oncogenesis herpesviruses and

Proc. Nat. Acad. Sci. USAVol. 69, No. 4, pp. 1056-1064, April 1972

Herpesviruses and Oncogenesis

GEORGE KLEIN

Department of Tumor Biology, Karolinska Institutet, Stockholm, Sweden

ABSTRACT Herpesviruses have been implicated inoncogenesis. They have been found to be etiologic agentsin certain chicken, primate, and frog neoplasms. Thereis suggestive evidence that herpesviruses may contributeto the genesis of certain human lymphomas.

Only a few years ago, herpesviruses were believed to lackoncogenic activity. This situation has changed radically,mainly as a result of the following developments:

(i) The Epstein-Barr virus (EBV), a previously unknownmember of the herpes group, has been discovered (1) in cellcultures derived from Burkitt's lymphoma (BL), identifiedas a causative agent of infectious mononucleosis (2, 3), andis strongly suspected as a possible causative or contributoryagent to the genesis of certain human lymphomas (4, 5);

(ii) Marek's disease, a highly infectious chicken lympho-matosis, was conclusively linked with a herpesvirus (MDHV)as the causative agent (6, 7);

(iii) A herpesvirus isolated from squirrel monkeys, H.saimiri, (HVS) invariably causes lethal lymphatic leukemiaor reticulum cell sarcoma in a number of primates (8, 9);

(iv) A herpesvirus is the most probable etiological agentof the Lucke frog kidney carcinoma (10);

(v) More tentative suggestions have been made aboutpossible links between herpes simplex type 2 and human cer-vical carcinoma (11) and between EBV and nasopharyngealcarcinoma (12-14).In this brief review, the human EB virus is considered first.

This is followed by some potentially relevant aspects of thework on MDHV, HVS, and some of the other potentiallyoncogenic herpesviruses. Some general considerations of theway in which herpesviruses may act as oncogenic agents con-cludes the article.The Epstein-Barr virus (EBV)This agent was first demonstrated by electron microscopy(1) in a lymphoblastoid cell line derived from Burkitt's lym-phoma. Subsequent antigenic studies (15) have shown thatit is not identical with any previously known herpesvirus.Addition of virus-carrying, irradiated cells (16) or of viralconcentrates (17) to normal adult peripheral white cells orto lymphoid cells of fetal origin (18, 19) converted the recipi-ent cells into established lines capable of indefinite prolifera-tion as stationary suspension cultures. Adult peripheral whitecells may convert into such lines without the addition of ex-traneous virus or of virus-containing cells, but the derivedlines carry EBV as a rule (18, 20). Unless EBV or an EBV-source was added, fetal lymphoid cells could never be estab-lished as lines, in spite of the very persistent efforts of workers

Abbreviations:EBV, Epstein-Barr virus, MD or MDHV, Marek'sdisease herpesvirus; BL, Burkitt's lymphoma; HVS, herpesvirussaimiri.

who were regularly successful in establishing cell lines fromadults (18, 19, 21). In addition to reaffirming the importantrole of EBV for the continuous propagation of human lym-phoblastoid lines, this experiment also argues against verticaltransmission.The relationship between EBV and infectious mononucleo-

sis was discovered (2) when a laboratory technician whoseserum was negative for anti-EBV antibodies developed thedisease and subsequently became seropositive. In a subsequentretrospective study, the sera of students, stored in the YaleUniversity serum bank, sampled before and after they hadinfectious mononucleosis, were compared for antibodies toEBV. All sera taken before the disease were negative, whereasthe sera of the same donors after they had had infectiousmononucleosis were positive. The etiological link suggestedby these studies was conclusively established by a subsequentstudy (3). College students with positive anti-EBV titerswere protected from infectious mononucleosis during obser-vation periods of 4-8 years, whereas 10-15% of the seronega-tive students contracted the disease and became seropositive.This implies that antibodies against EBV can protect againstinfectious mononucleosis.The age-dependent distribution of anti-EBV titers in re-

lation to socioeconomic groups, studied in normal popula-tions in various countries by numerous investigators, hasalso elucidated the reasons why infectious mononucleosis isessentially a "college disease." Anti-EBV titers are high inthe newborn, due to the passive transfer of antibody fromthe mother. This is followed by a rapid decline during thefirst 6-12 months. The first major rise in the incidence ofseropositiv-e children occurs around the age of 4-5 years. Inthe low socio-economic groups seroconversion continues pro-gressively, and 70-80% of the population becomes positivebefore puberty. In the high socioeconomic groups, there is a

plateau, and a second rise occurs in the later teens or in earlyadolescence. It seems that primary EBV-infection of seronega-tive adolescents is required to produce the symptoms typicalof classical, heterophile infectious mononucleosis, although"silent" seroconversions, without recognized disease, can beregistered in adolescents, although with a lower frequency(3). It is not known whether the infection of young childrenleads to any particular disease syndrome; relatively mild,noncharacteristic forms of infectious disease may well occur,but typical infectious mononucleosis is absent.The relationship of EBV to Burkitt's lymphoma (BL) is

more difficult to assess. All African BL patients with histo-logically confirmed disease were seropositive, most of themat high titers (4). There was an 8-fold difference in the geo-metric mean titer between the BL patients and African con-

