important for their normal differentiation. Useof purified neutralizing factor in cultures of developingembryos and differentiating tissues, such as the pancreas,should help elucidate these possibilities.

If the above concept is true, production of infecuousendogenous virus would not be required, and evolutionshould select for viral expression without significant virusreplication. Therefore, the small amount of virus-likeparticles which we and others have observed by electronmicroscopy or detected by biochemical assays in normaland malignant human tissues would be expected. Theymight represent human X-tmopic viruses which need not beinfectious to have a function. Alternatively, since they retainreverse transcriptase,7 they may be infectious for only aselect group of heterobogous cells.

The wide host range of xenotropic viruses and the ease ofphenotypic mixing suggest that these agents may also beresponsible for directly transferring information among thespecies and thereby affect evolution (4, 62). It may havebeen this phenomenon that brought endogenous virusesinto some mammalian hosts (4, 10). Moreover, certain genetic material from host cells, perhaps oncogenic, might bepicked up by these C-type viruses as they infect and replicate in cells from heterologous species. By this process anX-tmopicvirus might become an ecotmopic virus or vice versa(62).

In essence, our present information on endogenous C-type viruses is in a â€˜â€˜waxingmoonâ€•phase. The viruses werefirst discovered because of their association with cancer(Chart 4a). Now that new concepts and techniques haveenabled the recognition of other classes of these viruses innormal as well as malignant tissues, their mole must beconsidered in greater depth. As more light is shed on ourgeneral knowledge of C-type viruses, we believe their function will extend to maturation processes such as embryogenesis and differentiation (Chart 4b). Rapid growth, forinstance, appears to be a common characteristic of deveb

7J. Leong,J. Nelson,andJ. Levy,manuscriptin preparation.

2965AUGUST 1977



Acad.Sci.U.S.,71:3304-3308,1974.Datta, S. K. , and Schwartz, R. S. Genetics of Expression of xenotropicVirus and Autoimmunity in NZB Mice. Nature, 263: 412-415, 1976.Dirksen, E. R.. and Levy, J. A. virus-like Particles in Placentasfrom Normal Individuals and Patients with Lupus Erythematosus. J.NatI. Cancer Inst. , in press.Dmochowski,L., andGrey,C. E. ElectronMicroscopyof TumorsofKnown and Suspected Viral Etiology. Texas Rept. Biol. Med., 15: 704-753,1957.

24. East, J., Prosser, P. R., Holborrow, E. J., and Jaquet, H. AutoimmuneReactions and Virus-like Particles in Germ-free NZB Mice. Lancet, 1:755-757, 1967.

25. Ellerman, V., and Bang, 0. Experimentelle Leukamie bei Huhnemn.Zentr. Bakteriol. Parasitenk. Abt. I (a) Ref., 46: 595â€”609,1908.

26.Elliott,A.Y.,Fraley,E.E.,Cleveland,P.,Castro,A.E.,andStein,N.Isolation of RNA Virus from Papillary Tumors of the Human RenalPelvis. Science, 179: 393-395, 1973.

27. Fiori, M., and di Mayorca, G. Occurrence of BK Virus DNA in DNAObtained from Certain Human Tumors. Proc. NatI. Acad. Sci. U. S., 73:4662-4666, 1976.

28. Fraley, E. E., Elliott, A. Y., Castro, A. E., Cleveland, P., Hakala, T., andStein, N. Ribonucleic Acid Virus Associated with Human UrothelialTumors: Significance for Diagnosis and Treatment. J. Urol., 111: 378-381, 1974.

29. Freeman,A.E.,Price,P.J.,lgel,H.J.,Young,J.C.,Maryok,J.M.,andHuebner, R. J. Morphological Transformation of Rat Embryo CellsInduced by Diethylnitrosamine and Murine Leukemia Viruses. J. NatI.Cancer Inst., 44: 65-78, 1970.

30. Gabelman, N., Scher, W., and Friend, C. Alterations in MacromolecularSynthesis and Cellular Growth in Mouse Embryo Fibroblasts Infectedwith Friend Leukemia Virus. Intern. J. Cancer, 13: 343-352, 1974.

31. Gallagher, R. E., and Gallo, R. C. Type C RNA Tumor Virus Isolatedfrom Cultured Human Acute Myelogenous Leukemia Cells. Science,187: 350-353, 1975.

32. GaIlo, R. C., Miller, N. R., Saxinger, W. C., and Gillespie, D. PrimateRNA Tumor Virus-like DNA Synthesized Endogenously by RNA-dependent DNA Polymerase in Virus-like Particles from Fresh Human AcuteLeukemic Blood Cells. Proc. NatI. Acad. Sci. U. S., 70: 3219-3224,1973.

