Genetics of Resistance to Virus-Induced Leukemias

GENETICS OF RESISTANCE TO VIRUS-INDUCED LEUKEMIAS Daniel Meruelo’ and Richard Bach

.

lrvington House Institute. Department of Pathology. New York University Medical Center. New York New York

I . Introduction ...................................................... I1. The Initial Link between Viruses and Leukemias ...................... 111. Characteristics of the Retrovirus Family .............................. A. Genome Organization .......................................... B. Similarities with Transposons .................................... C. Replication ................................................... D. Gene Expression ............................................... E . Transformation ................................................ F. Assembly ..................................................... G. Polymorphism ................................................. H . Proximity of Proviruses, Histocompatibility, and Lymphocyte Antigen Loci ....................................................... IV. Expression in Inbred Mouse Strains of Antigens Associated with MuLV . . . A . Glx and GCSA ................................................. B . X.l .......................................................... c. G(ruo~l1,G(ERLD), and G(AKSU) ..................................... D . PC.l ......................................................... E . TL........................................................... F. ML .......................................................... G . Other Antigens ................................................ V. Genetics of Susceptibility to Viral Infection ........................... A . Genes Affecting Virus Spread .................................... B. Adsorption and Penetration ...................................... C . Fu-I:Restriction of Integration ................................... D. Availability and Replication of Target Cells ........................ E . Transformation ................................................ F. Immune Surveillance against Viral Infection and Transformation . . . . . . VI. Prospects for Control of Human Leukemia ............................ References .......................................................

107 108 109 109 111 111 114 117 117 119 124 132 132 134 135 135 136 137 137 138 138 139 140 143 151 161 173 176

1. Introduction

This article shall be concerned primarily with the genetics of susceptibility to leukemia in mice. the relevance of viruses as etiological agents. and the relationship of this information to leukemias and lymphomas in man . The studies that formed the foundations of present 1

Leukemia Society of America Scholar. 107

ADVANCES IN CANCER RESEARCH. VOL. 40

Copyright Q1.1983by Academic Press. Inc . All rights of reproduction in any form reserved. ISBN 0-12-006640-8

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day knowledge were begun several decades ago, with the period prior to 1960 being characterized by the gradual acceptance of the fact that viruses could cause cancer. This period also gave rise to the idea that genetically controlled factors could affect the degree and overall susceptibility to and latency of the disease. The 1960s and 1970s saw feverish activity, first defining the genes involved, and then probing cellular and molecular questions regarding their mode of action. Simultaneously, viruses were studied intensively to the extent that much of their molecular structure has now been elucidated. However, despite this intense activity, progress to date has not yet solved two fundamental questions: (1)Are viruses involved in human leukemias and lymphomas? (2) How can the knowledge on hand be applied to control or arrest the malignant process? It is hoped that this article summarizes and focuses the current state of knowledge in the field. While neither of the above stated questions can yet be answered, there is much reason to be excited and optimistic. The article shall first cover the virology of leukemia-inducing and related viruses, second discuss issues of specificity in virus-host genomic interaction, and third describe current knowledge of host genes conferring resistance or susceptibility to virus-induced neoplasia.

11. The Initial Link between Viruses and Leukemias

The proposition that leukemias are virus induced was suggested in studies of the infectious transfer of leukemias in chickens by Ellerman and Bang (1908) and in mice by Gross (1951). At that point in time, filtration had become an important tool in the study of virus diseases. If an extract prepared from diseased tissues could be filtered without losing its pathogenic potential (i.e., when the filtrate reproduces symptoms of the same disease following inoculation into a susceptible host) it was generally assumed that such a disease was caused by a virus (Gross, 1970). It was only the advent of the electron microscope that made it possible to visualize these pathogenic agents. Their electron microscopic morphology led to the use of the term “type C particles” for the leukemia viruses. In addition, the general class of oncogenic RNA tumor viruses was given the name “oncomaviruses.” However, many type C virus isolates do not show oncogenic activity. For this reason, a more general term was sought. All of these viruses have reverse transcriptase activity, hence the more recent term “retrovirus.”

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Ill. Characteristics of the Retrovirus Family

The designation “retrovirus” includes a variety of agents related to murine leukemia viruses. These have been classified into categories according to oncogenic properties. Broadly speaking, these classes include the sarcoma viruses, which induce rapid connective tissue neoplasms in vivo and transform fibroblasts in tissue culture; the acute leukemia viruses which cause rapidly identifiable hematologic neoplasms and generally possess some form of cell transforming potential in vitro; and the lymphatic leukemia viruses, whose inoculation can result in lymphocytic leukemia or lymphoma only after a long latency period and classically lack the ability to transform cells in tissue culture. In general, there are avian and mammalian examples of each of these categories. Structurally, the common characteristics shared by members of this family are genomes of diploid single-stranded RNA and a virion nucleotide polymerase capable of RNA-directed DNA synthesis. The basic genetic anatomy of the nondefective lymphatic leukemia viruses consists of three genes which code for products associated with the viral replicative cycle, termed gag, pol, and env.‘ The sarcoma and acute leukemia virus classes retain this basic framework while either adding an additional gene (Rous sarcoma virus, Fig. l),or acquiring new genetic material at the expense of replicative gene sequences, as has been demonstrated for Abelson-MuLV (Goff et al., 1980). These acquired sequences, apparently derived from cellular genes (Bishop, 1981), are thought responsible for neoplastic transformation by these viruses and have therefore been called oncogenes (see Section V,E,3).

A. GENOMEORGANIZATION The diploidy of retroviruses is unique among the genomes of known animal viruses and this property may explain the high recomgcnom IC IrrmnoL

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DANIEL MERUELO AND RICHARD BACH

bination frequency obtained with these viruses (Weiss et aZ., 1973; McCarter, 1977). Figure 1 illustrates the composition, structure, and topography of a haploid subunit of one well-characterized retrovirus genome, the avian sarcoma virus (ASV). The general features outlined in this figure are applicable to most, if not all, retroviruses. The 5' termini of both subunits of the dimer are capped by the structure 5'-m7 GpppGm (Furuichi et al., 1975; Rose et al., 1976)and the 3' ends are polyadenylated (- 200 residues) (Bender and Davidson, 1976; King and Wells, 1976). About 10 specific sites within the 3' half of the genome, containing adenosine residues, are methylated (Furuichi et al., 1975; Beemon and Keith, 1976; Dimock and Stoltzfus, 1977). These fea" tures, i.e., polyadenylation, capping," and a low level of internal methylation, are common to eukaryotic mRNAs. It is not surprising therefore that the retrovirus genome can serve as a messenger for the synthesis of virus-specific proteins. Host cell tRNATp is bound to the genome of ASV at a site 101 nucleotides distant from the 5' terminus of the genome (Taylor and Illmensee, 1975) and serves as primer for the initiation of DNA synthesis by reverse transcriptase in uitro (Dahlberg et al., 1974; Harada et al., 1975).The identity and location of the tRNA primer vary among retroviruses. Other host cell tRNAs are also bound to the haploid subunit of retroviruses although less firmly (Sawyer and Dahlberg, 1973).Their function(s) (if any) are not known, but their importance is unclear since many of the represented isoacceptor species are not abundant enough to be associated with all molecules of viral RNA (Sawyer and Dahlberg, 1973). The haploid subunits of the genomes of most retroviruses are terminally redundant (Fig. 2). For example, Temin's group (Shimotohno et al., 1980a; Shimotohno and Temin, 1980) has shown that spleen necrosis virus contains a 5 bp direct repeat of cellular DNA next to a 3 bp inverted repeat of viral DNA. The inverted repeats formed the ends of a 569 bp direct repeat of viral DNA. Comparable results have been obtained by investigators working with proviruses of Moloney murine 3'-LTR

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sarcoma virus (Dhar et al., 1980), mouse mammary tumor virus (Majors and Varmus, 1980), and Rous-associated virus 0 (Ju and Skalka, 1980). The lengths of the repeat differ in different viruses. The direct repeat of viral DNA is called LTR for long terminal repeat. These retroviral LTRs appear to function critically in the integration of proviral DNA into cellular DNA, and detailed analyses have revealed the presence of putative transcriptional promotor and polyadenylation signals (Ju and Skalka, 1980). WITH TRANSPOSONS B. SIMILARITIES

Several features of retrovirus organization, the presence of direct repeats of host DNA, inverted and direct repeats in the viral DNA, and unique sites on the virus DNA for insertion, are rather similar to those found in certain bacterial moveable genetic elements, namely, transposons (Shimotohno et al., 1980a). Other recently described moveable genetic elements of eukaryotic cells (yeast TY1 and Drosophila copia-see below) have these same features. In addition, all of these transposon-like structures end with the dinucleotides TG . . . GA (Allet, 1979; Kahnman and Kamp, 1979). Z Y I , copia, and retrovirus genomes have a 5 bp direct repeat of element or viral DNA flanking the inside ends of the two LTRs (Farabaugh and Fink, 1980; Dunsmuir et al., 1980; Gaffner and Philippsen, 1980). The similarities found are believed to be too strong to result merely from a chance coincidence. It should be noted that the only cellular moveable genetic elements described to date for vertebrates are the endogenously found retroviruses. If the retroviruses are derived from cellular genetic elements as is now presumed (Temin, l970,1971b), these movable genetic pieces may play a role in cell differentiation by regulating genetic rearrangements. For example, control of yeast mating type (Cameron et al., 1979) and immunoglobulin structure (Brack et al., 1978; Seidman and Leder, 1978) has been shown to involve DNA rearrangements. Whatever their function, the origin of retrovirus from cellular moveable genetic elements may provide a clue for carcinogenesis for which a viral etiology is not apparent. That is, such cancers may result from processes similar to those involved in the evolution of retroviruses. We shall return to this issue later in this article.

C. REPLICATION The viral envelope glycoproteins are primarily responsible for adsorption and penetration of virus into cells as demonstrated by the fact

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that deletions in enu give rise to fully assembled, noninfectious virus particles (Duesberg et al., 1975). Similarly, temperature-sensitive conditional mutants in enu render virus particles noninfectious at the restrictive temperature (Vogt and Hu, 1977).The interaction between viral envelope glycoprotein and host celI receptors has been reconstructed in uitro with purified gp70 of MuLV and is highly specific (DeLarco and Todaro, 1976). The events that follow adsorption of the virus to the cell surface and onset of viral DNA synthesis are not well understood. Electron microscopy studies have suggested that the virus genome moves quickly after infection (within 10-60 min) into the nucleus of the host cell (Dales and Hanafusa, 1972). By contrast, biochemical data indicate that initial viral DNA synthesis starts in the cytoplasm of the cell and continues therein for the first 12-24 hr following infection (Varmus et al., 1974),after which integration occurs. It is presently not clear whether protein synthesis is or is not required as an early event in the establishment of infection by retroviruses. It has been shown that parental RNA associates with polyribosomes (Salzberg et al., 1977) and may, in some cases, be translated prior to the onset of (or in the absence of) viral DNA synthesis (Gallis et al., 1976). However, their data were obtained with extremely high multiplicities of infection and may not reflect the in vivo situation. The clearest experiments have shown that viral RNA may be expressed directly after introduction into the host cell by either microinjection (Stacey et al., 1977) or application in the presence of polycations. Figure 3 outlines the principal molecular events in the replication of retroviruses. The scheme is partly hypothetical but reflects current experimental findings. Following infection, virus-coded reverse transcriptase copies the virus-single stranded RNA genome into double-stranded DNA (Verma et al., 1976). Studies of reverse transcription in uitro indicate that DNA synthesis proceeds in a 5' to 3' fashion beginning at the priming tRNA molecule, polymerizing deoxyribonucleotides complementary to the RNA genome until the 5' end of the template is reached. The nascent single-stranded DNA molecule migrates then to the 3' end of the RNA template; there it partially pairs by virtue of complementary nucleotides with the string of approximately 60 bases immediately 5' to the poly(A)tail on the end of the genome (Coffin, 1979). After migrating to the 3' end of the RNA template, the nascent chain is elongated, presumably continuously, to the 5' end of the template producing a complete minus-strand DNA copy of the virus genome.

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The major stable products of synthesis are linear duplexes and closed circular duplexes (Fig. 3) (Shank et al., 1978; Hsu et al., 1978; Shank and Varmus, 1978); both forms are approximately the length of the haploid subunit of the viral genome. The mechanism for plus-strand DNA synthesis and for producing terminal repeats to form the double-stranded DNA molecule has not yet been elucidated. Nonetheless, a sequence just into the body of the virus from the repeat junction (in MuLV positions 516-560) is of interest. The upper strand of this sequence contains 15 pyrimidines in a row, followed by a 15 nucleotide sequence containing 12 purines and an 11 nucleotide sequence containing 10 pyrimidines (Sutcliffe et al., 1980). What makes this sequence of particular interest is that it occurs at the repeat junction, where the origin of plus-strand replication is thought to be localized (Mitra et al., 1979). When this sequence is examined closely, it can be shown to form a stem-and-loop structure (hairpin) (Fig. 4) similar to that for the single-stranded bacteriophage origins of replication (Sims and Benz, 1980).

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FIG. 4. Origin of second strand synthesis can be drawn as a hairpin structure. A schematic minus-strand loop containing an inverted repeat is shown as derived from data of Sutcliffe et ol. (1980) for Moloney leukemia virus. Base complementation is indicated by heavy lines.