trol populations of similar age and sex distribution. Patients

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Herpesviruses and Oncogenesis 1057

with many other neoplastic diseases, including lympho- andmyeloproliferative malignancies failed to show similarly reg-ular or high antibody titers, with the possible exception ofthe lymphocyte-depleted form of Hodgkin's disease (22, 23),poorly differentiated lymphocyte-lymphoblast lymphoma(24), and, the most notable exception of all, nasopharyngealcarcinoma (12-14). Hypo- and oropharyngeal carcinomasor other head and neck malignancies, including tumors local-ized to the nasopharynx, showed no significant elevation ofanti-EBV titers (25). The evidence concerning sarcoidosisis conflicting; there is clearly some elevation of titers, butit is less regular and more moderate than in BL (26, 27). Acuteleukemias show no elevation of anti-EBV titers; on the con-trary, the mean titers are lower than that expected in a con-trol population (28).

Hybridization techniques for the detection of EBV-derivednucleic acid are still in the early stage of development, butthere already are interesting results. First, DNA-DNA hy-bridization was done, with purified EBV-DNA (29, 30), butmore recently (31, 32) complementary RNA has been syn-thesized in vitro on purified EBV-DNA and used for detec-tion of homologous sequences in various cells. Both methodsshowed that so-called "EBV-negative" human lymphoblas-toid lines, including Raji as the most extensively studiedprototype, carried multiple copies of the viral genome (29,31, 32). None of the viral products demonstrated by immuno-fluorescence tests can be detected in this line or in severalother similar lines, although they carry a complement-fixingEBV antigen (33-35) and a soluble antigen demonstratedby immunoprecipitation (36). In fact, no human lympho-blastoid lines have been found so far that do not carry EBV-homologous nucleic-acid sequences. Human cell lines of otherorigin did not give any evidence of EBV-DNA. The presentpicture would therefore be compatible with the assumptionthat human lymphoblastoid cells cannot grow in continuousculture unless they carry the EBV genome, but more lineswill have to be tested for a definite conclusion.

Studies on tumor biopsies (30) have shown that EBV-DNAis consistently associated with BL and nasopharyngeal car-cinoma in vivo, whereas a number of "control tumors," derivedfrom EBV-seropositive patients with other malignancies,gave no evidence of detectable EBV-DNA associated withtumor.

Corroborative evidence, indicating a more regular associ-ation of the virus with BL and nasopharyngeal carcinomathan with other tumors occurring in seropositive patients,has come from a study on the effect of local irradiation oftumors on various EBV-associated antibody titers (37, 38).Irradiation of BL and nasopharyngeal carcinoma in vivo hasled to an increase of antibody titers against the EBV-deter-mined membrane antigen and early antigen, whereas theirradiation of various other tumors has failed to do so. Viralactivation followed by antigen release or simple antigen re-lease due to tumor disintegration may be possible explana-tions of this effect.What is the relationship between the virus and the Burkitt

lymphoma cell in vivo? Virus particles and viral capsid anti-gens are not found in biopsy specimens. After explantation,capsid antigens regularly appear in 3-5 days (39). The ma-jority of the established BL-derived cell lines that emergeare still "virogenic," i.e., a small number of cells keeps pro-

cells making at least certain viral proteins can be increasedby arginine deficiency (40), x-irradiation, or small doses ofcertain synthetic inhibitors, such as mitomycin C, IrdU,BrdU, actinomycin D, or cytosine arabinoside (41, 42).Not all virogenic lines release infectious virus. The best

source of the virus is P3HR-1 (43), a clonal isolate from thewidely used Jijoye line (44). In this line, the virogenic cellsproceed all the way to production of infectious particles.

Several other lines proceed only to the formation of viralcapsid antigens, but release little or no infectious virus. Stillother lines contain cells making the early products of the viralcycle (membrane antigen, and/or early antigen), but few orno cells with viral capsid antigen. Such lines may be desig-nated as "abortively virogenic" (Klein, G., to be published).The reasons for this abortiveness are unknown; it appearsthat the cells die before they reach the later stages of the viralcycle. Parallel immunofluorescence and autoradiographyhas shown that the appearance of early antigen leads to a

progressive inhibition of synthesis of host cell macromole-cules in abortively infected cells (5). Even in the virogeniclines, viral capsid antigen and viral particles usually occur

only in degenerating and dying cells (46, 47).Cloning of the lymphoblastoid lines gives rise to progeny

with characteristics closely similar to the parental line (48-51).Virogenic lines give rise to virogenic clones as a rule, activeat about the same level. Nonvirogenic lines yield nonviro-genic clones as a rule. Occasionally, however, negative clonesgive rise to virus-positive progeny (51). It has been concludedthat all BL-derived lines carry an inherent potential for EBVproduction and that the rate at which this potential is in-duced determines the viral positiveness of a particular line.Obviously, there must be a difference between the virogenicand nonvirogenic lines with regard to the association betweenthe viral genome and the cell genome, or the way in whichthe viral genome is repressed or activated, or its completeor defective nature.

