THE MURINE LEUKEMIA-SARCOMA VIRUS COMPLEX *

BY ROBERT J. IIUE1NER

LABORATORY OF VIRAL DISEASES, NATIONAL INSTITUTE OF ALLMEItY AND INFECrIoUs DISE -SES,

NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND

The development of in vitro (tissue culture) assay systems for murine leukemia(MuLV)l" 2 and sarcoma (MSV)3-7 viruses, the demonstration of the defectivenessof the MSV genome,3 and its rescue with various MuLV's7 have brought out cer-tain remarkable similarities between these viruses and those of the avian leukosis-Rous sarcoma complex.3' 7, 8, "a Subsequent studies of the murine viruses, whichI propose to outline briefly in this presentation, not only emphasize further theextent and significance of these similarities, but show also that the natural behavior,prevalence, and modes of spread of murine leukemia and sarcoma viruses followvery closely the patterns of the known avian tumor viruses.Avian Tumor Viruses.-During the many intervening years since Rous's discovery

of the Rous sarcoma virus (RSV) in 1911, much information has been obtainedconcerning the nature and behavior of the RNA tumor viruses. However, as istrue of all virus research, the modern era of RNA tumor virus research can be saidto begin with the development of in vitro test systems. Manaker and Groupereported altered foci in chick embryo cultures induced by RSV, following whichRubin developed a tissue culture test for the growth and assay of avian leukosisviruses (ALV).9' ga In reporting the resistance-inducing factor (RIF) test, Rubinshowed that ALV interfered with the focus-forming activity of PSV in chick em-bryo fibroblast (CEF) cultures.10 Arined with two simplified and reproducibleassay procedures, the focus-forming for RSV and the RIF for ALV, Rubin, Hana-fusa, Vogt, and others demonstrated the defectiveness of the Bryan strain of Roussarcoma virus,iOa 11 described its rescue in vitro with various leukosis viruses,12and defined to a considerable extent the natural history and immunological speci-ficities of the latter in infected chicken populations."3 13a

Subsequently my associates and I reported complement-fixing (CF) antigens inhamster tumors induced by the Schmidt-Ruppin strain of Rous sarcoma virus(SR-RSV). Using sera of tumored hamsters, we showed that these antigens werealso present in chicken sarcomas and in chick embryo cells infected with avianleukosis viruses. The CF antigens were not sedimented with virus particles andappeared to be soluble internal antigenic components shared by all the knownavian leukemia-sarcoma viruses.'4' 15 This latter finding provided the basis forthe development of the COFAL test (complement fixation for avian leukosis),'a test now widelv used for detection and assay of ALV infections in chickens.These observations stimulated further studies on the nature of the internal anti-

gens as structural components of the leukosis viruses;17 on their presence in "non-producer" avian and hamster sarcoma cells;'8-20 and on the transfer of the non-infectious RSV genome from sarcoma cells to normal cells by direct contact.21 22As a result of these studies it was soon established that ALV and RSV were similarto the influenza-like myxoviruses in that they contained anl internal group-specificantigen in addition to the outer envelope aiitigeim which had been shown earlierto be the determinant of serotype specificity. 12 This broad sharing of soluble virionantigens made it possible with the use of the COFAL test to detect, identify, and

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assay in tissue cultures all the established strains of ALV and many naturally oc-curring leukosis viruses as well.An in vitro Assay Systemfor Murine Leukemia Viruses.-The new in vitro methods

for detection and assay of the avian tumor viruses led in our laboratory to attemptsto develop similar procedures for murine leukemia viruses. An in vivo test de-veloped earlier by Rowe for certain murine leukemias based on interference with theovert effects of Friend leukemia virus in mice,23 while successful, proved to be im-practical for most purposes.