The double-stranded DNA molecule with repeated ends is thus the structure that probably integrates into the host chromosome. There it is inherited in a Mendelian fashion and acts as a substrate for transcription and generation of new virus particles. Retroviruses integrate at multiple sites in the host DNA (we shall return to this issue in Sections III,G,l and III,H,l-4). It is not known if the circular or linear form of viral DNA is integrated. However, presence of inverted repeats at the termini of either 5’ LTR or 3’ LTR suggests an analogy with the bacterial transposons (Kleckner, 1977) and suggests that the circular form is a better candidate for integration. How the circular form of the unintegrated viral DNA can be integrated has been the subject of several hypothetical models (Shapiro, 1979; Shoemaker et al., 1980). Viral DNA containing one LTR copy can integrate after generating two copies of LTRs in the models described by Shapiro (1979) or Shoemaker et al. (1980). However, only those molecules that contain two copies of LTR are able to transform or infect cells in the studies with cloned M-MSV DNA (Verma et al., 1980) and in ljitro reconstructed clones M-MLV DNA. D. GENEEXPRESSION The mechanisms that generate the final gene products of retroviruses are illustrated in Fig. 5, using ASV as a prototype. Of the four ASV genes, three are translated from mRNAs that contain the expressed gene at the 5’ end; g a g from 38 S mRNA, enu from 28 S mRNA, and src from 21 S mRNA. On the other hand, pol is probably expressed by the continuous translation from gag and pol in 38 S

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mRNA. This scheme is consistent with the notion that translation in eukaryotic cells initiates only at the 5’ termini of mRNA (Jacobson and Baltimore, 1968), and is well suited for independent expression of viral genes according to the relative need for gene products as is the case for plant (Hunter et al., 1976) and animal (Cancedda et al., 1975) viruses. The primary product of translation from gag is a polyprotein from which the individual polypeptides of the viral core are generated by cleavage (Vogt et al., 1975; Shapiro et al., 1976). The linear order of the individual core proteins within gag for ASV is illustrated in Fig. 5. The fact that cleavage of the polyprotein does not occur in certain host cells suggests that this processing is carried out by cellular enzymes (Eisenman et al., 1974), although enzymes associated with ASV and MuLV have been shown to cleave the precursor protein into the correct products (Helm, 1977; Yoshinaka and Luftig, 1977b). The nature of these virion-associated processing enzymes is unclear; in ALV such activities have been shown to be associated with p15 whereas in MuLV the protease activity apparently resides on a previously unrecognized protein (Yoshinaka and Luftig, 1977b). Particles of the gag polyprotein are often expressed on the cell surface in a glycosylated form that is neither cleaved nor incorporated into virions (Snyder et al., 1977; Ledbetter and Nowinski, 1977) giving rise to important antigenic determinants on the surface of leukemic cells in mice (see Section IV) (Snyder et al., 1977). A virus-specific RNA with the size and composition expected for a pol messenger has not yet been detected (Weiss et al., 1977; Hayward, 1977). The evidence, however, is consistent with the notion that pol is expressed from the continuous translation of gag and pol in 38 S mRNA. The molecular weight of the readthrough product is 180,000 (Pr 18OPO’) and contains the antigenic determinants and tryptic peptides of both the gag and pol proteins (Oppermann et at., 1977). The evidence supporting the readthrough model is as follows: (1)Pr 18OPo1 has been found in virus-infected permissive (Oppermann et al., 1977; Jamjoon et al., 1977) and nonpermissive cells and can be synthesized in vitro with either the viral genome (Kerr et al., 1976; Purchio et al., 1977) or 38 S RNA isolated from infected permissive cells as messenger; (2) kinetic analysis studies indicate that Pr 180P”’is not a precursor for any of the mature gag gene products (Oppermann et al., 1977), and that its turnover is related to the appearance of mature reverse transcriptase in virus particles (Oppennann et al., 1977); and (3) Pr 180P”’is present in extracellular virions where it gradually decays in concert with the appearance of the mature polymerase (Oppermann et

116

DANIEL MERUELO AND RICHARD BACH gp37-s-s-gp85

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al., 1977). The above data are, therefore, in line with the notion that reverse transcriptase is generated from a polypeptide precursor, and that this processing occurs in extracellular virus. The discovery of Pr 180p"'raises the issue of how readthrough translation is regulated in eukaryotes. In prokaroytes it is known to occur by suppression of a termination signal (Weiner and Weber, 1971).The fact that synthesis of Pr 180p"' in uitro with the genome of MuLV as messenger can be substantially augmented by the use of an amber tRNA isolated from yeast (Philipson et al., 1978), identifies the signal that terminates translation from gag but does not establish the mechanism by which Pr 180P"' is synthesized in the infected cell. Readthrough of amber codons is usually a very rare event in viuo, yet amber codons are not usually bypassed by means other than specific suppressor tRNAs (Philipson et al., 1978). Messenger RNA for en6 of various retroviruses has been identified by translation in uitro (Pawson et al., 1977) and in Xenopus oocytes (Van Zaane et al., 1977) and by microinjection of cells infected with a deletion mutant in enu (Stacey et al., 1977). It appears to be generated by RNA splicing, containing sequences from the 5' end of genomic RNA contiguous with enu homologous sequences (Shinnick et al., 1981).The primary product of translation from ASV enu is a protein of 70K MW (Pr 70""") (Moelling and Hayami, 1977). It has been shown to accumulate in infected cells in the presence of an inhibitor of glycosylation (Shapiro et al., 1976; Moelling and Hayami, 1977). While Pr 70""" has been shown to contain some carbohydrate residues (Moelling and Hayami, 1977), further glycosylation generates a second form, Pr 90""" (Moelling and Hayami, 1977; Famulari et al., 1976; England et al., 1977). The changed mobility of the polypeptide SDS gels

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results from the effect of carbohydrate residues on its electrophoretic mobility. When Pr 90”””is cleaved, it produces the mature envelope glycoproteins: gp85 and gp37 ofASV, gp70 and p15E of MuLV. Cleavage may be coordinated with migration of the glycoprotein to the surface of the infected cell (see Section 111,F).

E . TRANSFORMATION It should be noted that neoplastic transformation is not a necessary consequence of viral replication. A case in point are ASV and avian leukemia viruses (ALV), both of which replicate in avian fibroblasts, yet only ASV transforms these cells. In general, transformation by any single strain of virus is restricted to particular target cells (see Section V,D), whereas the host range for replication is less specific. While transformation can occur in the absence of viral replication, the efficiency of transformation of nonpermissive cells (e.g., mammalian fibroblast hosts for ASV) is usually quite low to (Temin, 1971a).It appears that a 21 S virus-specific RNA in cells infected with ASV encoding only src and “c” is likely to be the messenger for the transforming protein. Deletions in STC abolish the ability of the virus to transform fibroblasts but have no effect on viral replication and are, therefore, denoted “transformation defective” (tdASV). At least some strains of tdASV can induce lymphoid leukosis in birds (Biggs et al., 1973) and are, therefore, analogous to the naturally occurring avian leukosis viruses. Such deletions in src appear spontaneously in high frequencies for some strains of ASV (Vogt, 1971) or can be induced by mutagenesis (Biggs et al., 1973).

F. ASSEMBLY A model for the assembly of retroviruses has been proposed (Bolognesi et d.,1978) on the basis of several parameters, including arrangement of virion structural components in the assembled particles themselves, the fine genetic structure determined through numerous experiments, and available biosynthetic data. The model proposes the following: 1. Virus envelope components (e.g., gp70-pl5E) migrate to and are inserted on the cell surface at the site of virus budding, already in molecular complexes (i.e., polypeptide complexes). 2. Internal virus precursor molecules are then transported to the budding site where one end of the molecule is joined to the virus envelope complex, while the other end associates with the virus RNA.

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3. Virus assembly progresses with the proteolytic cleavage of such precursor molecular complexes. 4. The cleaved virion components associate with each other to yield the substructures normally found in the mature, budded particles (i.e., the envelope, inner coat, core shell, and ribonucleoprotein complex). The mechanism of virus assembly seems to be greatly dependent on the proper association or “bonding” interactions between structural components which occur during the budding process. Thus specific recognition and noncovalent association between certain envelope and internal virion molecules is required for virus maturation. Whether such interactions serve an “aligning” function is not established, however some viral molecules remain associated during precipitation despite their noncovalent association (e.g., p19 and gp35 are brought down together by antiserum to gp85, the major glycoprotein) (Schlesinger, 1976). In addition, Rohrschneider et al. (1976) have shown that avian p19 is critical for virus synthesis and assembly since mutants with defective p19 molecules fail to assembly correctly. The evidence that precursor cleavage is a late step in virus maturation is as follows: First, the sequence of the structural components from exterior to interior of the mature virus is in good correspondence with the order they are found on identified precursor molecules (Barbacid et al., 1976; Eisenman et al., 1974; Jamjoon et al., 1977; Arcement et al., 1977). Second, several groups (Naso et al., 1976; Van Zaane et al., 1977; Famulari et aZ., 1976; Shapiro et aE., 1976) have identified nonglycosylated gag precusor molecules which appears to have escaped proteolytic digestion and processing. In addition, gag precusor molecules and precursor-specific proteases have been found in Rauscher leukemia virus. After appropriate incubation of these particles, cleavages result to give rise to the appropriate structural components (Yoshinaka and Luftig, 1977a,b). Third, it is known that some virus structural components (e.g., avian p15) are probably involved in this type of proteolytic activity (Von der Helm, 1977). The above data are consistent with the cleavage of gag precursor molecules at the cell surface, at a late stage of virus maturation and budding. Consistent with this notion is the fact that gag processing is usually associated only with virus-producing cells (Eisenman et al., 1974). The gag-pol molecule readthrough products often seen may not result from transcriptiodtranslation errors, but may function as a means of incorporating a small number of polymerase molecules into the virion. Viral RNA does not appear to be required for virion assembly, since murine

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leukemia virus produced in the presence of actinomycin D lacks 60 S to 70 S RNA, but contains normal amounts of virion structural polypeptides, including polymerase (Witte and Baltimore, 1977). Finally, in considering assembly, it was stated that the first step in virus assembly is the insertion on the cell membrane of viral envelope glycoprotein complexes. This notion is somewhat controversial in light of studies by Hanafusa and co-investigators (Scheele and Hanafusa, 1971; Kawai and Hanafusa, 1973) which have shown that defective Rous sarcoma virus (RSV) particles can be synthesized in the absence of detectable gp85 or gp35. The studies by Hanafusa’s lab (1971, 1973) involved analysis by polyacrylamide gel electrophoresis and it could not be excluded that very small amounts of the glycoprotein or a fragment of the envelope products were present in cells budding the RSV particles. This is important because Witte and Baltimore (1977) have shown that very little of env glycoproteins are required for the viral assembly process to occur.

G. POLYMORPHISM 1. Host Range Progress in the early years following discovery of avian and murine type C viruses was hindered by the fact that the only available assay for these viruses was leukemogenesis or tumorigenesis. The subsequent development of substantially more potent virus variants for several retroviruses and the isolation of several exogenous viruses (Pincus, 1980) considerably shortened the time required to observe leukemogenic effects. Further impetus to the study of viral oncogenesis came as a wide range of techniques ensued for rapid analysis of the biological functions and molecular biology of type C viruses. Studies made possible by these advances quickly revealed several different categories of host range polymorphisms for these viruses. For example, it was discovered that certain murine type C viruses can infect mouse cells but cannot infect cells of other species (these are designated ecotropic), while others cannot infect mouse cells but can infect cells of heterologous species (xenotropic viruses) (Levy and Pincus, 1970; Levy, 1978; Aaronson and Stephenson, 1973). Those than can infect cells of both the mouse and heterologous species are called dualtropic or polytroic viruses. In addition, a distinct category of viruses with this (dualtropic) same property, called amphotropic viruses, has been isolated from wild mice (only) (Hartley and Rowe, 1976; Rasheed et aZ., 1976; Chattopadhyay et aZ., 1978). Further poly-

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morphism among the murine type C viruses has been recognized in their varying ability to replicate in cells of mice bearing different alleles at the Fu-1 locus and to express themselves in cells of different mouse tissues (organotropism). We shall return to polymorphism with regard to Fu-1 and organotropism later on in this article, but we would like to discuss the species type polymorphisms first. Biochemical studies have shown that the viral enu gene products are responsible for the type of host range variation which has classified viruses as ecotropic, xenotropic, polytropic, or amphotropic (Elder et al., 1977, 1978; Troxler et aZ., 1977). For example, polytropic viruses isolated from AKR mice show p30 peptide (gag gene product) maps similar to ecotropic AKR virus, but gp70 peptides (enu gene products) which differ from classical AKR virus in containing xenotropic gp70 sequences (Elder et al., 1977). The recombinant polytropic HIX virus, isolated from Moloney-MuLV virus-infected cells (Fischinger et aZ., 1975), also contains a p30 identical to its ecotropic Moloney-MuLV parental type, but a gp70 containing xenotropic virus determinants (Fischinger et al., 1978). The polytropic B-MuX virus, induced from BALB/c cells by iododeoxyuridine, again shows a p30 core protein similar to that of its ecotropic virus progenitor and a gp70 envelope protein which contains endogenous xenotropic determinants (Ihle et al., 1978). Indeed, an interesting characteristic of retroviruses is their high recombination frequency. Recombination increases polymorphism and occurs usually, but not always, in the enu gene. For example, analysis of gp70 tryptic peptides has shown, with one exception, that no two gp70s isolated from different viruses are identical (Elder et al., 1978). Almost every gp70 has distinct tryptic peptides. There are, nonetheless, resemblances which allow classification and subdivision of the viruses according to relatedness (Elder et al., 1977). In contrast to gp70, p30, which by mass is the major core protein, is highly conserved. However, not all p30s are identical, and thus far several distinct types have been defined in the mouse (Gautsch et al., 1981). Less is known about the relative structure of the polymerases, p12, p10, and p15, but available data suggest that polymerases p10 and p15 are relatively conserved. In contrast, p12, in accord with its specific RNA-binding function, is more polymorphic (Stephenson et al., 1974; Aaronson and Stephenson, 1975). 2. Polymorphism with Respect to Fv-1 As mentioned previously, replication by most murine type C viruses is subject to Fu-1 host range restriction (Rowe and Sato, 1973).