Obviously, the only truly representative malignant BLcell is the in vivo tumor cell itself. It is, therefore, very im-portant to consider its viral antigen status. With rare excep-tions, early antigen and viral capsid antigen are not foundin cells from biopsy (39); however, EBV-associated mem-

brane antigens are regularly found in biopsies (52-54). Sincethey appear on the majority of the cells, it may be assumedthat they are compatible with continuous cell proliferation.The relationship of membrane antigen to the viral cycle was

studied (55) in Raji cells abortively super-infected with EBV-concentrates (56-58). Membrane antigen appeared early,simultaneous with or before early antigen (57), and in morecells (55). Inhibition of DNA synthesis by cytosine arabino-side or IrdU did not prevent its appearance. Combined im-munofluorescence and radioautography indicated (45) thatthe appearance of membrane antigen was compatible withcontinued synthesis of host cell macromolecules. These find-ings do not disqualify membrane antigen from playing a roleanalogous to the membrane-associated antigens present onvirally induced experimental neoplasms. Such antigens are

regularly exposed on the outer surface of tumor cells inducedby both RNA and DNA viruses. They are group-specific,i.e., identical or crossreactive for all tumors induced by thesame virus; they can induce rejection reactions in vivo ca-pable of dealing with at least a limited number of syngeneic

ducing viral proteins and viral particles. The frequency of

Proc. Nat. Acad. Sci. USA 69 (1972)

tumor cells induced by the same virus (for reviews see 59-


62). In the EBV system it is puzzling, however, that mem-brane antigen is also a component of the viral envelope (63,64). Envelopment is one of the latest events in viral develop-ment, and the synthesis of envelope components may be ex-pected to be a late event in the cycle. As a possible analogywith the small oncogenic DNA viruses, such as polyoma orSV40, one may have expected, furthermore, that membraneantigens characteristic for the virally transformed cell wouldnot be present on the virion. It must be remembered, however,that polyoma and SV40 are nonenveloped viruses. Althoughthey change the cell membrane, as reflected by the appearanceof the distinctive transplantation-type antigens, there is noparticular reason why these antigens should become associatedwith the unenveloped virus particle. With an enveloped virus,the situation may be quite different. Both the outside and in-side membranes may be changed (65). At a later time, whenenvelopment occurs, the virus would take on the changedmembrane.The possible importance of virus-induced membrane

changes for the oncogenic function of herpesviruses has beenemphasized by Roizman (66). Long before any herpesviruseswere suspected of any such function, he showed (67) thatdifferent mutants of herpes simplex virus, characterized byslightly different envelope proteins, insert their distinctiveproteins into the outer plasma membrane of the infected cell.At the same time, the "social behavior" of the infected cellschanges in a way that is characteristic for each mutant.Mixed infection with two mutants may lead to the predomi-nance of membrane proteins from one virus type; the change insocial behavior follows the corresponding pattern.

Cells infected with lytic herpesviruses, such as herpes sim-plex virus, invariably die. It is noteworthy, however, that UV-inactivated herpes simplex virus type 2 was recently reportedto induce malignant transformation in hamster fibroblasts(68). The transformants grew as highly malignant tumorsafter inoculation into hamsters. The sera of the tumor-bearinganimals contained antibodies that neutralized the virus andreacted with the membrane of the transformed cells (69).Indirectly at least, this suggests that part of the viral genomemay be present in the cells and is perhaps responsible for theirmalignant behavior. It may be recalled that UV-inactivatedherpes simplex virus was also found capable of introducing avirally determined enzyme (thymidine kinase) into anenzyme-deficient cell line maintained on selective medium(70).There are other reasons why membrane changes in tumor

cells deserve attention, particularly when they are due to anoncogenic virus or to a virus regularly associated with thetumor. Growth-regulating impulses, including humoral, longrange and contact, short range signals, have to be transmittedthrough membrane-associated receptors. The outer membraneof tumor cells is reportedly changed in a number of ways. Adisruption of normal membrane architecture due to theinsertion of viral-determined products may, if compatible withcontinued cell life and proliferation, create a state of de-creased responsiveness to growth control, i.e., neoplasia.

Suggestive as these arguments may be, they are obviouslyinconclusive as far as the etiology of BL is concerned. Thepossibility remains that EBV is merely a passenger in thetumor, although it is difficult to see, in that case, why itshould not show a corresponding association with other

EBV is irrelevant for the genesis of BL, a number of seronega-

tive patients would be expected. In the young age groups atrisk, there is a substantial proportion (10-15%) of seronega-

tive children, even in the high endemic regions of Africa.These considerations, together with the proven ability of thevirus to cause at least a self-limiting lymphoproliferative dis-ease in vivo and its remarkable tendency to stimulate lymphoidcell proliferation in vitro, are at least in line with an oncogenicpotential. The regular presence of viral nucleic acid in thetumor cells is also consistent with this possibility. Perhaps themost important argument comes from the analogies with theoncogenic animal herpesviruses, discussed below.