Since murine leukemia viruses were known to persist in cultures of leukemic tis-sue24 and to grow to a limited extent in covert fashion in normal mouse embryofibroblast (MEF) cultures,25 a test modeled along the lines of the COFAL testseemed to offer the best solution. Eventually, Hartley, Rowe, and I developedsuch a test; we found that sera from rats carrying transplantable lymphosarcomasinduced by the Rauscher and Gross leukemia viruses or from rats immunized bythese and other leukemia viruses grown in MEF tissue culture would react in CFtests with specific antigens present in MEF cells infected with these same viruses.This led to standardized procedures for producing specific rat antisera, for assayingmurine leukemia viruses in tissue culture, and for measuring CF and neutralizingserum antibodies to the latter. 2, 25The initial test procedure, which might be termed the "complement fixation test

for murine leukemia," or the COMuLV test, while useful for assaying the growthin MEF cultures of well-recognized strains of leukemia such as the Gross (G+)virus on the one hand and Friend, Moloney, and Rauscher (FMR) viruses on theother,26 was relatively insensitive for detecting naturally occurring leukemiaviruses.1 2 While the various immune rat sera used to detect viral growth containedCF antibodies to sedimentable infectious virus particles, they did not react withsoluble antigens that we suspected must also be present in the supernates. Thuswe were unable in our early studies to confirm in the MuLV's the presence of agroup-specific internal (CF) antigen similar to that found earlier in the ALV-RSVcomplex viruses. However, in 1966 Geering, Old, and Boyse26 reported a gel pre-cipitation test which revealed a group-reactive antigen released by ether treatmentfrom murine leukemia viruses, which appeared to be shared by both theG+ andthe FMR groups of murine leukemia viruses. We readily confirmed this observa-tion in complement fixation tests using G + rat sera provided by Old and sera of ratsin our own laboratory carrying tumors induced by the murine sarcoma virus (MSV).7

Prevalence of Murine Leukemia Viruses as Determined by the COMuLV Test.-Sera from inbred Fisher rats carrying fibrosarcomas produced by MSV and bypseudotypes of MSV developed CF and neutralizing antibodies to the correspondingstrains of virus.3' I Following several serial transplantations of the sarcomas intonewborn or weanling rats, neutralizing antibodies no longer appeared in sera andinfectious virus was less evident in the tumors.27 Complement-fixing antibodies,however, were frequently present, and some were highly reactive with antigenicpreparations of the well-known murine leukemia viruses and with preparations ofmany murine tissues and tissue cultures suspected to contain naturally occurringleukemia viruses.27With the use of highly reactive rat sera we found that MSV and MuLV prepara-

tions usually contained large amounts of a group-specific antigen separable by

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TABLE 1DEMONSTRATION OF SOLUBLE COMPLEMENT-FIXING" ANTIGENS IN MSV(RLV)

WITH THE USE OF MSV RAT ANTISERUM

1st Centrifugation

2nd Centrifugation without ether

2nd Centrifugation after ether RLQ

RStarting suspension"Upper supernatant5Lower supernatantPelletf

( PelletUpper supernatantLower supernatantPellet

(PelletUpper supernatantLower supernatantPellet

I Se IMSV

IRS (V)b (V + S) C16 16

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development of clinical leukemia or lymphoma might then be regarded as the re-sult of the additive effects of environmental factors such as physical and chemicalcarcinogens; thus such determinants would activate viral leukemias in the sameway that various other inciting agents precipitate other types of viral activity.Additional determinants would be infection early in life with high dosage of virusand/or genetically determined host susceptibility. On the other hand, oncogenicmutants might also influence the onset and frequency of the neoplastic expressionsof the leukemia-sarcoma viruses.

Recent Observations on the Murine Sarcoma Viruses.-Following the developmentof tissue culture assay systems for murine leukemia and sarcoma viruses, we demon-strated that the genome of the latter persisted in a defective noninfectious state intransplantable sarcomas induced in newborn hamsters by MSV.7 We found alsothat the defective genome could be rescued and rendered fully infectious whenMSV-induced hamster tumor cells (MSV-HT) were grown in mixed tissue cultureswith CHIEF cells which were superinfected with the Moloney, Rauscher, and Friendvariants of leukemia virus. The newly reconstituted pseudotype MSV's-MSV-(MLV), MSV(RLV), MSV(FLV)-were found to have the serological specificitiesof their helper leukemia viruses and to be as active in producing sarcomas in miceand transformed foci in MEF as the original MSV virus (Tables 4 and 5).More recently, other pseudotypes were produced by using tissue-culture-grown