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Viruses, therefore, are designated as N-tropic or B-tropic according to their preferential growth on either N-type (Fu-1" homozygous) or Btype ( Fv-lb homozygous) mouse cells (Pincus et al., 1971b). The viral determinant of susceptibility to this restriction system has been biochemically assigned to the gag gene product (Hopkins et al., 1977; Schindler et al., 1977; Gautsch et al., 1978). N-tropic virus strains replicate 100-1000 times more efficiently in cells derived from NIH Swiss mice than in BALB/c cells (Pincus et al., 1971b). Conversely, B-tropic viruses replicate 30- to 100-foldbetter in BALB/c cells than in NIH cells (Pincus et al., 1971b). Fu-l permissiveness is governed by a single genetic locus on chromosome 4,whose two alleles (Fu-1" and Fu-lb) exert dominant restriction upon infection (Rowe et al., 1973). Early studies on the mechanism of Fv-l restriction showed, by using vesicular stomatitus virus (VSV) pseudotypes, that Fv-l did not influence virus adsorption or penetration. For example, VSV genomes encapsidated into coats derived from either N- or B-tropic viruses could infect normal cells of either Fu-1 type equally well (Huang et al., 1973; Krontiris et al., 1973). It was, therefore, concluded that an intracellular process specific to the type C virus infectious process must be affected by the Fu-l gene product. To study the mechanism of Fu-l restriction, several experiments were conducted. These revealed that viral DNA synthesis in Fu-l restrictive cells proceeds at normal levels (Jolicoeur and Baltimore, 1976; Sveda et al., 1976), but viral RNA synthesis is curtailed by Fu-l restriction (Jolicoeur and Baltimore, 1976). Association of viral DNA with cellular DNA is inhibited in restrictive cells, indicating that the Fu-l gene product interferes with integration of viral DNA into cellular DNA (Jolicoeur and Baltimore, 1976). Recently, it was demonstrated that a restrictive Fu-l gene product does not affect the production of linear viral DNA, but does markedly interfere with the appearance of supercoiled, closed circular viral DNA, possibly a crucial intermediate in proviral integration (Jolicoeur and Rassart, 1981). Fv-l restriction is, however, not absolute, since it can be overcome by infection ofa single cell with two or more virus particles (Pincus et al., 1975; Declkve et al., 1975; O'Donnell et al., 1976). The abrogation of Fu-l restriction by high multiplicity of infection has shown that infection by a single particle of the wrong tropism, even if it does not replicate, is sufficient to allow replication of coinfecting virus of the same tropism (Duran-Troise et al., 1977). The viral gene product determinants for N- and B-tropic host range appear to reside in the p30 structural protein by peptide mapping analysis (Hopkins et al., 1977; Schindler et al., 1977; Gautsch et al., 1978),although certain N- and B-

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DANIEL MERUELO AND RICHARD BACII

tropic isolates were found to differ in other structural proteins by use of isoelectric focusing (Pfeffer e t al., 1976). The viral determinants of N-, B-, and NB-tropism appear to be allelic on the basis of oligonucleotide mapping (Faller and Hopkins, 1977) and RNA sequencing (Rommelare et al., 1979). The gene product of this viral locus is present in infecting virions, and in Fv-l-restricted cells it participates in an interaction between newly synthesized viral DNA and the Fu-l gene product in some way which prevents successful integration of viral DNA into cellular DNA. Studies to identify the cellular gene product involved in Fv-1 restriction have shown that soluble extracts from uninfected Fv-l -restrictive cells are able to transfer resistance to Fv-1-permissive cells, if added shortly prior to or after virus infection (Tennant et al., 1974). Very little of this gene product, a cytoplasmic RNA molecule (Yang et al., 1978), can be found in cells. This scarcity of the material probably accounts for the fact that only relatively weak levels of resistance have been obtained in transfer experiments and for difficulties hampering progress in achieving a molecular understanding of the mechanism of Fv-1 restriction. While no mutants have been generated either in vivo or in vitro which fail to show FG-1restriction of either N- or B-tropic viruses, the study of Fv-l function might be helped by availability of Fv-1 congenic mice and the existence of several mouse cell lines which show an Fv-1 nonrestrictive phenotype (Hartley and Rowe, 1975). 3. Organotropism Newborn mice infected with murine type C viruses such as Gross, AKR, and RadLV viruses, develop a specific thymus-derived leukemia after a latency of several months (Gross, 1970; Kaplan, 1967). The development of leukemia follows the appearance of high titers of infectious leukemia virus in blood (Lilly et al., 1975). Using mice infected with Moloney-MuLV, molecular hybridization experiments have shown that virus-specific DNA sequences can be found only in target lymphoid organs, i.e., the thymus and the spleen (Jaenisch et al., 1975; Jaenisch, 1976).Virus-specific sequences are not detected in nontarget organs, such as kidneys, liver, brain, testes, muscle, and lungs. It would appear that injection of MuLV into newborn animals leads to specific infection of a restricted set of target cells, such as thymus-derived lymphocytes or other lymphocytic cells. It is probably through replication in the latter cells that virus makes its way into the bloodstream or infects mammary epithelial cells which then secrete high titers of infectious virus into the milk (Jenson et al., 1976).

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Other tissues, such as the germ line cells, very rarely seem to be infected. Shoyab and Baluda (1975) have made similar observations in chickens infected with avian myeloblastosis virus. In mice, McCrath et al., (1978a) have observed target specificity with mammary tumor virus (MMTV). Organotropism of infection, as described above, applies to the in uivo interaction, i.e., the infection of animals with virus and not necessarily to the in oitro situation. Thus cells which cannot be infected in uivo are known to support the replication of virus in uitro. For example, fibroblast cultures infected with Moloney-MuLV (M-MuLV) in vitro replicate virus efficiently and can be used to prepare virus stocks. In the animal, however, fibroblasts are not usually susceptible to infection and replication of M-MuLV. A second caveat in defining organotropism is that this tropism for infection can be different from that for transformation by any one virus. For example, while both T and B cells are susceptible to productive infection by MoloneyMuLV, virus-induced transformation (leukemia) is restricted to thymus-dependent T cells (DeclBve et al., 1974; Waksal et al., 1976; Baird et al., 1977). This tissue tropism (organotropism) has he€ped identify an additional polymorphism (to those described in earlier sections of this article) among viruses. For example, the polytropic viruses (PTV) of HRS/J mice differ from the HRS/J prototype ecotropic virus (ETV-1) in their tissue tropism. At least four isolates of PTVs are highly thymotropic in both HRS/J and CBA/J mice (Green et al., 1980). By contrast, ETV-1, when tested in virus-free CBA/N mice, infects thymus, spleen, and bone marrow to about the same extent (Green et al., 1980). Another example is provided by study of isolates of radiation-induced leukemia virus. Several type C viruses with distinctively different cytotropisms have been recovered from mice of strain C57BLKa (Dec k v e et al., 1976, 1978; Lieberman et al., 1979). The radiation leukemia virus (RadLV) and its tissue culture version (RadLVNL3) induce thymic lymphomas after inoculation into C57BWKa hosts, whereas three other isolates, designated BL/Ka (B), BUKa (N), and BL/Ka (X), are devoid of leukemogenic activity (Declgve et al., 1976; Lieberman et al., 1977). RadLV and RadLVNL3 are B tropic (for Fo-1) and thymotropic because they replicate preferentially in thymic lymphocytes of adult mice. The other three viruses are designated fibrotropic since they productively infect fibroblasts but not thymocytes ( i n uitro) of appropriate genotype: Fu-lbbfor BWKa (B), Fu-1"" for BWKa (N), and nonmurine cells for BL/Ka (X). The mechanism by which tissue-specific restriction operates ap-

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DANIEL MERUELO AND RICHARD BACH

pears to be distinct from that of Fu-1. For example, viral DNA from Band N-tropic virus-infected cells can transfect fibroblasts of either Fu-lbb or Fu-I"" genotype, because the viral integration block of Fu-1 is skipped. Blocks in the virus infection process such as Fu-1 are by-passed by transfection. On the other hand, the capacity of thymocytes and fibroblasts of different genotype to undergo transfection with DNA isolated from cell lines previously infected with the Becotropic, B-fibrotropic, or B-thymotropic-RadLV isolates i s not equal (Kopecka et al., 1980). For example, transfection of fibroblasts is not achieved with DNA from a thymotropic virus-producing cell line (Kopecka et al., 1980). Thus, viral organotropism is determined by mechanisms not surmountable by transfection.

H. PROXIMITY OF PROVIRUSES, HISTOCOMPATIBILITY, AND LYMPHOCYTE ANTIGENLOCI 1. A Hypothesis Several hypotheses have been postulated to account for the mechanism(s) responsible for organotropism. One of these is that tissuespecific activation of endogenous viruses might be a consequence of the specific chromosomal integration site of the virus. Expression or repression of genetic elements of different viruses might be under control of different cellular loci involved in normal tissue differentiation, These loci might exert their effect in a cis-specific manner, controlling gene expression downstream (Jaenisch and Berns, 1977). Thus, leukemogenic endogenous viruses might be integrated at chromosomal sites of the mouse which are not expressed early in embryogenesis. As a result of normal tissue differentiation, virus-related loci might become activated in cells of the lymphatic-erythropoietic lineages. If this were the case, integrated viral genomes would remain silent in all other organs and cells of the developing and adult mouse in which the specific differentiation loci remain silent (i.e., all nonlymphoid organs). Such a cis-acting mechanism for the regulation of tumor virus expression in avian cells has been suggested by the data of Cooper and Silverman (1978).The different patterns of virus gene expression seen in various mouse strains could also be explained by the simple assumption that in each instance a virus gene is integrated at a different chromosomal region which is active at specific stages of development or in certain tissues but not at other stages of development or in other tissues. We would like to propose that the speci$c integration sites are spec$cally near histocompatibility loci (minor and major) and lymphocyte differentiation loci encoding Ly antigens. We shall develop this hypothesis further shortly.

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This hypothesis to account for organotropism is reasonable in view of recent experiments by Jaenisch et al. (1981), in which strains of BALB/Mo mice were derived which carry the exogenous Mo-MuLV genome as stable Mendelian genes. Mice carrying virtually identical proviral sequences integrated at different chromosomal loci showed different phenotypes of virus expression, placing emphasis on the regulatory function of adjacent regions of cellular DNA (see Section 111,H74).By extension, this work implies that although proviruses may integrate randomly into cellular DNA during infection, the site of integration is not inconsequential for virus expression. This hypothesis is also compatible with recent experiments by Dina and Penhoet (1978), which suggest that the various integrated copies of Mo-MuLV and MSV in infected fibroblasts are expressed at different rates. How could one account for differential rates of expression? The answer might lie-in the chromatin structure. There are some suggestions that actively transcribing chromosomal sites have a different chromatin structure. Consistent with this notion is the fact that the chromatin structure of the actively transcribed Moloney-MuLV genomes in target cells of BALBfMo mice shows altered sensitivity to digestion with DNase I compared to the repressed Moloney-MuLV genome in nontarget tissues (Breindl and Jaenisch, 1979).Groudine et al. (1978) have made similar observations for the integrated genomes of several endogenous and exogenous avian tumor viruses. In addition, it has been demonstrated previously (Hayward and Hanafusa, 1976) that endogenous and exogenous viral genomes, which are presumably integrated at different chromosomal sites, are subject to different control mechanisms.

2. Studies with RadLV The genetic mapping of endogenous viruses in different inbred mouse strains is in its infancy. Thus it would appear premature to draw firm conclusions about the relation of chromosomal integration of a virus and its expression on the differentiated cell. Nonetheless, our studies with radiation leukemia virus (RadLV) have revealed some interesting, relevant findings. Association between malignant disease and the murine major histocompatibility complex H-2 (Fig. 6) was first noted by Gorer (1956)and Gross (1970), who observed that all strains showing a high incidence of spontaneous or induced leukemia had the same H-2 haplotype. Since then, H-2 associations with susceptibility or resistance to virally induced leukemias have been extensively demonstrated (Meruelo and McDevitt, 1978).

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SUBREGIONS REGIONS

DANIEL MERUELO AND RICHARD BACH

H-2K

I r - U Ir-I9 10-4 la-5 Ir-1C

-A

-K

la-3 Sip --

10-1

-B -J -E -C I -

Ss

5 -

H-20 H-2L

-1

0 -

00-100-2 TLo

TL -

FIG.6. Partial genetic fine structure of mouse chromosome 17 (linkage group IX) encoding H-2-TLa complex. The complex is divided into five main regions: K, I, S, D, and TLa.These regions are divided by marker loci H-2K, Ir-1, Ss(S l p ) , H-2D, and TL. The boundaries of each region are defined by inba-H-2 recombinations. The I region has been subdivided into five subregions by recombination: A, B, J, E, and C defined by marker loci Is-1A (la-l), lr-lE, Ia-4, IQ-5and Is-1C (la-3), respectively. By convention, the K end of the complex is the segment to the left of Ss. The D end is the segment to the right of Ss. Alleles are alternate genes at defined loci, and haplotype designates the specific combination of all alleles at all loci within the complex characterizing a given mouse strain. Allele and haplotype designations are noted in lower case letters, whereas regions, subregions, and marker loci are in capital letters. The TL region is subdivided by marker loci QQ-1,Qa-2, and TLa.

We have been investigating one of these associations after (Meruelo

et al., 197%) demonstrating that resistance to RadLV-induced neoplasia is associated with gene(s) in the D-region of the H-2 complex.