If EBV is involved in BL in a capacity more important thanthat of accidental passenger, it will be necessary to understandthe difference between the infectious process that leads toseroconversion, without disease, or to infectious mononucle-osis, or to malignant lymphoma. One possibility is that dif-ferent virus subtypes are responsible. There is no evidence foror against this; the absence of fully permissive target cellsin vitro has inhibited a search for viral mutants, and the anti-genic studies so far performed tended to emphasize the com-

mon features of the virus carried in lymphoblastoid lines ofdifferent origin, and there was no effort to look specifically forsubtype differences.Another alternative is that the same virus is associated with

the benign and malignant diseases, and the difference dependson genetic and/or environmental cofactors. Burkitt, whooriginally interpreted his findings concerning distribution ofthe high endemic regions as suggestive of an oncogenic virustransmitted by an insect (71), has more recently modified hisview to accommodate an ubiquitous virus, such as EBV,associated with an insect-transmitted cofactor required forthe manifestation of a malignant potential such as chronicholoendemic malaria (72). Conceivably, malaria might act byproviding additional stimulus for lymphoid proliferation or as

an immunosuppressive influence permitting a self-limitingdisease to turn into a malignant tumor. At the moment, thereis no factual basis for distinguishing between these alterna-tives. The study recently initiated in the West Nile district ofUganda (73) may provide more definite epidemiologicalanswers for the involvement of EBV in BL.

Before leaving the subject of EBV and BL, it may be appro-

priate to comment on the remarkable immunological behaviorof the tumor in vivo. It is quite clear that the tumor is rela-tively highly antigenic in the human patient (for review see

ref. 5). This is manifested in the documented occurrence ofspontaneous regressions (74) and the substantial proportion(15-20%) of long-time survivors after chemotherapy, some ofthem perhaps permanently cured (75-77). The differentEBV-associated antibodies relate to the course of the diseasein individual patients in different ways. Antibodies to earlyantigens (78, 79), and possibly to soluble, immunoprecipitat-ing antigens as well (80, 81), are more frequently present and

have a higher titer in tumor-bearing patients than in regres-sors. In remission patients, the risk of recurrence is higher in

anti-early antigen positive than in negative patients (78, 79).Membrane reactive antibodies behave quite differently. Theyare at a high and constant level in regression patients, as a rule,but tend to fall to low levels before recurrence (80 and

Gunven, P., Klein, G., Henle, W. & Henle, G., to be pub-lished). It is not presently known whether this fall is the

malignant lymphoproliferative diseases as well. Also, if

1058 Kleinl

cause or the consequence of tumor recurrence.


Marek's disease (MD)

This is a highly contagious, malignant lymphomatosis inchickens that creates major economic problems for the poultryindustry. It is caused by a herpesvirus, MDHV, that can begrown in chick or duck embryo fibroblasts where it causescytopathic changes. Unlike EBV, MDHV lends itself fairlywell to ordinary virological studies (for reviews see refs. 6 and7). Recently, a related virus has been isolated from turkeysthat is apathogenic in chickens and is partially crossreactivewith MDHV (82). Vaccination with turkey herpesvirus isefficient in reducing the incidence of MD in infected flocks, inspite of the fact that virus production and shedding is notreduced.One of the most interesting features of MDHV is the range

of its interactions with different host cells (83). In the tumor,no intracellular viral antigens can be demonstrated by im-munofluorescence; membrane antigens have not been studied.This is in line with the fact that no infectious virus has beenisolated from the tumor, nor were any virus particles seen inthe lymphoma cells under the electron microscope. Neverthe-less, the viral genome was clearly present because the diseasecould be transmitted with intact viable cells, inducing neo-plastic proliferation in the cells of the recipient. In certainlymphoid tissues, e.g., the bursa of Fabricius, viral antigenscan be demonstrated, but the cycle is abortive since it does notproceed to virus maturation. It is sufficient, however, toproduce a cytopathic effect. Although the relation of the viralantigens to the viral cycle and the virion has not been defined,the situation is reminiscent of the abortively infected earlyantigen-positive, viral capsid antigen-negative cells in theEBV system that may show, in certain cell lines, a progressiveinhibition of synthesis of macromolecules, leading to cell deathbefore the appearance of later viral functions.For many years the unsuccessful attempts to isolate in-

fectious virus from the affected lymphoid tissues appeared asan unresolved paradox in relation to the highly contagiousnature of the disease. This was recently resolved by the dis-covery (84) that the feather follicle is an abundant source ofinfectious virus. Virus production occurs in the superficiallayers of the stratified squamous epithelium. This is the onlyknown location where virus maturation and envelopment iscompleted. The virus remains infectious on storage under con-ditions in which the cells die. Maturation in the keratinizingcells is reminiscent of the Shope papilloma system, in whichinfectious nucleic acid, but not mature virus, can be isolatedfrom the growing basal epithelium. Infectious virus is pro-duced only in the keratinizing cells of the permissive hostspecies.

It is of particular interest for the discussion regarding EBVand human tumors that MDHV can cause a self-limitingdisease, resembling in some respects infectious mononucleosis,or progressive malignant disease, or no disease at all. Neo-plastic disease is a relatively rare event in relation to thenumber of cells infected. The factors that determine the differ-ence between the self-limiting and the malignant form are notknown. Host resistance is an obvious candidate (85). Neo-natal thymectomy increases the incidence of the neoplasticdisease in genetically resistant birds, but not in susceptiblestrains (86). This suggests that genetic resistance may func-tion by enabling the host to limit proliferation of tumor cellsby immunological mechanisms, presumably cell-mediated

The study of the neoplastic cell inMD in the analysis of thevirus-cell relationship has been hampered by the fact that no

cell lines have been established in vitro. This may be related tothe notorious difficulty in the establishment of continuouschicken cell lines. Different types of virus-related antibodieshave been identified by immunofluorescence, immunoprecipi-tation, or virus neutralization (7, 83, 87, 88), but the relationof the corresponding antigens to the viral cycle and the possi-ble role of the different antibodies for host defense or for tumorenhancement have not yet been clarified.