Gross leukemia virus and a strain of leukemia virus derived from normal cells ofAKR newborn mice.29 Both pseudotypes-MSV(GLV) and MSV(AKRLY')-were derived by superinfection of mixed hamster tumor (MSV-HT) and MEF cellswith tissue-culture-grown preparations of the respective leukemia viruses. Neu-tralization tests in MEF tissue cultures using type-specific rat antisera describedpreviously" 2 revealed that despite certain differences MSV(GLV) and MSV-(AKRLV) were antigenically closely related to each other, but were quite distinctfrom the pseudotype MSV's induced by Friend, Moloney, and Rauscher viruses(Table 5). Sedimentation and treatment with ether showed that these pseudo-types also contained the group-specific soluble antigen described above. Theseand other investigations confirmed results obtained earlier showing that the MSVpseudotypes, like the RSV pseudotypes, carried type-specific outer envelopes identi-

TABLE 4INFECTIOUS VIRUS AND CF ANTIGENS IN EXTRACTS OF MOUSE SARCOMAS INDUCED IN

NEWBORN NIH MICE BY PSEUDOTYPE MOLONEY SARCOMA VIRUSESFocus- CF

Mouse Type of forming antigenPseudotypea Derivation passageb extracts titer titer Tumor InductiondMSV(MLV) Mixed TC + MLVe P0 Crude 103.1 4 NT NT4i4i P1 Conc. 105-0 16 13/13 8 days

it HT-1 + MLV (in vivo) P0 Crude 10'.7

ROBERT J. HUEBNER

TABLE 5ANTIGENIC CHARACTER OF MSV PSEUDOTYPES DETERMINED BY SERUM

NEUTRALIZATION TESTSNeutralizing Antibody Titera vs:

Rat antiserum MSV (RL) MSV (ML) MSV (FL) MSV(GL) MSV(AKR)Controlb Od 0 0 0 0Rauscherb 40 0 20 0 0Moloneyb 0 40 0 0 0Friendb 0 0 40 0 0Grossc 0 0 0 40 80AKRc 0 0 0 0 160C58c 20 20 20 320 320a Reciprocal of serum dilution giving 67% or greater reduction in number of MSV foci.b Sera of rats immunized with control tissue culture fluid or murine leukemia viruses grown in tissue

culture.c Sera of rats bearing transplanted tumors from leukemias induced by indicated viruses. The serum

versus the C58 (G +) virus was kindly supplied by Dr. Lloyd Old.d 0 =

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TABLE 6In vitro AND In vivo ATTEMPTS TO RESCUE MSV GENOME FROM HAMSTER

TUMOR (HT) CELLSHT MSV (-)

Tissue culture experimentsHT cells + MuLV-yield MSV (-)HT cells + MEF cells-yield MSV (-)HT cells + MEF cells + MuLV-yieldMSV (+)

In vivo experiments (NIH Swiss mice)HT cells only into NIH mice-yield MSV (-)HT cells + MuLV into NIH mice-yield MSV (+)HT cells into AKR mice-yield MSV (+)

MSV (8th passage, BALB/c); newborn hamsters; 9th-30th tissue culture passage of HT cells.MSV = Moloney sarcoma virus; (-) = virus-free; (+) = positive for MSV.MEF = cells cultured from special NIH strain Swiss mice.MuLV = murine leukemia virus (several strains).The NIH Swiss mice were MuLV-free.The AKR mice were positive for MuLV.

TABLE 7SIMILARITIES BETWEEN AvIAN AND MURINE-SARCOMA VIRUSES

Focus formationD)efective sarcoma genomeRescue with leukemia virusIn vitroIn vivo

AntigensType-specific envelopeGroup-specific internal

Replication by budding at plasma membraneHost range (determined by helper virus envelope)Pathogenesis determined by sarcoma genome, not by helper virusInterference by leukemia viruses with sarcoma virusTransfer of genome from nonproducer sarcoma cells to normal cells

without helper virus

Avian Murine+ ++ +

+ ++ ++ ++ ++ ++ ++ ++ +

tion neutralization test described earlier1' I provides perhaps the simplest procedureavailable for distinguishing the antigenically different murine leukemia viruses.