Pertinent to the question of organotropism (as explained below) and relevant to the mechanism of action of H-2D genes in conferring resistance to RadLV is the finding that dramatic changes in the quantitative expression of cell surface H-2 antigens (particularly H-2D) occur following intrathymic RadLV inoculation (Meruelo et d., 1978). Studies measuring incorporation of [35Slmethionine strongly suggest that changes in expression of H-2D molecules reflect increased synthesis of this determinant after virus infection rather than simple uncovering of additional “buried” H-2D molecules (Meruelo et al., 1978). Several hypotheses to account for the observed induction of H-2D antigen(s) expression by RadLV have been suggested and tested (Meruelo, 1980). Our findings support the notion that increased H-2 expression results from RadLV integration at or near H-2D or genes that affect H-2D transcription. Thus our studies have shown (1)that ultraviolet inactivation (5020 ergs/mm2) or X-irradiation (400,000 rads) is sufficient to prevent RadLV-induced increases in synthesis and expression of H-2D antigens (Meruelo and Kramer, 1981). It has been shown (Decleve et aZ., 1977%)that such treatments are sufficient to lower infectivity drastically without affecting any of the viral proteins or viral penetration of target cells. Therefore, the effect of these treatments must be on the viral RNA so that it remains untranscribed or

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defective DNA copies result. (2) Intrathymic inoculation of RadLV in resistant mice leads to increased H-2D expression as long as these mice are of the Fu-lbb genotype. Mice of the Fu-1"" genotype which restricts RadLV replication fail to show increased H-2D antigen expression. The Fu-1 locus has been shown to restrict viral replication by blocking integration (Jolicoeur and Baltimore, 1976). (3)Inoculation of hybrid mice (resistant by susceptible F1) with RadLV leads to increased cellular expression of resistant but not of susceptible haplotype antigens. Thus the inductive mechanism augmenting H-2D synthesis does not operate in a trans mode (Meruelo and Kramer, 1981). (4) Agents that prevent integration of closed circular double-stranded DNA into the host genome, such as ethidium bromide (Guntaka et al., 1975) and fluorodeoxyuridine (FUdR) (Sveda et al., 1976), inhibit RadLV-induced increases in H-2D antigen expression (Meruelo and Kramer, 1981). Thymidine, which blocks the effect of FUdR on virus integration (Sveda et al., 1976), also blocks the effects of FUdR on RadLV-induction of H-2D antigen expression (Meruelo and Kramer, 1981). The findings described above encompass several concepts related to organotropism. First, RadLV integration after infection may occur randomly, but the interaction with or integration at one site must occur consistently, since RadLV infection always results in deregulation of H-2D synthesis in resistant mice. Second, such an interaction must occur early on and remain stable for a long period, since deregulation of H-2D synthesis is routinely observed by 36 hr after virus infection and persists for at least 12 weeks (Meruelo et al., 1978). Third, insertion at such a site must be kinetically important since H-2D synthesis and expression is enhanced. Furthermore, the integration site must be different between susceptible and resistant mice, since enhanced synthesis of H-2, which we currently attribute to increased transcription, occurs in resistant but not susceptible mice. Since resistant and susceptible are congenic pairs differing only at H-2D, one can go further and speculate that the integration of RadLV must occur at H-2D, or that H-2D has an effect on distant integration sites. The former possibility, which is easier to conceive (mechanistically), implies that H-2D DNA sequences determine the viral integration site(s). Recognition of some H-2D sequences will lead to integration and promotion of transcription, whereas other H-2D sequences will not lead to recognition and integration. Not yet discussed is the fact that overt leukemia, which occurs approximately 100% in susceptible mice and to a much lesser degree in resistant mice (Meruelo et al., 1977b), decreases H-2 synthesis to un-

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DANIEL MERUELO AND RICHARD BACH

detectable levels (Meruelo et al., 1978). Our preliminary results suggest that H - 2 methylation (Meruelo et al., unpublished) and rearrangements (Meruelo et al., 1983c) induced by RadLV transformation are responsible for the shutdown of H-2K and H-2D synthesis. While the results do not yet directly demonstrate the specific integration of RadLV related information directly at H-2, they suggest this very strongly. More recently examining a cosmid library of H - 2 genes we have found viral sequences proximal to H - 2 genes (Meruelo et al., unpublished). Therefore, the total evidence clearly supports the theory that specific viral integration sites are located at histocompatibility loci and loci encoding lymphocyte differentiation antigens. We shall provide additional data in the next section.

3. Differentiation-SpeciJc Loci and Genes Related to Leukemia Viruses and Leukemogenesis If the above findings are relevant to organotropism, one would expect to find additional examples of similar interactions. Figure 7 provides information suggesting that viral integration may occur often at or near loci coding for differentiation-specific determinants. “Productive” viral loci are those recognized to be of importance in some aspect of leukemogenesis or viral infection or induction. For example, the locus coding for xenotropic murine leukemia virus inducibility ( B m - I ) has been mapped to the same location on chromosome 1 (Kozak and Rowe, 1980a) as the major lymphocyte activating determinant 1

2

7

4

a

9

17

0

H-22 H-24

-H-2 -Tla

- H-31

-~-32

H-15

Ly-15

F s - -H-7 H-29

Fu-1 H-20

ECO/BC

FIG.7 . Correlation between virus-related mendelian loci and chromosomal sites rich in differentiation antigens.

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locus (Mls) (Festenstein et al., 1977). The recombination frequency between Pep-3 and Mls is 0.18 0.04 (Festenstein et al., 1977), and that between Pep-3 and Bxu-l is 0.20 k 0.05 (Kozak and Rowe, 1980a). Both loci are on the noncentromeric side of the Pep-3 locus. A locus on chromosome 4 affecting expression of xenotropic virus structural components, XenCSA (Morse et al., 1979),is flanked by loci coding for minor histocompatibility antigens H-15, H-16, H-20, and H-21, and by lymphocyte determinant(s) Ly-22.2 (Meruelo et al., 1983a).Aku-l, a locus encoding ecotropic viral genetic information on chromosome 7, is flanked by loci coding for minor histocompatibility antigens H-22 and H-24 (Rowe and Hartley, 1972). Fu-2, a locus that confers total resistance to the erythroleukemic disease induced by Friend virus (FV) infection, is situated adjacent to the locus for minor histocompatibility -7, H-7, on chromosome 9 (Lilly and Pincus, 1973; Axelrad, 1966). TL, a locus coding for antigenic determinants on thymocytes and leukemia cells, has been mapped less than 2 recombination units to the left of H-2D on chromosome 17 (Boyse et al., 1964, 1966). Ever since thymus leukemia antigens (TL) were first described, suggestions have been made about their possible origin from a viral genome integrated in chromosome 17. This was generally supported by the fact that TL- to TL+ phenotypic conversion was always diagnostic of malignancy (Stockert et al., 1971). Recently, this notion has come into question (Old and Stockert, 1977) principally because the molecular weight of the antigens on SDS gels is 45,000rather than the molecular weight expected for any known viral glycoproteins. However, Elder et aE. (1978) have shown that the enu product of MuLV, although 70,000 in molecular weight, is derived by glycosylation from a 45,000-molecular weight protein. Further support for the notion that TL may be somehow related to MuLV has been provided by Gazdar et al. (1977) and Ruddle et al. (1978). Their work on somatic cell hybrids indicates that all hybrid clones capable of replicating ecotropic MuLV retain mouse chromosomes 5, 15, and 17. These chromosomes may contain genes important in virus replication or cell-virus interaction. A locus involved in susceptibility to murine irradiation leukemogenesis, El-I, is adjacent to minor histocompatibility locus H-30 on chromosome 2. This region of chromosome contains in addition loci coding for minor histocompatibility antigens H-3, H-13, and H-30, and differentiation antigens Ly-6, Ly-8, Ly-11, Lym-11, H9/25, ThB, DAG, and ALA-1 (Meruelo et al., 1981, 1982). In addition another locus affecting susceptibility to RadLV induced leukemia is found adjacent to H-3 (Meruelo et al., 198313).Furthermore, in parallel with observa-

*

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DANIEL MERUELO AND RICHARD BACH

tions that histocompatibility and differentiation loci are closely associated with genes affecting irradiation and RadLV leukemogenesis, Haughton and collaborators have shown (G. Haughton, personal communication) that H-4-H-2 interactions are critical for the development of B cell lymphomas. Among unmapped loci, Ac-1, a locus controlling susceptibility to Abelson virus-induced lymphogenesis in mice, is closely linked to H 36 (Risser et al., 1978), a minor histocompatibility locus mapping on chromosome 2 (Meruelo et al., 1982). These observations receive further support from our recent observations. Several polymorphic DNA restriction fragments by bridging with xenotropic and ecotropic envelope viral probes map adjacent to minor histocompatibility and lymphocyte antigen coding loci. Viral restriction fragments are associated with Ly-17on chromosome 1, H-30, H-3, and H - 1 3 on chromosome 2, H-16 on chromosome 4, Ly-21 on chromosome 7, H - 2 8 on chromosome 3, and H - 3 8 (chromosome location undetermined) (Meruelo et d.,1983d). It is, therefore, worthwhile to explore the concept of organotropism further. 4. The Integration Sites of Endogenous und Erogenous Moloney

Murine Leukemia Viruses Perhaps the strongest data supportive of the mechanism(s)proposed to account for organotropism come from the studies of Jaenisch and collaborators (1981).The integration site of a virus can be characterized by digestion of cellular DNA with a restriction enzyme that does not cleave within the viral genome itself. When coupled with separation of digested DNA fragments on agarose gels and hybridization with specific viral probes (“blotting” technique; Southern, 1975), such studies can serve as a sensitive method to compare integration sites of viruses and to investigate the specificity of integration as first exemplified by characterization of the integration site of DNA tumor viruses by Botchan et ol. (1976) and Ketner and Kelly (1976). The integration site of a number of type C viruses has now been studied using these techniques. Such studies have generally established the principle of random integration of viruses in host DNA. While Battula and Temin (1977) concluded that reticuloendothelius virus (REV) integrates at a unique site upon infection of chicken cells by infectivity studies, molecular hybridization has shown that multiple integration sites exist for REV (Battula and Temin, 1978; Keshet and Temin, 1978). The discrepancy between infectivity and hybridization experiments can probably be explained by postulating the existence of integrated subgenomic fragments which are not infectious

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but are detectable by hybridization. Similar studies by Steffen and Weinberg (1978) using rat cells infected with Moloney-MuLV and by Hughes et al. (1978) with chicken cells infected with avian sarcoma virus (ASV)have shown that viral genomes (or genome fragments) are found integrated at multiple sites. At the genetic level, the basis for the difference in expression of these genes is poorly understood. This is partially due to the presence of multiple copies of viral sequences in almost all mouse strains. Progress in this area, however, has recently been made by Jaenisch and coworkers (1981). These workers have introduced the well-defined exogenous Moloney leukemia virus (M-MuLV) into the germ line of mice, and prepared probes for M-MuLV which do not cross-hybridize with endogenous viruses (specific cDNA, Berns and Jaenisch, 1976; Jaenisch, 1977). After introducing the M-MuLV into the germ line of mice and preparing appropriate probes, Jaenisch et aZ. developed several new substrains of mice, BALB/Mo, which carry the exogenous MMuLV as an endogenous gene (Jaenisch et al., 1981). These sublines differ only in the fact that the M-MuLV genome is associated with different chromosomal sites as determined by restriction analysis. Thus, when the M-MuLV-specific cDNA probe was used, no labeled band was detected in EcoRI digested DNA isolated from uninfected mice (Jaenisch et al., 1981). In contrast, in DNA extracted from the nontarget organs of BALB/Mo mice, a single DNA fragment of 16 x 106 daltons was detected (van der Putten et al., 1979; Jahner et al., 1980). One of the substrains generated (BALB/Mo) carries M-MuLV specific sequences at a single Mendelian locus, designated Mou-1, and located on chromosome 6 (Jaenisch et al., 1978; Breindl et al., 1979). The presence of this dominant gene is associated with early viremia and high incidence of leukemia. Other BALB/c substrains of mice carrying M-MuLV at other distinct genetic loci were derived experimentally by similar germ line integration procedures (Jahner and Jaenisch, 1980). These three new substrains were designated as Mou-2, Moo-3 and Mov-4 (Jahner and Jaenisch, 1980). Each of these new substrains carry a single M-MuLV genome (integrated on a different chromosome (Jahner and Jaenisch, 1980). Mice carrying the Moo3 gene develop early viremia and die rapidly of leukemia, whereas animals transmitting the Mow2 gene express virus only occasionally, and then late in life (Jahner and Jaenisch, 1980). The M-MuLV proviruses integrated at the M o w 2 and M o o 3 loci are identical as judged by restriction enzyme analysis. In addition, the viruses activated from Mou-2, Mou-3, and BALB/Mo ( M o u - I )

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are identical by biological and biochemical criteria. The differences in expression thus appear to be determined by the chromosomal location of the virus, and cannot be explained b y arguments that differences in expression are due to defective virus genomes being associated with some chromosomes and not others. (However, the M-MuLV genome in Mou-4 mice was shown to have a partial deletion and no virus expression was observed in these animals.) Expression of the same endogenous virus in specific tissues and not in others, as observed in several systems (Cooper and Silverman, 1978; O’Rear et al., 1980), might be explained by the existence of cisacting cellular control elements which activate adjacent cellular genes during differentiation in specific tissues. This notion is supported by preliminary experiments (Jahner and Jaenisch, 1980; Jaenisch et al., 1981) in the M-MuLV system described above. Thus, transfection experiments performed with liver DNA from Mou-1,Mou-2 and Mou-3 mice indicated that XC plaques are induced only with Mou-3 DNA. This observation is compatible with a cis-acting control element determining the expression of the adjacent provirus, since the regulation of virus expression observed in uivo is not disrupted, despite the fact that DNA is sheared into several fragments to enhance transfection.

IV. Expression in Inbred Mouse Strains of Antigens Associated with MuLV

A. GIx AND GCSA The first definition of an antigen associated with MuLV and expressed at least under certain circumstances in normal cells became possible with the recognition that rat antisera to syngeneic MuLVinduced leukemias detected a broader spectrum of MuLV antigens than had been seen with mouse antisera (Geering et al., 1966). The probable explanation for this observation is that rats, which appear to lack endogenous MuLV, are not only highly susceptible to leukemia induction by MuLV, but also able to make a strong humoral response against these antigens because they are not part of the “self” repertoire. However, precisely for this reason, MuLV induced leukemias in rats are strongly immunogenic and readily rejected on transplantation unless they are passaged in immunologically immature recipients. Alternatively, the strong immunological response can be overcome by transplantation of sufficiently large numbers of leukemia cells in adult animals. This results in progressively growing tumors in hosts whose

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sera contain high levels of cytotoxic neutralizing and precipitating MuLV antibodies. The GIx antigen system was first identified using one such rat serum (Stockert et al., 1971). More recently, the development of methods to concentrate and purify MuLV has permitted preparation of heteroimmune sera in rabbits and goats to intact virus and to isolated structural components. Another early reagent, a mouse anti-murine Gross MuLV-induced tumor cell line, led to detection of the Gross cell surface antigen (GCSA) (Slettenmark-Wahren and Klein, 1962). A cell line, designated ESG2, was one of many obtained from a large series of C57BL mice injected as newborns with Gross virus (Old and Stockert, 1977). Immunization against antigens reacting with E S G2 cells was done indirectly. C57BW6 mice were injected with a transplantable AKR spontaneous leukemia, K36. The C57BL antiserum showing highest titer against ESG2 was absorbed with a variety of Gross MuLV induced and spontaneous leukemias arising in mice of high leukemia incidence and shown to detect a common antigenic determinant shared by ESG2, K36 cells and all leukemias induced by MuLVGross and spontaneous leukemias (Old and Stockert, 1977). Furthermore, the reactivity of this serum against tissues of normal young mice from different inbred strains indicated a high correlation between occurrence of antigen in spleen and other lymphoid tissues and incidence of spontaneous leukemia. Thus, high incidence strains, AKR, C58, PL, and CSH/Figge, were antigen positive, whereas low incidence strains, e.g., C57BL, A, and BALB/c, lacked the crossreacting antigen. Because of this relationship of the antigen to the leukemia incidence associated with Gross virus among many inbred mouse strains, the antigen was named G (Gross) cell surface antigen (GCSA) (Old et al., 1965). It soon became clear, however, that GCSA was found not only in lymphoid tissues of high leukemia incidence strains, but was also found in normal and malignant tissues of low incidence strains. Therefore, it became apparent that expression of GCSA among low incidence strains indicated widespread infection of mouse populations with MuLV. Further evidence favoring the link of GCSA with MuLV was provided by electron microscopy analysis which revealed an excellent correlation between GCSA expression and occurrence of MuLV particles in both normal and tumor tissue (Old and Stockert, 1977). Although initially it appeared that G I and ~ GCSA were identical antigenic determinants, further study has shown that this is not so. For example, anti-GIx antibody [(W/Fu X BN)FI rats anti-W/Fu leukemia cells] is cytotoxic not only for normal thymocytes from the high leuke-