Herpesvirus saimiri (HVS)

This herpesvirus was isolated from squirrel monkeys in 1968(89). It induces a uniformly fatal lymphoma or reticulum celltumor in various primates, including several species of mar-

mosets, owl monkeys, and cinnamon ringtail monkeys(8, 9, 90). In marmosets, the disease develops after remarkablyshort latency periods, with an accumulation of lymphoblastsand reticulum cells at the site of inoculation within 7 days(9, 91). Mortality is 100%, even in animals inoculated as

adults; this is most unusual for any oncogenic virus. Cotton-topped marmosets die within 25-35 days with generalizedreticulum cell infiltrate, mainly involving the liver, spleen,lymph nodes, thymus, and adrenals. White-lipped marmosetsdie after 42-116 days with generalized lymphoblast infiltrationand leukemia. Virus particles or viral antigens cannot bedemonstrated in the neoplastic cells. Tumor filtrates are notinfectious, but inoculation of whole, viable cells induces thedisease in recipient cells, as shown by sex chromosomemarkers. The virus can be recovered regularly by cocultivatingthe neoplastic cells with susceptible indicator lines. Non-lymphoreticular cell lines from a variety of primates, includingsquirrel monkeys and marmosets, support productive infectionand respond with cytopathic changes. At the inception ofleukemic disease in the marmoset, millions of white cells are

required to recover the virus by cocultivation, but at theheight of the leukemia, as few as three cells are sufficient (9).Long-term lymphoblastoid cell lines have been established insuspension culture, derived from primary lymphoid explantsof diseased animals (9, 92, 93). These resemble the EBV-carrying human lines with regard to general growth andmaintenance properties, and also because only a small numberof cells produce viral antigen with little or no release of in-fectious virus. Still another point of resemblance is theabsence of viral antigens from the neoplastic lesion in vivo andtheir very prompt appearance, within 24 hr after explantation.Corresponding findings were made with regard to EBV inprimary explants of Burkitt lymphoma biopsies (39).

Originally, it was stated (8) that the squirrel monkey, thenatural host species of HVS, is not susceptible to the onco-

genic effect of the virus. This statement may have to be re-

examined, however, in view of the fact that the majority ofsquirrel monkeys had antibodies to the virus (9, 94). Further-more, by cocultivation with susceptible cells, virus could beisolated from their peripheral white cells. This is reminiscentof the anti-EBV seropositivity of normal human adults or

patients after infectious mononucleosis and the regular out-growth of EBV-carrying lymphoblastoid lines from peripheralblood under appropriate conditions. In analogy with thepathogenicity of EBV in seronegative individuals, the poten-tial oncogenicity of HVS in its natural host species would have

rejection.


to be tested in seronegative animals, if such can be found or


produced by isolation rearing. It would be obviously mostimportant to examine this question, since the natural host-virus relationship is most likely to provide meaningfulanalogies for the analysis of the interactions between EBVand man.

The Luck6 frog tumorThe Luck6 frog kidney carcinoma is the oldest known tumorassociated with a herpesvirus. The etiological role of the her-pesvirus is still not conclusive, however, although there isstrong suggestive evidence. The main difficulty is that thereare no in vitro systems for the study of the virus and tumor-derived cell lines. Furthermore, at least two other viruses havebeen isolated from the Lucke tumor, but there are no equallystrong reasons to consider them as potential etiologic agents(95-99). A strong argument for an etiological link between theLucke herpesvirus and the tumor was recently provided bythe demonstration (98) that fractions from tumors containingboth inclusion bodies and virus particles were oncogenic infrog embryos, whereas similar fractions from virus-negativetumors were inactive.The maturation of the herpesvirus associated with Luck6

tumor shows a remarkable temperature dependence. Virusparticles are seen only in hibernating winter frogs and disap-pear during the summer. Tumors growing in summer frogscontain no viral particles (95, 97, 99). An inverse relationshipappears to exist, furthermore, between tumor growth andviral maturation (99). Metastatic growth occurs only at hightemperature. Spontaneous regressions at low temperature maybe due to virus maturation, leading to cytopathic changes incarrier cells.A natural system where virus production is temperature

sensitive and malignant transformation occurs at a nonper-missive temperature may provide a remarkable tool for thestudy of viral functions required for transformation andproductive infection, respectively, provided that appropriatecell lines can be established for studies in vitro.McKinnell (97) has performed a remarkable experiment by

transplanting cell nuclei derived from a Lucke tumor inducedin a triploid frog into enucleated eggs. Several of the eggsdeveloped into triploid tadpoles. If the nucleus was reallyderived from a neoplastic cell and if these cells harbored theHV-genome, this would imply either that no irreversiblenuclear changes have occurred in the tumor cells or that thechanges that have occurred fail to affect a large number ofnormal differentiation processes. The latter alternative wouldsupport the view that the oncogenic effect is a rare accident ofvirus-cell interaction that can take place only within a veryrestricted range of target cells representing special types ofdifferentiation. It would also be in line with the demonstrationthat oncogenic viral genomes can be maintained in revertantcells isolated from virally transformed clones, or in virus-induced tumor cells whose malignancy has been suppressed bythe introduction of the genome of a normal cell by hybridiza-tion.