Since the AMLV-MSV internal (S) antigens are generally produced in higher titerthan the envelope (V) antigens, they are therefore easily detected in the COMuLVtest, an observation that, as described above, has greatly facilitated the detectionand assay of prevalent leukemia viruses.The murine leukemia viruses have been found by Sarma et al.32 to interfere with

MSV focus formation in tissue cultures, thus behaving very much like the ALV-RSV complex viruses. This observation was utilized to develop a standard assayprocedure for the known leukemia viruses. This new test appears to have a sen-sitivity nearly equal to that of the COMuLV test.

Recently we found that host and host cell susceptibilities to infection with variousMSV's are genetically determined. Cell penetration apparently is determined bythe envelope furnished by the helper MuLV. Thus the host range as well as thedevelopment of neutralizing antibodies in the infected host are helper-dependentproperties of the MSV's. The pathogenic behavior of the sarcoma genome, on theother hand, appears to be helper virus-independent in that the tumors produced byall the pseudotypes have the same cytopathic appearance. 0

General Considerations.-One of the more obvious yet frequently overlooked factsin considering causes of disease is that very few etiological agents behave exclusivelyin highly specific fashion. Studies of the whole "icebergs" including the hidden(subclinical) activities of most infectious agents show that the clinical manifesta-tions regarded as most characteristic nearly always represent relatively unusual

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events in the natural histories of such agents. More often thait not, therefore,epidemiological studies based exclusively on highly specific clinical manifestationsof widespread agents are likely to result in incorrect conclusions which hinder ra-tional control efforts.

In the past, misconceptions about the prevalence of virus infections in relation topoliomyelitis or respiratory diseases, for instance, were anything but helpful. Untilrecently, similar misconceptions about the rarity, specificity, and uniqueness ofanimal leukemia viruses may have obstructed progress in developing more usefuldetection and assay systems for these viruses. Similarly, recent emphasis on theincreasing incidence of lung cancer induced by chemical carcinogens found in to-bacco tars may have tended on the one hand to obscure the importance of such car-cinogens in causing other cancers and on the other the importance of other carcino-gens in lung cancer.

In addition, recent investigations suggest that multiple carcinogenic agents(physical, chemical, and viral) may combine to produce certain tumors.33 Carcino-genesis resulting from the interactions of two or more chemicals, two viruses, virusesplus chemicals, irradiation and viruses, are known to occur. However, almostnothing has been done as yet to determine to what extent such synergistic phe-nomena occur naturally in general populations. The recent development of in vitrolaboratory assay systems for viruses and environmental pollutants now should makesuch efforts possible.

Conclusion.--The newer in vitro techniques for detecting and assaying the leu-kemia and sarcoma viruses of chickens and mice have produced radical changesin previous concepts concerning the natural behavior of these viruses. Like manyother viruses, leukemia virus infection appears to be widespread, yet in most naturalcircumstances it rarely results in clinical disease during the normal lifetime of theinfected animal. Indeed, one might conclude from studies of the prevalence ofALV's and MuLV's that they represent the most common virus infections of themouse and the chicken. If this is so, as I pointed out previously for other patho-genic viruses, ecological studies of a limited or uncontrolled nature could be expectedto uncover such infections as frequently in association with good as with ill health,despite the fact that under certain natural conditions they do cause clinically ob-servable disease.34

Finally, with the tools at hand, meaningful field studies can now be done to de-termine more specifically the roles of the avian and murine leukemia-sarcomaviruses in producing leukemia and other neoplastic illnesses in their natural hosts.The many similarities exhibited by these "natural" models in two different classesof animals suggest that they may represent expressions of a general biological pat-tern likely to be expressed also in man and his domestic animals.