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mia incidence, GCSA' strains but also for thymocytes from some GCSA- strains (Old and Stockert, 1977). The original designation G(129) (Old and Stockert, 1977) was changed to GIxwhen it appeared that one of the two genes responsible for antigen expression resided in linkage group IX of the mouse (chromosome 17). However, this linkage has been questioned (Old and Stockert, 1977). Although the studies described above clearly suggested that GIX and GCSA were antigens coded for MuLVs proper it was only recently that this has been shown directly. Advances in the biochemical analysis of MuLV and new methods to define cell surface molecules has permitted demonstration that both GIx and GCSA are in fact viral structural components incorporated into the cell surface. GIX is a typespecific antigen of the major envelope glycoprotein of MuLV, gp70 (Obata et al., 1975; Tung et al., 1975). Even more recently, comparisons of GIx+ viral gp70 and GIX- gp70 (Rosner et al., 1980) and of RNase T1 oligonucleotides from the genomes of these viruses (DonisKeller et al., 1980) have suggested that GN phenotype relates to the glycosylation of gp70. These studies reveal that Glx- gp70 has one additional glycosylation site compared to GIx+ gp70; the GIX-phenotype may result from masking of the GIX antigen by the extra oligosaccharide chain. GCSA is related to the internal core proteins of MuLV, p30 and p15, which occur as glycosylated polyproteins on the surface of infected cells (Tung et al., 1976a; Snyder et al., 1977). B. X.l The X . l system was first defined by the rejection of certain X-rayinduced BALB/c leukemias (Sato et al., 1973) in BALB/c hybrids. While resistance to these X-ray-induced lymphomas was not demonstrable in BALB/c mice, it was easily seen in (BALB/c x C57BL/6)Fl hybrids. It was subsequently shown that this hybrid resistance to leukemia transplants was under control of an H-2 linked Zr gene (see Section V,F,2,a) derived from the C57BL/6 parent. Furthermore, humoral immunity (responsible for resistance) could be shown to recognize an antigen, designated X.1, in the inoculated BALB/c leukemia RL6 1cells. This antigen is present on BALB/c, A, and AKR leukemias and is unrelated to GCSA or GIX(Tung et al., 1976b). X.l is present in normal tissues of high leukemia strains, but unlike GCSA and GIx is present in very low levels. While the presence of X.l in the normal tissues of high leukemia incidence strains and its induction in leukemias of X.l- strains suggests an association with MuLV, more direct

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evidence is still needed to show that X . l is an MuLV-related antigen (i.e., induction in MuLV-infected cells).

c*G(RADA1)7 G(ERLD)>and G(AKSL.2) Since these early studies, several additional MuLV-related cell surface antigens systems have been detected with the aid of murine normal sera. It appears that sera from normal mice, particularly F1 hybrids and random-bred Swiss mice, provide a rich source of antibodies reactive with MuLV-related cell surface antigens. Three re(Old and cent examples are the discovery of G ( R A D A ~ ) and G(ERLD) Stockert, 1977), and G(AKSLP) (Stockert et al., 1979). The notation used indicates their relation to MuLV-Gross with the subscript designating the prototype leukemia cell lines used in their definition. Randombred Swiss mice are the source of G(RADAI) antibody, and (C57BL x 129) FI mice are the source of G(ERLD) antibody. Both G(mDA1) and G(ERLD)are found in normal and leukemic lymphoid tissues of strains with a high incidence of leukemia, and both can be induced in fibroblasts by infection with N-tropic MuLV (Old and Stockert, 1977). Sera from a normal AKR-Fv-lb and (C3H X AKR)F1 mice was used to define the G(NSL.2)antigen. This determinant is also found in lymphoid tissue from high leukemia-incidence strains, but appears to be related specifically to dualtropic virus; it could not be induced by most ecotropic or xenotropic MuLV, but was expressed by fibroblasts infected with several different dualtropic MuLV (Stockert et al., 1979). The strain distribution patterns of these antigens clearly distinguishes them from one another as well as from GIX,GCSA, and X.l. The relationship of G(ERLD) and G ( R A D A ~ to ) MuLV structural components has been investigated, and it is suggested that they are associated with envelope glycoproteins (Old and Stockert, 1977).

D. PC.l Antisera prepared against the BALB/c myeloma MOPC-70A in H-2 compatible DBA/2 were shown to react with BALB/c myeloma cells but not with normal thymocytes or thymic leukemias (Takahashi et al., 1970). However, despite its lack of reactivity with T normal or malignant cells, the serum could be shown by absorption to react with normal BALB/c cells. The distinctive tissue distribution of the antigen recognized when compared with other known surface antigens indicated that a novel antigen was detected. Since normal and malignant

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plasma cells (myelomas) expressed this antigen, it was called PC.l. After the PC.l system was defined, it was observed that the serum of BALBlc mice, the prototype PC.l+ strain, had cytotoxic antibody to BALB/c myelomas and that this BALB/c antibody appeared to be detecting an antigen with a strain and tissue distribution that was identical with PC.l (Herberman and Aoki, 1972). Similar antibodies were subsequently found in the sera of a number of other mouse strains, both PC.1+ and PC.l-. The widespread distribution of naturally occurring antibody to PC.1+ myelomas and the presence of MuLV in high frequency in myelomas suggested to some investigators that PC.l was coded for by MuLV. However, a PC. 1-inducing virus might also be expected to occur in myelomas arising in PC. 1- strains. To date, only one such instance has been reported (Herberman and Aoki, 1972), but the issue remains controversial and some authors believe that PC.l is the product of a conventional Mendelian gene (Old and Stocked, 1977). A new surface antigen (PC.2) expressed exclusively on plasma cells, and distinct from PC.l, has been recently defined (Tada et al., 1980). Anti-PC.2 antibodies are not directed at MuLV associated antigens.

E. TL The TL system of cell surface antigens was recognized during the course of a study of radiation-induced leukemias of C57BL mice (Old et al., 1963). Among several C57BL antisera, one, prepared against ASL1, a transplantable A strain spontaneous leukemia, had a high titer against a transplantable, radiation-induced leukemia, ERLD. This serum was used to define the TL system of antigens. While the antiserum appeared to be specific for leukemia cells in C57BL/6 mice, it reacts with normal as well as leukemic cells derived from A strain animals. Among normal cells, however, only thymocytes expressed the antigen. In normal mice, T L is inherited as a Mendelian dominant trait. Linkage studies have mapped the TL locus, designated TZa,on chromosome 17, approximately 2 units to the left of the D end of the H-2 complex (Boyse et al., 1964). The anomalous appearance of TL+ leukemias in mice of TL- strains is thought to indicate that all mice possess the structural gene for TL but that in TL- strains, the gene is not normally expressed (Boyse and Old, 1969; Old and Boyse, 1973). During leukemogenesis, however, the TZa locus must be derepressed or activated to account for the expression of TL antigens on the surface of leukemia cells (Stocked et al., 1971). The latter suggestion led to

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the postulate that the Tla locus might encode an integrated viral genome (Stockert et al., 1971). Although this has not been formally excluded, current understanding of the TL system makes it most unlikely (Old and Stockert, 1977).

F. ML Antisera to DBN2 leukemias prepared in histocompatible mice and absorbed in vivo to remove alloantibody led to definition of the ML system (Stuck et al., 1964). The absorbed antisera still retained cytotoxic activity for the immunizing leukemia and other DBN2 leukemias but not to spontaneous or induced leukemias of any other mouse strains. The identity of the antigen recognized was first suggested by the finding that the sera reacted with normal mammary tissue and spontaneous mammary tumors of mice infected with mammary tumor virus (MTV). This restriction of antigen to leukemias of DBN2 mice and MTV-infected cells prompted the designation ML for mammaryleukemia. There is currently little doubt that the ML antigen is coded directly or indirectly by MTV (Old and Stockert, 1977). However, the reason for ML appearance in DBN2 leukemias is not clear. Molecular hybridization experiments have shown that genetic information related to MTV is present in all mouse strains (Varmus et al., 1973). However, expression is restricted to certain strains (MTV strains) and in these strains to certain tissues (as discussed previously in Sections III,G,3 and 11I,H7l.-4). Therefore, the detection of ML antigen in leukemia cells might indicate derepression of MTV genetic information in malignant lymphoid cells. Host regulatory factors would have to account for the fact that such derepression occurs only in DBN2 mice and, as recently reported, in GR mice (Hilgers et al., 1975). Alternatively, DBN2 and GR mice might express a unique leukemogenic virus that arose through genetic interaction with MTV. In fact, unique restriction fragments for MTV have been detected in GR mice (Michalides et al., 1981). Further biochemical definition of the ML antigen, examination of its relation to MTV structural components, and analysis of viruses obtained from ML+ leukemias are needed to definitively explain the above observations.

G . OTHERANTIGENS A diverse array of MuLV-related cell surface antigens have now been identified in mouse leukemia and there is every indication that the list will continue to grow. In this article we have listed only a few

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of these because we wish to make the reader aware of the existence of the class of antigens rather than attempt to provide a total compendium. The complexity and diversity of such antigens parallel that seen for MuLV envelope antigens (as alluded to in Section II1,G) (Aoki et al., 1974) and reflects the extensive MuLV polymorphism that has been previously discussed. The MuLV-related surface antigens do not appear to be transformation-specific, because they are also found on MuLV-infected but still untransformed cells. By contrast, avian and feline oncomaviruses express transformation-specific surface antigens that appear to be unrelated to viral structural proteins (Stephenson et al., 1977; Rohrschneider et al., 1975). Some investigators are actively pursuing detection and characterization of such antigens. V. Genetics of Susceptibility to Viral Infection

A considerable number of genes with the capacity to regulate or modify the replication and/or oncogenic process induced by C viruses have been identified. Although the detailed mechanisms by which they act are not fully understood for any of them, genes regulating neoplasia by retroviruses do so by acting at many different points in the development of the disease. We shall review the salient genes described to date. A. GENESAFFECTING VIRUSSPREAD To achieve substantial expression of type C viruses, several steps are required. First, one needs activation of chromosomally integrated viral genomes. Second, the infectious spread of virus from one cell to another within the population must occur. Exemplifying these steps is expression of virus in AKR mice. The ecotropic viral genomes represented by the Ako-l and Aku-2 loci are expressed as high levels of infectious virus in the tissues of AKR mice (Rowe et aZ., 1972). However, quantitative studies have revealed that only about 10%of spleen and thymus Iymphocytes initially produce virus (Mayer et at., 19781, followed by infectious spread of the virus pioducer trait to many other cells in the organ (Mayer et aZ., 1978). Similar findings have been obtained in ljitro (Rowe, 1972). Therefore, viral expression often occurs by exogenous infectious rather than by simultaneous activation of preexisting integrated viral genomes in many cells. Genes affecting induction and spread are, therefore, critical in susceptibility to leukemia. Induction genes generally represent integrated viral genomes and many of these have been defined including Akv-1, Akv-2 (Rowe et

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al., 1972; Kozak and Rowe, 1980a), Bxu-l (Kozak and Rowe, 1980a), Aku-3 (Rowe and Kozak, 1980),Aku-4 (Rowe and Kozak, 1980), Dbv (Jenkins et al., 1981), Blv (Jenkins et al., 1981), Fgu-l (Kozak and Rowe, 1982), Cu (Kozak and Rowe, 1979),Nxu-l and Nzu-2 (Datta and Schwartz, 1976,1977), Fgv-2 (Kozak and Rowe, 1982), C58u-2 (Kozak and Rowe, 1982), and Seu-l (Kozak and Rowe, 1982). Genes affecting virus spread are described below. B. ADSORPTIONAND PENETRATION Infection of cells by virus first requires successful adsorption and penetration. It has been shown by somatic hybridization that murine chromosome 5 contains a gene required for ecotropic virus infection (Gazdar et al., 1977; Oie et al., 1978) and coding for a cell surface receptor for ecotropic virus adsorption (Ruddle et al., 1978). Successful infection by pseudotype virus [consisting of a vesicular stomatitis virus (VSV) genome packaged into an ecotropic type C virus coat] of hamster-mouse hybrid cells is possible only in the presence of mouse chromosome 5, indicating an interaction between the host gene product(s) and the type C virus coat in adsorption. Likewise, host range restriction of xenotropic viruses by mouse cells appears to result from a virus penetration block, as even concentrated preparations of various xenotropic viruses show no replication in murine cells (Levy, 1978). Several experiments investigating blocks to xenotropic replication have shown an absorption/penetration block. For example, pseudotype particles with vesicular stomatitis virus cores and a xenotropic virus envelope were shown not to replicate (Besmer and Baltimore, 1977). The precise block has been more fully defined in experiments measuring absorption of xenotropic virus to nonpermissive mouse cells and permissive human and mink cells (Levy, 1978). In these experiments, residual titration experiments showed that absorption was similar in permissive and nonpermissive cells; therefore, the block operates at the level of virus penetration. Further experiments along these lines have shown that hybrids of mouse and human cells can be infected by xenotropic viruses only when somatic hybrid cells contain a complete complement of human chromosomes and very few mouse chromosomes (Gazdar et al., 1974; Scolnick and Parks, 1974), suggesting that an additional type of intracellular restriction to xenotropic virus replication may exist, regulated by the presence of murine DNA. However, the restriction in cells with murine genes has been explained by interference to viral penetration by viral genome glycoproteins on the surface of the so-

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matic cell hybrids derived from murine DNA (Besmer and Baltimore, 1977; Levy, 1978). Consistent with the blockage of penetration concept is the fact that embryo cells from some wild mice are susceptible to xenotropic virus infection. Susceptibility is dominant since hybrid mice of these wild strains with the usual inbred mouse strains are susceptible (Stephenson and Aaronson, 1977). Inbred mouse strains generally available must, therefore, lack the wild-type allele. In addition to restrictions by cellular membrane proteins or receptors, other factors can block adsorption of penetration by inactivating virus infectivity. For example, the sera of most normal mouse strains have been found to inactivate xenotropic type C virus infectivity (Aaronson and Stephenson, 1975), the NIH strain being a notable exception. This inactivating activity is not mediated by immunoglobulins (Levy et al., 1975; Fischinger et al., 1976), but rather by a serum lipoprotein (Levy, 1978). This lipoprotein is a high-density, triglyceride-rich lipoprotein, and can be converted from high-density to very low-density lipoprotein in uitro.