Other herpesviruses suspected of oncogenic action

A number of less extensively investigated or less firmly estab-lished cases where herpesviruses are suspected of a possibleoncogenic action may be briefly mentioned. For EBV, a sero-logical association has been found in relation to nasopharyn-geal carcinoma (12-14) and the sarcomatous form of Hodgkins

Burkitt lymphoma. It is noteworthy that the same high EBV-associated antibody titers are found in poorly differentiated oranaplastic carcinomas of Chinese, African, American, Swedish,and French origin (13, 14). Hypo- or oropharyngeal car-cinomas or other unrelated tumors localized to the naso-pharynx are, on the other hand, not associated with regularlyhigh EBV-titers (13, 14, 25). This and the regular demonstra-tion of EBV-DNA in biopsies from nasopharyngeal carcinomas(30) secures the position of this carcinoma as an EBV-associated tumor, although it is hard to see how "a non-ethnicvirus can cause an ethnic tumor" (100). As in BL, one mayenvisage different viral subtypes or the same virus actingtogether with genetic and/or environmental cofactors, butthere is no rational basis for choosing between the alterna-tives. Even less is known about the role EBV may play for thelymphocyte-depleted form of Hodgkins disease (22, 23).A serological association may exist between infection with

the genital form (type 2) of the herpes simplex virus andcarcinoma of the cervix (11), but whether both are conse-quences of a common third factor, sexual promiscuity, or arecausally interrelated cannot be decided as yet. Tests for herpessimplex virus antigens in cervical carcinoma cells have not yetgiven conclusive results. It is of interest, on the other hand,that UV-inactivated herpes simplex virus type 2 has inducedfocus formation in hamster fibroblasts (68, 69). Subsequently,the transformed cells grew as highly malignant tumors inhamsters. Concurrently, virus neutralizing and membrane-reactive antibodies appeared in the sera of the tumor-bearinganimals, suggesting the presence of genetic informationderived from herpes simplex virus in the tumor.

Recently, a rabbit herpesvirus has been isolated, capable ofinducing a self-limiting, lymphoma-like proliferation (101).Although this is obviously not a malignant tumor, it may berecalled that polyoma virus also induces self-limiting disease inrabbits, in contrast to most other species for which it is onco-genic and in which it induces highly malignant tumors.Together with the well-known virally induced multiple,benign rabbit tumor, known as the Shope papilloma, this maybe viewed in relation to the unusually strong, delayed hyper-sensitivity (DHS) and graft rejection responses that charac-terize the rabbit.Herpesviruses and lymphoproliferative disorders:Some comparative aspectsSome properties of the three main herpesviruses, EBV,MDHV, and HVS, considered in this review are listed inTable 1. All three can enter into a remarkable, nonpermis-sive, proliferation-stimulating, and, in part at least, onco-

genic association with lymphoid cells. EBV and MDHV dothis in their natural host species. HVS can do it in a numberof nonnatural hosts; evidence is not yet conclusive with re-

gard to the natural host. Susceptible target cells, capableof supporting the full viral cycle, have been found for MDHVand HVS; they are nonlymphoreticular cells.The lymphoproliferative lesion in vivo does not contain

viral particles, and filtrates are noninfectious. The virus can berecovered by cocultivation with susceptible cells, however, inthe two systems where susceptible cells are available (MDHVand HVS). In the third, EBV, its presence is indicated bynucleic acid hybridization. In the two systems where long-term lymphoblastoid lines can be established (EBV and HVS),viral antigens appear in a minority of the cells promptly afterexplantation. In all probability, these cells cannot divide any

1060 Klein

disease (22, 23) that is as pronounced and consistent as in


more and must eventually die. In MD an analogous situationis found in the abortively infected lymphoid cells of some

extratumoral tissues, notably the bursa and the thymus, withthe production of viral antigens and degenerative changes.Most established lymphoblastoid lines produce little or no

infectious virus; the antigen-positive cells die before virusmaturation has been completed. In the EBV system there are

some exceptional lines, however, that produce a certainamount of infectious virus. The proportion of cells that enterinto the virogenic cycle in this and other lines can be increasedby arginine deficiency, small doses of x-irradiation, or variousDNA inhibitors. At least superficially this is reminiscent ofthe induction of lysogenic bacteria. Conceivably, an inte-grated virus genome is excised or, alternatively, a noninte-grated viral genome is activated upon interference with thesynthesis and/or the integrity of the host cell DNA. In non-

virogenic lines that carry the viral genome, this event may beprecluded because either the viral genome is defective or

necessary enzymes are lacking, or as a result of repressor

action.The largely nonpermissive and only very occasionally

virogenic association of the viral genome with the lymphoidtarget cells responsible for the proliferative disease in vivo is

thus fairly well established. This is important in relation to thequestion, particularly clearly formulated in MD (86), whetherlymphoid proliferation is "intrinsic" in nature, i.e., due to theaction of the viral genome inside the lymphoid cells, or

"extrinsic," i.e., a reactive proliferation of lymphoid tissue inresponse to other virus-infected cells. The pertinence of thisquestion is apparent, e.g., in relation to the cutaneous lesionsin MD, where lymphoid proliferation occurs in direct associa-tion with the productively infected feather follicles, or to thebroad proliferative response in infectious mononucleosis.