* This work was partially supported by the Etiology Area, National Cancer Institute, NIH.1 Hartley, J. W., W. P. Rowe, W. I. Capps, and R. J. Huebner, these PROCEEDINGS, 53, 931

(196.5).2 Rowe, W. P., J. W. Hartley, and W. I. Capps, NOCI Monograph No. 22 (1966), p. 15.I Hartley, J. W., and W. P. Rowe, these PROCEEDINGS, 55, 780 (1966).4 Moloney, J. B., in Some Recent Developments in Comparative Medicine (London: Academic

Press, 1966), 251.5 Harvey, J. J., Nature, 204, 1104 (1964).6Ting, R. C., Virology, 28, 783 (1966).

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7Hluebner, R. J., J. W. Hartley, W. P. Rowe, W. T. Lane, and W. I. Capps, these PNRocE:-INGS, 56, 1164 (1966).

8 Huebner, R. J., in Perspectivee in Leukemia, Proceedings of the Leukemia Society, Inc. (NewOrleans, La.: in press).

8a Huebner, R. J., in Carcinogenesis: A Broad Critique, Proceedings of the Twentieth AnnualSymposium on Fundamental Cancer Research (Houston, Texas, M.D. Anderson Hospital andTumor Institute, in press).

9 Manaker, R. A., and V. Groupe, Virology, 2, 838 (1956).9a Rubin, H., and P. K. Vogt, Virology, 17, 184 (1962).10Rubin, H., these PROCEEDINGS, 46, 1105 (1960).1Oa Vogt, P. K., and H. Rubin, Virology, 13, 528 (1961).11 Hanafusa, H., T. Hanafusa, and H. Rubin, these PROCEEDINGS, 49, 572 (1963).12 Ibid., 51, 41 (1964).13 Rubin, H., Bacteriol. Rev., 26, 1 (1962).18a Vogt, P. K., and R. Ishizaki, in Viruses Inducing Cancer-Implications for Therapy (Salt

Lake City: University of Utah Press, 1966), p. 71.14 Huebner, R. J., D. Armstrong, M. Okuyan, P. S. Sarma, and H. C. Turner, these PROCEED-

INGS, 51, 742 (1964).15Armstrong, D., M. Okuyan, and R. J. Huebner, Science, 144, 1584 (1964).16 Sarma, P. S., R. J. Huebner, and D. Armstrong, Proc. Soc. Exptl. Biol. Med., 115, 481 (1964).17 Bauer, H., and W. Schafer, Z. Naturforsch., 20b, 815 (1965).18 Vogt, P. K., P. S. Sarma, and R. J. Huebner, Virology, 27, 233 (1965).19 Kelloff, G., and P. K. Vogt, Virology, 29, 377 (1966).20 Payne, F. W., J. J. Solomon, and H. G. Purchase, these PROCEEDINGS, 55, 341 (1966).21 Hanafusa, H., and T. Hanafusa, these PROCEEDINGS, 55, 532 (1966).22 Sarma, P. S., W. Vass, and R. J. Huebner, these PROCEEDINGS, 55, 1435 (1966).23 Rowe, W. P., Science, 141, 40 (1963).24 Manaker, R. A., P. C. Strother, A. A. Miller, and C. V. Piczak, J. Natl. Cancer Inst., 25, 1411

(1960).25 Hartley, J. W., W. I. Capps, W. P. Rowe, and R. J. Huebner, unpublished data.26 Geering, G., L. J. Old, and E. A. Boyse, J. Exptl. Med., 124, 753 (1966).27 Huebner, R. J., W. T. Lane, H. C. Turner, and J. W. Hartley, unpublished data.28 Schaffer, F. L., and C. E. Schwerdt, in Viral and Rickettsial Infections of Man (Philadelphia:

J. B. Lippincott Co., 1965), p. 119.29 Hartley, J. W., W. P. Rowe, W. I. Capps, and R. J. Huebner, unpublished data.30 Igel, H. R., and R. J. Huebner, unpublished data.31 Lieberman, M., N. Haran-Ghera, and H. S. Kaplan, Nature, 203, 420 (1964).32 Sarma, P. S., M. Cheong, J. W. Hartley, and R. J. Huebner, Virology, in press.33 Symposium: Conference on Epidemiologic Approaches to Cancer Etiology, Cancer Research,

25, 1271 (1965).34 Huebner, R. J., Ann. N. Y. Acad. Sci., 67, 430 (1957).

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