C. Fu-1: RESTRICTIONOF INTEGRATION Mouse chromosome 4 carries a gene closely linked to the Gpd-l locus that can interfere with infection of mouse cells by type C virus at a postpenetration step (Rowe and Sato, 1973). The mechanisms by which Fu-1 mediates restriction of virus spread have been alluded to earlier (see Sectioin 1117G,2). Restriction does not involve a membrane-receptor phenomenon and occurs after virus absorption and penetration, as “pseudotype” viruses with cytopathic vesicular stomatitis cores and N- and B-tropic type C virus envelopes showed similar cytopathic effects (Huang et al., 1973; Krontiris et al., 1973). Fu-1 resistance is relative and can be overcome at high input viral multiplicity of infection (Pincus et al., 1975; Declbve et aZ., 1975; O’Donnell et al., 1976). Once productively infected, Fu-1 -resistant and -sensitive cells produce virus in comparable amounts with similar latent periods (Pincus et al., 1975; O’Donnell et al., 1976); however, the length of the viral latent period varies with the multiplicity of infection, independent of Fu-1-mediated effects (O’Donnell et al., 1976).Abrogation can be affected using viruses inactivated by heat or X-irradiation (Bassin et al., 1978), suggesting that the virus need not replicate to overcome Fu-1 resistance. Early studies suggested that Fu-l restriction operates at a point prior to proviral integration (Jolicoeur and Baltimore, 1976).Jolicoeur and Rassart (1981) have shown recently that while synthesis of linear

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proviral DNA is unaffected by Fu-l, accumulation of closed circular DNA is impaired in resistant cells. They hypothesized that the Fu-l gene product may either block circularization of linear viral DNA directly or promote synthesis of a defective linear DNA which cannot circularize (Jolicoeur and Rassart, 1981).It is presumed that the supercoiled closed circular intermediate may be a critical species in the process of proviral integration. Tennant et al. (1974) have indicated that cytoplasmic fractions derived from Fu-l resistant cells transfer relative resistance to sensitive cells with two-hit dose-response relations (Tennant et al., 1974). Treatment with ribonuclease but not with deoxyribonuclease or proteases is able to abolish transfer of resistance (Tennant et d., 1976), and RNA preparations have been shown to transfer specific Fu-l resistance (Yang et al., 1978), implying that some RNA species are involved in Fu-I resistance. At least two Fu-l alleles have been described. Probably more than two exist since analysis of leukemogenesis in AKR X R F hybrid mice (both Fu-I"") has shown significantly lower leukemia incidence in hybrids of R F types Gpd-lamice than in those of the Am-type Gpd-lb (Mayer et al., 1978). Since Fu-l is linked to Gpd-I, and since both R F and AKR mice are supposedly Fu-I"", the Fu-l alleles of RF and AKR mice must differ. There is also accumulating evidence that the Fu-l" alleles in DBN2 and NIH mice are different. Cells of DBN2 mice show greater sensitivity to B-tropic viruses and somewhat reduced sensitivity to N-tropic viruses than other Fu-1" strains (Pincus et al., 1971a). In addition, DBN2 cells do not behave as NIH, C3H, and C57BL cells in sharing reduced efficiency in infectious center plating of certain B-tropic viruses (Pincus et al., 1975). Furthermore, B-tropic viruses which have been passaged on NIH cells so that they can grow in Fu-In or Fu-Ib cells with almost equal ease (i-e., with titers in NIH and BALB/c cells within 0.5 loglo of one another and single-hit titration patterns in both cells), do not grow efficiently in DBN2 cells, again suggesting a different allele at Fu-l for DBN2 versus NIH cells. Despite the almost universal restriction of virus replication in uiuo and in uitro associated with Fv-1, two cell lines have been identified which do not show evidence of Fu-I resistance. One of these is a subline of NIH 3T3 cells termed 3T3FL (Gisselbrecht et al., 1974), and a second group consists of a series of cloned cell lines derived from fetal mouse embryos, termed SC-1, SC-2, etc. (Hartley and Rowe, 1975).The SC-1 line known as IIIGA shows optimal sensitivity to both N- and B-tropic viruses in tissue culture assays, and has been used extensively for both isolation and titration of ecotropic viruses. SC-1

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cells have shown a stable phenotype of increased sensitivity to Btropic virus through greater than 100 passages (Hartley and Rowe, 1975). Its initial phenotype, however (the uncloned cell line), was found to be Fu-I", and many of the clones generated were insensitive to B-tropic virus (Hartley and Rowe, 1975). The interactions of N- and B-tropic viruses with Fu-ln and Fu-lb cells clearly constitute a reciprocal system, but N- and B-tropic viruses do not appear biologically equivalent for several reasons. First, all high-leukemia strains recognized, including AKR, C58, and C3H/Fg, are Fu-I", and show spontaneous infection with N-tropic viruses only. By contrast, B-tropic viruses are usually found in older mice and Fu-l mouse strains (e.g., BALB/c and C57BL/6) and are not generally associated with high frequencies of spontaneous leukemia. Second, Ntropic viruses are readily inducible by iododeoxyuridine from cells of most murine strains, as is xenotropic virus, whereas B-tropic virus is induced with difficulty in only a few strains (Stephenson and Aaronson, 1976). Third, while the phenotypes of N- and B-tropic viruses are in general quite stable, it is possible to broaden the host range of some viruses from B-tropic to NB-tropic after passage at high multiplicities of infection in NIH fibroblasts; however, broadening of host range for N-tropic viruses has never been found despite similar high multiplicity passage in BALB/c cells (Hopkins et al., 1976). Along with this extensively studied effect on exogenous infection of murine cells by MuLV, Fu-1 may also act at other levels in viral replication and expression. This has been suggested by work of Fenyo et al. (1980), who studied production of N-tropic virus in segregating populations of somatic cell hybrids. In these experiments, L cells (FuI") constituitively producing N-tropic "L virus" were fused with C57BL ( Fu-Ib) cells. The presence of chromosome 4 derived from C57BL (and, therefore, the restrictive Fu-Ib allele) in hybrids completely suppressed virus production, whereas loss of that chromosome during segregation resulted in reappearance of N-tropic virus production. These data would indicate that Fo-I restriction may have an affect in cells already carrying integrated proviral DNA. The in uitro data concerning Fu-1 function are reflected in studies of experimental leukemogenesis in mice. Thus, the susceptibility of mouse strains to Friend virus-induced disease (Lilly, 1967), RadLV and BE-L virus-induced leukemias, and Gross virus leukemia (Decleve and Kaplan, 1977) correlates largely with the permissive Fu-I allele. Studies of spontaneous leukemia in genetic crosses of AKR mice have also implicated the Fo-l gene as the major determinant of leukemia susceptibility (Nowinski et al., 1979).

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D. AVAILABILITY AND REPLICATION OF TARGET CELLS As stated earlier, infection and integration are not enough to ensure transformation. For example, MuLV infect fibroblasts in vitro, but such cells are not transformed either in vivo or in vitro. Transformation seems to require the availability of certain “target” cells whose growth regulatory program can be decontrolled by viral infection. Identification of these cells and the factors or genetic influence that mark them for transformation would significantly aid in understanding malignancy. Therefore, the subpopulations that serve as target cells for malignant transformation have been the subject of intense investigation. For example, radiation-induced leukemia appears to require an abundance of immature lymphoblastic cells that are present in the periphery of the thymic center during the first 2 weeks of life and decrease sharply in number thereafter (Kaplan, 1961; Axelrad and Van der Gag, 1962). After irradiation, the thymus has been shown to undergo injury characterized by a profound depletion of lymphoid cells followed by a period of regeneration, during which the same type of immature lymphoblastic cells are once again present for a period of several days (Kaplan and Brown, 1957). This short period of lymphoblastic cell regeneration may be critical for the restoration of thymus susceptibility to leukemogenesis in irradiated animals (Kaplan and Brown, 1957). A few relevant studies reflecting on target cell availability for leukemogenesis are described below in an effort to understand the role of these cells in the disease.

1. The Hairless Gkne During maintenance of the inbred mouse HRS strain, which carries the recessive hairless gene, hr (chromosome 14), it was observed that hairless homozygotes of this strain have a higher incidence (72%) of spontaneous lymphoma than their normal-haired littermates (20%) (Meier et al., 1969). Further study indicated that normal and hairless littermates had high titers of ecotropic type C virus in their tissues. On the other hand, hdhr mice have much higher titers of xenotropic virus activity in their thymuses shortly before developing lymphoma (Hiai et al., 1977). (The situation is analogous to the discussion of AKR leukemia in Section V,E,l.) Higher leukemia incidence in hrlhr mice may be related to an abnormality in differentiation associated with differentiation of the T lineage. At three months of age, hrlhr mice show increased Ly-1,2,3 and Ly-2 positive T cells, and decreased Ly-1+,2-,3- T lymphocytes when compared to their normal ( + / h r )littermates (Reske-Kunz et al.,

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1979). Whether this finding explains an immunological defect related to the inability of the hairless homozygote to suppress xenotropic virus expression and lymphomagenesis or provides a different ratio of target cells for xenotropic virus replication has not been investigated.

2. Fv-2 and Recruitment of Target Cells The recessive allele of Fv-2, FG-2‘ confers almost total resistance to the erythroleukemic disease associated with Friend virus infection. The mechanism of FG-2resistance is not yet understood, but it has been suggested that it might result from a reduced capacity of spleen virus (SFFV) to replicate in Fv-2’ cells (Blank et al., 197613).For example, it has been shown that Fu-2‘ impairs recruitment of neighboring target cells into infectious centers (Steeves et al., 1978). Similar restriction on SFFV replication is seen by other genes which reduce the number of erythroid target cells available for virus replication, such as loci involved in hereditary anemia, W (dominant spotting), S1 (steel), and f (flexed) (Meruelo and McDevitt, 1978). These loci probably have their effect because they alter the quality or quantity of target cells available to the virus. 3. T Lymphoma Retroviral Receptors and Control of T-Lymphoma Cell Proliferation

McGrath et al. (1980) have proposed a hypothesis for lymphoma induction based on the assumption of “fit” between a particular TMuLV and a particular subset of target cells in the thymus of susceptible hosts. Exact fit is expected to occur only on rare subsets of thymic cells whose receptors have specificity for the envelope glycoprotein of transforming MuLVs. According to their view, rare subsets of cells expressing receptors coded for by cellular genes are infected, transformed, and give rise to clonal progeny all bearing the same type of viral receptors. Furthermore, such receptors might represent antigenspecific receptors normally expressed by T lymphocytes. McGrath et al. (1980) have provided experimental data which they interpret to support this hypothesis. The evidence entails four principal observations. First, all cells in a particular thymic lymphoma bind T-MuLVs, whereas only 0.2-2% of normal thymocytes bind T-MuLVs (McCrath and Weissman, 1979). Second, binding appears to be fairly specific for the particular T-MuLV produced by the T lymphoma and is highly specific in competitive binding assays with even closely related T-MuLVs (McGrath et al., 1978a; McGrath and Weissman, 1979). Third, T-MuLV recognizes T cell receptors via their env glyco-

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protein products (McGrath et al., 1978b). Fourth, within the thymus of a T-MuLV infected host, only incipient T-lymphoma ,cells (not preleukemia cells) express cell-surface binding sites for leukemogenic TMuLV (McGrath and Weissman, 1979; McGrath et al., 1978~). These findings are of great interest in understanding the nature and availability of specific target cells for viral-induced leukemogenesis. Clearly depletion of target cells for a particular oncogenic virus should lead to enhanced resistance to the disease by the host. 4. Abelson Murine Leukemia Virus Target Cells in Mouse Bone Marrow Shinefeld et al. (1980)have reported the characterization of a monoclonal antibody which detects a surface antigen expressed by the bone marrow target cell of A-MuLV. Treatment of bone marrow cells with this antibody and complement results in a diminution of greater than 95% of the A-MuLV-derived in vitro transformed loci. The surface antigen detected by this antibody is also expressed on A-MuLV-transformed lymphoid cell lines, thymocytes, and some peripheral lymphocytes. This antigen is not expressed, however, by the pluripotent hematopoietic stem cells defined by the spleen colony-forming assay. The antigen detected is not a virally encoded product (Shinefeld et al., 1980).

5. Target Cell of Rauscher-MuLV Transformation Is a Pre-B Lymphoid Cell The phenotypic characteristics of Rauscher-MuLV-induced lymphoma cells appear to be those of immature B (pre-B) cells, which have been reported to synthesize small amounts of intracellular IgM, but to lack detectable surface immunoglobulin (Cooper et al., 1976; Owen et al., 1976, 1978; Raff et al., 1976). Recent evidence indicates that such cells express intracellular heavy chains in the absence of light chains (Burrows et al., 1979; Siden et al., 1979). Among cell extracts of different Rauscher-MuLV lymphoma lines tested in the competition immunoassay for mouse p chain, the level of p chain ranged from 30 to 700 nglmg of soluble cell protein (Premkumar et al., 1980). In contrast, neither spontaneous nor Moloney-MuLV-induced tumor cells contained detectable p chain. Neither kappa nor lambda chain was detectable (less than 1 ng/mg cell protein) in any of the Rauscher-induced lymphoma cell lines analyzed (Premkumar et al., 1980). These results suggest that the target cell for transformation by Rauscher-MuLV is a pre-B lymphoid cell.