Although the occasional involvement of an extrinsicmechanism as a contributory factor cannot be entirely ex-

cluded, the regular association of the viral genome with thecells responsible for the lymphoproliferative lesion is entirelyanalogous with the behavior of other oncogenic viruses andspeaks for an intrinsic mechanism. For EBV, the in vitro"transforming" effect of EBV on normal lymphocytes in theestablished lines points in the same direction. The way inwhich an extrinsic mechanism may contribute is illustrated byan experiment (102) showing that the simultaneous exposure

of tuberculin-sensitized lymphocytes to antigen and to EBVincreases the probability of converting them into establishedlines, compared to the virus alone.

In Burkitt's lymphoma, the demonstrated uniclonality ofthe disease (103) is a strong argument against reactive lym-phoid proliferation. One may ask why this tumor should beuniclonal even in patients with multiple tumors if a virus likeEBV, which promotes lymphoid cell proliferation in vitro withhigh frequency, is responsible for its induction. It must beremembered, however, that virally induced neoplastic trans-formation is by no means synonymous with the outgrowth oftransformed cells into a frank tumor in vivo. Polyoma virustransforms appropriate target cells in vitro with a high effi-ciency, for instance, and there is evidence of many incipientneoplastic foci in neonatally inoculated animals as well, butonly one or a few manage to eventually grow into a tumor.Concomitant immunity, demonstrable as the resistance ofanimals with progressively growing antigenic tumors againstthe new implantation of small tumor inocula with the same

antigenicity, provides an efficient mechanism that can explainhow the successful emergence of one virally transformed,antigenic clone may lead to the suppression of all othersinduced by the same virus (for review see ref. 104). The pecu-

TABLE 1. Comparative aspects of the main lymphoproliferative disorders induced by herpesviruses

Virus EBV MDHV HVS

Cell susceptible to productive Unknown Chick or duck fibroblast Squirrel monkey, marmoset, andinfection other primate cell lines of fibro-

blastic or epithelial origin.Pathogenicity in vivo Self-limiting lymphoprolifera- Self-limiting or malignant lym- In natural host species: no identi-

tive disease (infectious mono- phoproliferative disease, de- fied disease, but high antibodynucleosis) on primary infec- pending on host genetics and levels. In several New Worldtion of seronegative adoles- other factors. primates: highly malignantcents; suspected role, alone or lymphoma or reticulum cellwith cofactors, in Burkitt tumor.lymphoma.

Virus-cell relationships in vivo Lymphoid target cell: nonper- Lymphoma cell: nonpermissive, Lymphoma cells: nonpermissive,missive, proliferative, possibly proliferative, oncogenic. Lym- proliferative, oncogenic. Sus-oncogenic. No other known phoid cells in bursa and thy- ceptible cell lines of non-lym-target cell. mus: abortive, cytopathic. phoreticular origin may have

Feather follicle: productive in their in vivo counterparts, notkeratinizing cells, cytopathic. yet identified.

Recoverability of virus from fil- No No Notrates of target lymphoid tis-sue in vivo

Presence of viral genome in tar- Suggested by nucleic acid hy- Yes, recoverable by cocultiva- Yes, recoverable by cocultiva-get lymphoid tissue bridization. tion. tion.

Establishment and virogenic Yes, virogenic, abortively viro- No established lines. Yes, abortively virogenic, carrystatus of lymphoblastoid lines genic, or nonvirogenic but recoverable viral genome.in vitro harbor viral genome.

Effect of virus on normal lym- Conversion into lines, carriers of Not known Not knownphoid cells in vitro viral genome.


Proc. Nat. Acad. Sci. USA 69 (197j )

liar distribution of BL, with well-localized solid tumors and thelack of lymph node involvement, would also be in line withthis view. In fact, the only conceivable reason why a lym-phoma would remain restricted to solid lumps, in spite of thedemonstrated ability of its cells to spread freely throughoutthe body, lies in the restricting influence of a host response.

If immune mechanisms can restrict the growth of all but onevirally transformed clone in the tumor patient, it is reasonableto postulate that in many similarly infected individuals theywill restrict neoplastic growth altogether, or make it self-limiting, like infectious mononucleosis. The spontaneousregressions described (74) are the rare, visible manifestations ofwhat must happen at the microscopic level with a very muchhigher frequency. It becomes very important to understand thedifference, in terms of immune mechanisms and/or cellularsusceptibility to them, between the self-limiting and themalignant disease. In MD both forms occur, and their relativeproportions depend, among other things, on genetic factors.In genetically resistant strains, thymectomy increases theincidence of the malignant form, indicating that resistance isdetermined by thymus-dependent, presumably cell-mediatedimmunity. In genetically susceptible strains with a highincidence, thymectomy has no effect. The mechanism of thelack of reactivity in susceptible strains is not known; byanalogy with other systems, it could be due to a lack of recog-nition of the relevant antigen, or to an overwhelming of thereaction, e.g., by viremia or some malfunctioning at theeffector level.