33. Gardner, M. B., Rasheed, S., Rongey, R. W., Charman, H. P., Alena, B.,Gilden, R., and Huebner, R. J. Natural Expression of Feline Type CVirus Genomes. Prevalence of Detectable FeLV and RD-i 14 GS Antigen, Type-C Particles and Infectious Virus in Postnatal and Fetal Cats.Intern. J. Cancer, 14: 97-105, 1974.

34. Geder, L., Lausch, R., O'Neill, F., and Rapp, F. Oncogenic Transformation of Human Embryo Lung Cells by Human Cytomegalovirus. Science,192:1134-1137,1976.

35. Gerber, P., Whang-Penn, J. , and Monroe, J. H. Transformation andChromosome Changes Induced by Epstein-Barr Virus in Normal HumanLeukocyte Cultures. Proc. NatI. Acad. Sci. U. S., 63: 740-747, 1969.

36. Gold, P., and Freedman, S. 0. Specific Carcinoembryonic Antigens ofthe Human Digestive System. J. Exptl. Med., 122: 467-481 , 1965.

37. Gross, L. Spontaneous Leukemia Developing in C3H Mice FollowingInoculation in Infancy, with AK-Leukemia Extracts, or AK-Embryos.Proc. Soc. Exptl. Biol. Med., 76: 27-32, 1951.

38. Gross, L. Oncogenic Viruses. New York: Pergamon Press, 1970.39. Hampar, B., Aaronson,S. A., Derge, J. G., Chakrabarty, M., Showalter,

S. D., and Dunn, C. Y. Activation of an Endogenous Mouse Type CVirus by Ultraviolet-Irradiated Herpes Simplex Virus Types 1 and 2.Proc. NatI. Acad. Sci. U. S., 73: 646-650, 1976.

40. Hartley, J. W., and Rowe, W. P. Naturally Occurring Murine LeukemiaViruses in Wild Mice: Characterization of a New â€œAmphotropicâ€•Class.J. Virol., 19: 19-25, 1976.

41. Hartley,J. W., Wolford, N. K., Old, L.J., and Rowe, W. P. ANewClassof Murine Leukemia Virus Associated with Development of Spontaneous Lymphomas. Proc. Natl. Acad. Sci. U. S., 74: 789-792, 1977.

42. Hehlmann, R., Kufe, D., and Spiegelman, S. RNA in Human LeukemicCellsRelatedto theRNAofaMouseLeukemiaVirus.Proc.NatI.Acad.Sci.U.S.,69:435-439,1972.

43. Hellman, A., and Fowler, A. K. Hormone-Activated Expression of the C-Type RNA Tumor Virus Genome. Nature New Biol., 233: 142-144, 1971.

44. Henle, W., Diehl, V., Kohn, G., zur Hausen, H., and Henle, G. HerpesType Virus and Chromosome Marker in Normal Leukocytes afterGrowth with Irradiated Burkitt Cells. Science, 157: 1064-1065, 1967.

45. Hino, S. , Stephenson, J. R., and Aaronson, S. A. Radioimmunoassaysfor the 70,000 Molecular-Weight Glycoproteins of Endogenous MouseType C Viruses: Viral Antigen Expression in Normal Mouse Tissues andSera. J. Virol., 18: 933-941 , 1976.

46. Hirsch, M. S., Black, P. H., Tracy, G. S. , Leibowitz, S., and Schwartz, R.S. Leukemia Virus Activation in Chronic Allogeneic Disease. Proc. NatI.Acad. Sci. U. S., 67: 1914-1917, 1970.

47. Huebner, R. J. The Endogenous Origin and Transmission of RNA ViralGenomes That Code for Cancer. Recent Results Cancer Res., 54: 63-


J. A. Levy

oping embryos and cancers. If a C-type virus were thestimulus for this growth, their expression in these seem- 21.ingly opposite stages of development would not be surpris- 22.ing. Embryonic antigens in animal tumors including humanones, in fact, may be of viral origin (14, 36, 101). In this 23sense, these viruses serve as double agents producing bothbeneficial and detrimental effects as they function in earlyand late developmental stages (62) (Chart 1). A paradoxcould be involved, then, in trying to prevent cancer byantiviral approaches. Any attack on the virus might lead toserious disturbances in other life processes.

Acknowledgments

The author thanks Eleanor Haas, Dr. Joanne Leong, and Dr. Stuart Levyfor their critical review of this manuscript.

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1977;37:2957-2968. Cancer Res Jay A. Levy DevelopmentEndogenous C-type Viruses in Normal and ''Abnormal'' Cell

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