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6 . Target Cells for Virat-Induced Leukemogenesis Are InfEuenced by the Organ Microenvironment Datta and Schwartz (1978) have studied the expression of ecotropic and xenotropic viruses, as well as the incidence of leukemia in the F1 progeny of crosses between AKR and NZB mice. These (AKR x NZB) Fl hybrids (ANFJ inherit four autosomal dominant loci which control expression of ecotropic and xenotropic viruses. Aku-1 and Akv-2, which determine the expression of ecotropic virus (Rowe, 1972; Chattopadhyay et id.,1975),are inherited from the AKR parent; and Nzv-1" and N z G - ~ "which , determine the expression of xenotropic virus (Datta and Schwartz, 1976, 1977), from the NZB parent. Despite the presence of these four loci, the incidence of leukemia in the ANFl mouse is very low and markedly delayed (Holmes and Burnet, 1966; Datta and Schwartz, 1978). In addition, restriction of ecotropic and xenotropic virus expression in ANFl is limited to thymocytes and peripheral T-lymphocytes. Datta and Schwartz (1978) have postulated that the thymus-specific restriction of virus expression (reduction in available target cells) in the ANFl mouse accounts for its unexpected resistance to leukemia. These investigators have postulated that the mechanism of retrovirus expression restriction in T lymphocytes of the ANFI mouse resides in the radiation-resistant thymic stroma (thymic reticuloendothelial elements) or in radiation-sensitive prothymic or thymic lymphoid cells. Datta et al. (1980) have shown that there is a specific augmentation of the expression of MuLV antigens and of ecotropic and xenotropic viruses in ANFl thymic lymphocytes when they were allowed to differentiate in irradiated, leukemia-prone AKR mice. By contrast, these phenotypic changes did not occur when ANFl thymocytes differentiated in irradiated ANFl and C57BR hosts, which have very low incidence of spontaneous leukemia (Datta and Schwartz, 1978; Rowe, 1972; Chattopadhyay et al., 1975). At least three interpretations are possible for these results. The first, postulated by Datta et al. (1980), is that the thymic microenvironment exerts a major influence on the expression of retroviral genes by thymocytes. This is supported by their argument that the thymic reticuloendothelial environment is solely responsible for the differentiation and maturation of bone marrow precursor cells to thymocytes (target cells) in lethally irradiated, bone marrow-restored chimeras (Von Boehmer and Sprent, 1976; Zinkernagel et al., 1978; Fink and Bevan, 1978; Sprent, 1978). Two other interpretations of their results are possible, one consistent with the hypothesis of McGrath et al. (1980) and the other with

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the findings of Teich and Dexter (1978). Both hypotheses would conclude that infection of ANFl cells by AKR virus in the ANFl- AKR chimeras provides proliferative potential to specific cells within the population giving them neoplastic potential. Teich and Dexter (1978) have presented strong evidence favoring the notion that murine leukemia virus infection of bone marrow cells in vitro can alter the program of differentiation. In fact, specific viral gene functions can, according to these investigators (Teich and Dexter, 1978),be defined in terms of capacity to induce specific differentiation properties. Furthermore, it is said that these effects are under control mechanisms of virus infection and replication by the cell analogous to the in uivo situation. On the other hand, the hypotheses of McGrath and Weissman (1979) and Lee and Ihle (1979) would postulate that chronic blastogenesis of ANFl lymphoid cells results in response to AKR viruses in the new chimera environment and the eventual proliferation of a malignant clone. Zielinski et al. (1982) have argued vigorously against alteration of differentiation and/or selective proliferation by MuLV infection. They argue that, as contrasted to the ANFl + AKR situation, there is no change in MuLV antigen expression or production of xenotropic virus in AKD/2 + AKR or ANFl-+ C57BR and AKR + ANFl chimeras. That is because C57BR and ANFl are nonproducer or low virus expressing strains, and AKD/2 donor mice lack the NZB genes needed for xenotropic virus expression. Furthermore, AKR or AKDI2 cells that were allowed to differentiate in AKR hosts did not generate either polytropic or xenotropic virus, although they did produce ecotropic viruses (Zielinski et aZ., 1982). In those chimeras the donor cells possess and express ecotropic viral genes, and they are not known to restrict the replication and spread of polytropic recombinant viruses (Hartley et al., 1977); yet, augmented expression of xenotropic and ecotropic viruses and generation of polytropic viruses (at 2 months of age) occurred only in the ANFl+ AKR chimeras. However, in the latter respect, the results of Zielinski et al. (1982) are different from those of Kawashima et al. (1976a,b). The latter authors have observed the amplification of MuLV antigens by thymocytes of syngeneic AKR + AKR chimeras. However, the difference reported by the two groups is age related. Kawashima et al. (1976a,b) did not observe MuLV antigen amplification in 2-month-old recipients, but only in 6-month-old preleukemic recipients, whose thymuses expressed xenotropic virus in high titers, as well as polytropic (MCF) particles. Zielinski et al. (1982) found MuLV antigen amplification in ANFl+ AKR chimeras when AKR recipients were as young

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as 2 months. At this age, the AKR thymus expresses only ecotropic virus. Zielinski et al. (1982) propose that the amplifying “signals,” which are unlikely to be either ecotropic, xenotropic, or polytropic virus, increase with time so that by the age of 6 months, the phenomenon can be detected in AKR thymocytes as observed by Kawashima et al. (1976a,b). Zielinski et al. (1982) concluded from their experiments that the functional status of the thymic reticuloendothelium is of particular importance in determining the expression of integrated retroviral genes by thymocytes, and the ANFl thymus lacks all of these properties despite the presence of target cells susceptible to transformation. 7. Ly-11.2: A Cell Surface Antigen of Znterest for Target Cell Studies Related to Leukemogenesis The above described results in different leukemia models reveal two important concepts. First, specific target cells exist for oncogenic viruses (e.g., Abelson, Rauscher, etc.). Second, the proliferation of such target cells before and after viral infection is affected by the microenvironment in which such cells replicate (e.g., the thymus epithelium). In contrast to the high selectivity of each virus for a particular target cell subpopulation, one subpopulation of T cells, those bearing Ly11.2, seems to proliferate rapidly during the preleukemic period in response to most thymoma-inducing agents (Meruelo et al., 1980a). Thus C58/J and AKWCum mice develop spontaneous leukemia some time after the fifth month of age (Meruelo et aZ., 1980a). Four split doses of X-irradiation (175 rads weekly) of 4- to 6-week-old mice have been shown to cause leukemia with incidences ranging from 75 to 100% (Kaplan, 1967; Pazmiiio et al., 1978). Intrathymic inoculation of RadLV into 3- to 6-week-old mice has been shown to induce leukemia with an incidence and latency period that depend on the strain of mice used (Kaplan, 1967). Whether leukemia arises spontaneously (as in A W J and C58/J mice) or is induced by RadLV inoculation or Xirradiation, a marked elevation in the expression of Ly-11.2 in the bone marrow and thymus is observed as animals progress from the healthy normal state to the preleukemic stage (Meruelo et al., 1980a). As the animal progresses to the more severe phase of the disease, a sharp drop in Ly-11.2 expression is observed (Meruelo et al., 1980a). The observed changes in Ly-11.2 are associated with leukemogenesis because no such changes occur in normal mice as they age (Meruelo et al., 1980a).The observed changes in Ly-11.2 expression during the

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preleukemic stage is due to an increase in the percentage of Ly-11.2 bearing cells. For example, during the preleukemic stages, the number of Ly-11.2 positive cells in the thymus increases from less than 5% to greater than 45% (Meruelo et al., 1980a). Several possibilities may account for the observed changes in Ly11.2 expression during leukemogenesis. One is that Ly-11.2 represents a virus-coded or virus-associated determinant. A comparison of the strain distribution of Ly-11.2 with that of other virally coded antigens or related traits (Meruelo et al., 1980b) shows that Ly-11.2 has a unique strain distribution when compared with Abelson, FE, XenCSA, X.1, G(ERLD),GIx, TLa, G(AKSLO), and G ( u D A 1 ) . In addition, no correlation can be found between Ly-11.2, Fv-1, and PC.1 phenotypes. Furthermore, a variety of tumor cells have been tested for Ly-11.2 expression and found negative (Meruelo et al., 1980a). Of these cells, several are actively producing complete, infectious virus and express viral components (gp70, p30, p15, p12, p10) on their cell surface. These data would tend to argue against a viral origin for Ly-11.2. Another possibility that could account for the observed changes in Ly-11.2 expression would be that during leukemogenesis, a particular functional subclass of T lymphocytes proliferates more rapidly than the rest. For example, Lee and Ihle (1979) have shown that during leukemogenesis, AKR-derived lymphocytes show an increased capacity to make a blastogenic response to purified viral antigens such as gp71. The response starts out low and peaks during the preleukemic period much in the same manner as Ly-11.2. However, changes in Ly11.2 occur only if leukemogenesis is under way, whereas increased blastogenic capacity occurs in mice that will not develop the disease (Lee and Ihle, 1979). Ly-11.2 antigens are present on prothymocytes, but not on several other functional lymphocyte subpopulations, including helper, suppressor, or cytotoxic T cells. The only subpopulation of cells involved in immune surveillance that have been found to bear Ly-11.2 to date, and hence that may proliferate in an effort to defend the host from neoplastic cells, have been natural killer (NK) cells (Meruelo et al., 1980a). Interestingly, in contrast to the increased percentage of Ly11.2 cells found in mice following leukemogenic fractionated X-irradiation, NK activity decreases sharply (Meruelo et al., 1980a). Another possibility that would account for the observed changes in Ly-11.2 expression during leukemogenesis is that Ly-11.2 is present in transformation-sensitive lymphocytes (TSL). During the early stages when these lymphocytes are proliferating, Ly-11.2 expression

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increases, but as the host’s immune responses begin to operate, the proliferating lymphocytes escape by shutting down Ly-11.2 expression. We have found that Ly-11.2 cells are singularly susceptible to infection by RadLV. During the first week after intrathymic inoculation of this highly leukemogenic virus, greater than 90% of virusinfected cells are Ly-11.2 positive (Meruelo et al., 1980a). In contrast, very few (10-20%) RadLV-infected thymocytes display other cell surface T cell phenotypes (e.g., Ly-1, Ly-2, Ly-3, Thy-1, etc.) (Meruelo et al., 1980a). The significance of this observation is strengthened by the fact that for a transient period of time (first 20 days) and then again in the preleukemic stage, Ly-11.2 antigen expression increases sharply on thymic cells of mice genetically susceptible to RadLV, but not in mice resistant to the virus (Meruelo et al., 1980~). If we presume Ly-11.2 to represent target cells for leukemogenesis, we are faced with the dilemma of explaining the distinct target found by others (described previously) for various MuLVs. At the moment, we have no explanation that accounts for this paradox. However, the importance of Ly-11.2 bearing cells in leukemogenesis cannot be easily discounted. We have mapped a major locus, Ril-1, conferring resistance to radiation-induced and RadLV-induced leukemogenesis to a site on chromosome 2 not distant from the locus coding for Ly-11.2 (Fig. 8). Furthermore, this chromosome has now been shown to contain the Abelson c-onc (Goff et al., 1981; P. D’Eustachio, personal communication). (A tentative location for this cellular oncogene is shown in Fig. 8.) Thus, a variety of loci involved in distinct types of leukemias are shown to map to the same chromosome, and this chromosome contains the locus coding for Ly-11.2. It is, therefore, clear that for the moment the concept of target cells for ieukemogenic MuLVs has to be qualified not only in terms of the role of the microenvironment, but also with regard to the fact that a T cell subpopulation singularly susceptible to viral infection proliferates rapidly during the preleukemic period in response to distinct oncogenic agents. B2M

Rii-l

e-jJ

H-30

Ly-8 ,The

Lr 6 I

I

H:13 a 1

1

FIG.8. Map of the H-30-Agouti region of chromosome 2. Markers shown are primar-

ily lymphocyte and erythrocyte differentiation antigens coding loci. Only selected unrelated loci have been included in the map.

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E . TRANSFORMATION 1. Recombinant Viruses as Etiological Agents: The Evidence in Favor The role of endogenous MuLV in leukemogenesis is as yet controversial. The observations to be dealt with are as follows. First, let us examine the AKR disease. High titers of endogenous N-ecotropic MuLV are found in a wide variety of tissues of AKR mice throughout life (Rowe and Pincus, 1972; Kawashima et al., 1976b; Nowinski and Doyle, 1977). Expression of ecotropic MuLV seems essential for leukomogenesis and virus expression is required early in life (Meier et al., 1973; Lilly et al., 1975; Huebner et al., 1976; Nowinski et al., 1976; Schwarz et al., 1979). Thus a high degree of correlation was seen between ecotropic virus titer at 6 weeks of age and incidence of spontaneous leukemia in (BALB/c X AKR) X AKR backcross mice (Lilly et al., 1975). Furthermore, an Fv-l restriction greatly reduced the final incidence of leukemia. Thus, AKR x Fu-lbhybrids expressed much less virus at 6 weeks of age and a greatly reduced incidence of leukemia when compared with AKR X Fv-In hybrids (Rowe and Hartley, 1972). Again, other studies have shown that mice can be protected against leukemia if antibodies to MuLV are administered in the early neonatal period (Huebner et al., 1976; Schwartz et al., 1979; Nobis and Jaenisch, 1980). In contrast with the above information, some studies have shown that ecotropic virus expression in neonatal life does not invariably lead to leukemia. In (AKR X C3H) and (AKR X RF) hybrids, 100% of mice show spontaneous ecotropic virus expression at 6 weeks of age, yet only 22% of these hybrids come down with the disease (DuranReynals et al., 1978).Thus, neonatal virus expression appears necessary, though hardly sufficient, for development of leukemia. However, recent evidence has begun to downplay the role of ecotropic virus as the etiological agent per se. At 5-6 months of age, preleukemic changes in the AKR thymus seem to manifest themselves as amplified expression of MuLV-related cell surface antigens. These changes correlate with appearance of xenotropic MuLV (Kawashima et al., 1976a,b; Nowinski and Doyle, 1977). Along with these changes there appears to arise a novel type of recombinant viruses, having both xenotropic and ecotropic host range. These recombinant viruses, known as mink cell focus-inducing (MCF) (Hartley et al., 1977), are thought to be representative of genetic envelope recombinants between N-ecotropic and xenotropic MuLV (Hartley et al.,