Some potentially fruitful fields for comparative studies

As it will have appeared from this review, none of the systemsprovides a complete picture of the oncogenic action of herpes-viruses and the relevant virus-cell and cell-host relationships,but each system has its own merits and difficulties, with themajor gaps localized to different areas. Comparative studiesmay therefore complement each other in a useful way, inspite of the difficulties involved in extrapolation. The followingare some of the many areas where joint or parallel experimentsmight be useful.

It is obviously very important to distinguish betweenoncogenic and virogenic functions. As far as the herpesvirusesare concerned, there is no exception to the rule so far thatall productively infected cells must die. The oncogenic func-tion must therefore be different from, or a subset of, the func-tions required for virus production. For its identification, astudy of the tumor cell in vivo may be crucial, since this is themost unambiguously defined neoplastic cell and it produces novirus. Nonvirogenic lines that carry the viral genome in vitrowould be equally interesting, if it would be certain that theyrepresent the neoplastic cell; since such lines are presentlyrestricted to the human EBV system, however, where neo-plastic behavior cannot be tested, this approach is not as goodas the study of the tumor cell in vivo. As far as such a study isconcerned, it will be recalled that EBV-associated membraneantigens were present on BL biopsy cells in vivo, whereas thevirally determined intracellular (early and viral capsid)antigens were absent. It would be important to know whetheranalogous membrane changes can be found in MD and in theHVS-induced lymphoma as well. If so, herpesvirus-inducedmembrane changes would become important candidates forthe oncogenic function. It would be important, furthermore, to

membrane changes and viral envelope, as well as to study thechemical nature of the membrane change, its relationship tothe viral cycle, and its compatibility with continued cellproliferation. Another important aspect is the role played bynew membrane antigens in host sensitization, leading to theresistance phenomena associated with the various diseases.Clonality studies of the type that have been already per-formed on BL would be important for MD and the HVS-lymphoma since they would give information about howmassive viral transformation becomes expressed in the form ofa malignant tumor. They can also help in deciding definitelybetween intrinsic and extrinsic mechanisms. While poly-clonality would not exclude intrinsic origin, uniclonality wouldbe very hard to fit with a purely extrinsic (reactive) patho-genesis. Although it may be difficult to find useful X-linkedisozyme markers for this purpose in the various animalspecies, production of chimeric chicks by embryonic parabiosis(105) and the regular occurrence of natural chimeric twins inmarmosets (9) may facilitate such an approach.The reasons why infectious mononucleosis regresses in man,

in spite of extensive tumor-like changes in many internal or-gans (106), while some MD lesions regress and some progress,and why most Burkitt lymphomas progress, although someregress (with or without chemotherapy), require an im-munological explanation. The importance of studies on cell-mediated immunity, and particularly on the thymus-derivedsystem, has already been emphasized. One may add the studyof humoral antibodies directed against specific antigens re-lated to the oncogenic and the virogenic function as anotherimportant parameter. The indications, in BL, that antibodiesdirected against the membrane antigen may play a role intumor regression and recurrence, whereas antibodies to theearly antigen may reflect residual tumor mass and perhapsviral activation, suggest the fruitfulness of such an approach,alone or in combination with studies on cell-mediated im-munity. Corresponding antigen-antibody systems in MD andHVS still remain to be defined, but there is every reason toexpect that analogous systems exist.The remarkable success of chemotherapy in a substantial

proportion of BL cases has been interpreted as the combinedeffect of therapy and host response. This assumption could betested in MD. The availability of genetically susceptible andresistant chicken strains would make this into a particularlyinformative study. So far, there has been practically no use ofMD-infected chicks for model experiments related to thehuman problems, since nearly all work has been focused on thepractically important problem of prevention. Since turkeyherpesvirus vaccination appears to have brought this problemclose to its solution, it may be hoped that more attention willbe paid to the possible significance of the chicken disease forman from other viewpoints than consumption. This appearseven more justified by consideration of the fact thatMD is theonly known naturally occurring neoplastic disease induced bya virus that is highly contagious. In addition to geneticfactors, age, maternal antibodies before infection, patho-genicity of the virus strain, and social stress were alreadyshown to influence the incidence of MD. It is therefore obviousthat this may become a very fruitful area for model studieson factors influencing viral oncogenesis under natural condi-tions.The status of viral DNA in the neoplastic cells, and particu-

larly whether it is covalently linked with the host cell genome

1062 Klein

carry out comparative studies on the relationship between


or is merely carried along in a nonintegrated state, is obvi-ously of prime importance. The closely related problem ofviral induction in the virogenic lines including possible anal-ogies with lysogenic systems and the factors responsible forthe difference between virogenic and nonvirogenic associa-tions appear equally important.

These investigations were conducted under Contract no. NIH-69-2005 within the Special Virus Cancer Program of the NationalCancer Institute, National Institutes of Health, USPHS. Grantswere also received from the Swedish Cancer Society, and AkeWiberg's Foundation.

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oncogenesis herpesviruses and

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