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1977; Elder et al., 1977; Chien et al., 1978; Devare et al., 1978; Rommelaere et al., 1978; Lung et al., 1980; O’Donnell et al., 1980). Some of these recombinant MCF MuLVs have been shown to accelerate leukemia development after injection of newborn or young AKR mice (Nowinski and Hays, 1978; Cloyd et al., 1980; O’Donnell et al., 1980), suggesting that age-dependent formation of dualtropic recombinant viruses in thymus can account for at least part of the disease’s latent period. This notion is strengthened by the fact that neither ecotropic nor xenotropic virus accelerates the disease. In this light, earlier experiments showing that only extracts of thymus of older AKR mice could accelerate leukemia development (Kaplan, 1967; Nishizuka and Nakakuki, 1968; Hays and Vredevoe, 1977) may now be understood. Comparative study of MCF isolates obtained by Hartley et al. (1977) from individual leukemia mice has shown that these viruses generally fall into two groups. Class I accelerates leukemia, and in general were isolated from lymphomas arising in the thymus of AKR or C58 mice. Class 11, on the other hand, do not accelerate leukemia and were isolated from leukemias arising primarily in spleen and lymph nodes. In addition, Class I1 MCF viruses generally do not replicate efficiently in the thymus. The genomes of some of the cloned MCF viruses belonging to the two classes have been analyzed by RNase T1 fingerprinting and T1 oligonucleotide mapping and by restriction analysis using cloned viral probes. These studies have shown regions of identity between MCF viruses and ecotropic and xenotropic viruses (Rommelaere et al., 1977, 1978). From the fingerprints obtained, each of the MCF genomes examined appeared unique although related. All of the MCF viruses are altered in the 3’ third of the genome relative to their putative ecotropic parent (AKV). In addition, many of the new, presumably xenotropic, sequences they have acquired are held in common between them. Restriction studies have confirmed this general feature. Class I viruses share some oligonucleotides with AKV, but differ in the 3‘ terminal oligonucleotides (Rommelaere et al., 1978). Instead they have new MCF-specific oligonucleotides in this region. By contrast, Class I1 viruses share terminal T1 oligonucleotides with AKV in the 3’ end of the genome. One MCF, whose origin is unclear, has biological properties intermediate between the two classes, that is, it shares some T1 oligonucleotides with MCF (Class I property), but it has the same 3‘ terminal oligonucleotides as AKV (Class I1 property). Restriction analysis has shown that the principal difference between Class I and I1 MCFs is that the former share an XbaI site with ecotro-

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pic viruses that the latter lack. This XbaI site of the pathogenic MCF viral DNAs is located at 7.7 kb, in the coding region for p15E. However, the results of Chattopadhyay et al. (1982) suggest that the gp70 (and p15E) of at least some MCF viruses might be derived entirely from nonecotropic sequences. Furthermore, the env sequences, in such cases, might be found entirely in high-molecular-weight DNA of normal cells. This observation has suggested that embryo DNA of AKR mice contains preexisting (prior to any recombination event) copies of intact MCF like env gene sequences with a proviral structure. Evidence that recombination within the p15(E) region of MuLV may be significant for leukemogenicity is also supplied by the studies of Pedersen et al. (1981), where isolates of MuLV from a spontaneous AKR tumor which accelerated leukemia in AKR mice were found to share common oligonucleotide changes in the p15(E) coding region of the viral RNA. Thus, the emphasis on a role for MuLVs in leukemogenesis has shifted focus from the ecotropic to the MCF-like or recombinant MuLV. Molecular and biochemical studies have addressed the issue that some MCF viruses do not appear to be pathogenic; however, we shall return shortly to further arguments against a role for these viruses in the disease. Additional evidence favoring a viral etiology for leukemia are studies showing that MuLV infection of target cells is required for transformation. Thus, viral DNA sequences are amplified stoichiometrically in AKR thymomas (Berns and Jaenisch, 1976) as well as in lymphomas of BALB/Mo mice (Jaenisch, 1979) and often appear first in an unintegrated form (Jahner et aE., 1980) characteristic of acute virus infections (recall that approximately 10% of AKR cells are initially found to replicate virus and de novo infection appears to be required for virus expression in 100% of cells). Restriction endonuclease studies have further shown that newly integrated viruses are only partially homologous to the ecotropic MuLV-specific cDNA probes used (consistent with involvement of recombinant viruses), and that the resulting leukemias are of clonal origin, selected in some unknown way from a pool of infected target cells (Canaani and Aaronson, 1979; Steffen et al., 1979; van der Putten et al., 1979). Studies on the role of viruses in AKR leukemia have, in summary, established the following facts. Viremia in AKR mice, which occurs by spontaneous activation of ecotropic MuLV, is usually a first step in leukemogenesis and may provide a source of parental virus for the yet unspecified events of genetic recombination which ultimately yields dualtropic MCF viruses. The preleukemic changes of antigen amplifi-

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cation probably results from infection of susceptible target cells in the thymus by recombinant viruses. Antigen amplification in such cells probably accompanies viral DNA amplification. Infection of thymocytes may represent only an initiating event and other somatic alterations such as trisomy 15 (Dofuku et at., 1975; Klein, 1979, 1981) may actually be involved and account for the latent period of disease development. The involvement of MCF viruses in virally induced leukemia in uico has also been well studied in the Friend virus disease system. Work of Troxler et al. (1978)and Ruscetti et al. (1981) indicates that the pathogenicity of an ecotropic, molecularly cloned F-MuLV depends on its ability to efficiently generate high levels of replicating MCF virus in target tissues. In fact, Ruscetti et al. (1981) further showed that resistance to F-MuLV disease in certain strains of mice correlated with endogenous expression of MCF-related envelope glycoproteins, suggesting a viral interference mechanism. A final observation relevant to this discussion pertains to the finding by Fischinger et al. (1981) that a virus-negative cell line, NIXT, derived from an X-irradiation-induced Swiss mouse thymoma, expressed a glycoprotein which resembled MCF envelope glycoprotein by peptide mapping and viral interference data. Hence, recombinant viruses or expression of recombinant type glycoproteins may be critical factors in a variety of pathways for induction of murine leukemia. 2. Recombinant Viruses as Etiological Agents: Some Problems There are two basic difficulties with the notion that recombinant viruses are the causative agents for neoplasia as described above. First, in some instances (see below) leukemia occurs without evidence of viral replication. Second, the degree of viral recombination is not well correlated with the degree of oncogenicity in all cases (see below). Fractionated irradiation can cause leukemia (Kaplan and Brown, 1952). It is sometimes possible to isolate from irradiation induced tumors a recombinant virus(es) at the time of leukemia appearance (Deckve et al., 1977a; Haas, 1978). However, in contrast to the AKR lymphomas, most C57BL thymic lymphomas do not express viral antigens as detected by immunofluorescence or radioimmunoassay competition. The second problem discussed above is that a perfect correlation has yet to be found between recombinant virus structure and oncogenicity. Thus a high degree of oncogenicity has been found in some isolates, i.e., MCF247 (Hartley et a[., 1977), HIX (Fischinger et al.,

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1978), and C57BL/G-derived RadLV (Decleve et al., 1977b; Haas, 1978). However, the degree of recombination is not well correlated with the degree of oncogenicity in recombinant viruses of C3H origin (Devare et al., 1978). 3. Other Transforming Genes It is important to remember at this point that a viral association with lymphoma development is not obligatory (e.g., radiation-induced lymphomas-see above). Thus, it may be speculated that the crucial genes and gene products involved in oncogenic transformation in vivo are yet to be recognized. In this light, it is proper now to consider models for transformation based on other RNA tumor viruses and information regarding "cellular transforming" genes (see below). A general characteristic of RNA tumor viruses with transforming capacity is that they encode virus genomic sequences recombined with nucleotide sequences originating from cellular DNA. The substituted sequences are often found in the env and the c regions of the genome, but can stretch out to encompass the major portion of the viral RNA. The analysis of transformation competent sarcoma viruses has shown that viral RNA encodes the transforming gene proper, which has been termed STC. The analysis of src function has been possible because of the availability of viral mutants conditionally transforming at various temperatures (ts) (Stehelin et aZ., 1977; Hanafusa, 1977; Vogt, 1977) and the isolation and identification of the in vitro translation product of the src region (Beemon and Hunter, 1977; Purchio et al., 1977; Kamine et al., 1978; Levinson et al., 1978; Sefton et al., 1978). It is quite clear that src sequences constitute a gene whose product is responsible for, and maintains cellular transformation (Hanafusa, 1977; Vogt, 1977). The following specific information has been obtained for the avian sarcoma virus (ASV).A viral phosphoprotein of 60,000 daltons designated as pp60"" is detected in ASV-transformed cells of many species by immunoprecipitation of labeled cell extracts with serum from rabbits bearing ASV-induced tumors (Brugge and Erikson, 1977; Levinson et al., 1978; Sefton et at., 1978). Pp60"" is a transformation-specific phosphoprotein and not a viral structural antigen. Pp60"" is probably the product of src since translation in vitro of the region known to code genetically for src specified function results in the synthesis of a polypeptide of the same size and having a similar peptide map. In addition, normal chicken embryo fibroblasts contain a gene, c-STC, which is homologous to the avian sarcoma virus transforming gene src (Spector et al., 1978). Both genes

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code for a 60,000-molecular weight protein which is phosphorylated at serine and tyrosine residues (Collett and Erikson, 1978; Levinson et ul., 1978) and has the unusual function of phosphorylating tyrosine residues. The Rous sarcoma virus STC may be conceived as the model for virally associated transformation genes. In RSV STC, a cellular gene is combined with a retrovirus skeleton to generate a transforming virus. Similar findings have been made with at least seven additional avian transforming viruses: Fujinami, PRCII, avian erythroblastosis, MC29, avian myeloblastosis, and reticuloendotheliosis transforming viruses. These seven avian transforming viruses all have, as far as is known, non-cross-reactive cellular genetic information as a crucial component of their structure (Fischinger, 1980). For mammalian viruses, non-cross-reactive genes have been found in murine sarcoma virus, Abelson murine leukemia virus, and feline sarcoma virus. Recently, it has been shown that in the Harvey and Kirsten strains of rat-derived sarcoma virus, there is a segment of normal rat cell genetic information as well as a long stretch of sequence of a virus-like structure endogenous to rat cells (Fischinger, 1980). While these findings would suggest that in general these cell-derived proteins are probably responsible for viral transformation, hard evidence for their transforming potential exists in only the Rous sarcoma virus (RSV) system as described above. Nonetheless, transformation-defective mutants of Abelson virus have been isolated and shown to lack protein kinase activity (although they can act as acceptors in a trans-kinase reaction) (Reynolds et ul., 1980). Furthermore, mapping studies with fragments of the viral genome have located the murine sarcoma virus transforming activity in the cellular insert and similar evidence for Kirsten sarcoma virus has been gathered. Thus it seems increasingly evident that the putative transforming proteins are in fact responsible for changing the growth properties of infected cells. We shall return below to the question of whether leukemia viruses such as the MCFs may transform by a similar joining of the retrovirus skeleton to cellular oncogenes. However, it is important to realize that this concept may further stretch to apply to what is until now regarded as “nonviral” induced transformation, i.e., chemical carcinogenesis. The demonstration that the transforming genes of retroviruses are derived from cellular genes, or protooncogenes (Temin, 1974; Hayman, 1981; Bishop, 1981), has considerable implications. If certain cellular genes are oncogenic when placed under the influence of viral

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control elements, perhaps other genes, or indeed the same ones, mediate chemical carcinogenesis by being expressed at high levels as the result of carcinogen-induced mutations. Recent work from Shilo and Weinberg (1981) and Cooper et al. (1980) has shown that genes involved in chemical carcinogenesis can be detected by DNAmediated gene transfer experiments. Shilo and Weinberg (1981) have been able to induce transformation by transfecting into nontransformed mouse fibroblasts DNA from lines of mouse fibroblasts transformed in vitro by the chemical carcinogen 3-methylcholanthrene (MC). DNA from 15 transformed lines were tested and successful transfer of the transformed phenotype was obtained with DNA from five of these lines. Restriction endonuclease digestion of MC-transformed cell DNA was used to assess the number of transforming genes. The principle of this analysis is that a gene will be inactivated in transfection if an enzyme cuts within it, whereas enzymes that cut outside the gene will have no effect. In four of the cases tested, EcoRI and Hind111 abolish transformation, whereas digestion with B a d , XhoI, and SaZI had no effect, suggesting that the same gene is involved in transformation in each of the four cell lines. On the other hand, the transforming genes of other cell types, a rat neuroblastoma and a line of cells transformed by transfection with normal mouse DNA, exhibited distinct patterns of restriction enzyme sensitivity. The lines showing the same pattern were all derived by in vitro transformation of mouse fibroblasts with MC. Nonetheless, the DNA of many MC-transformed cells were not active in the transfection assay, and it is quite possible that only a subset of MC-responsive sequences was examined. The general picture that arises from these transfection experiments may be applicable to the virally associated oncogenes. The findings suggest that genes which during normal expression are not deleterious, and may even be vital for normal growth and development, can be oncogenic when joined to regulatory element which increase their expression. In these transfection experiments, transformation is induced when the DNA is sonicated to a small size before transfection, probably because sonication separates the transforming gene from regulatory sequences that normally control its expression. In the primary transfection, which is very inefficient, transformation occurs if the gene integrates close to a strong promoter. In a secondary transfection, a high efficiency is obtained because the lesion is now cis-dominant (Cooper et al., 1980). Viruses may work by similarly capturing such genes and promoting their transcription. This model has received direct support from experiments of Blair et al. (1981). They

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have cloned a protooncogene, the murine cellular src gene from normal mouse cells and shown that it does not transform when transfected into cells. However, when the cellular src gene is joined to sequences from the 5’ end of murine leukemia virus which contain the strong viral promoter, the resulting recombinant DNA molecules transform efficiently.

4 . Mechanisms for Leukernogenesis Let us now return to the issue of how “leukemia” viruses may cause leukemia. In view of the above discussion, it is difficult to resist suggesting that one of several mechanisms is responsible for their neoplastic effect. Important genes might be hidden in the virus, possibly at the 3’ end. This possibility is still viable, but the recent availability of the nucleotide sequence of the Moloney leukemia virus has not yielded data supportive of this notion (Shinnick et al., 1981).A second possibility is that the promoter function inherent in the terminal repeat structure (see Section I1,B) might, following integration in appropriate regions of host DNA, enhance downstream (or upstream) transcription and thus activate to high production cellular genes (oncogenes) that are normally regulated to low levels. A model consistent with this notion has recently been proposed for the induction of bursa1 lymphomas in chickens by ALV, and the evidence which suggests this model is as follows: lymphomas induced by ALV usually contain proviruses integrated at one or more sites. Nonetheless, at least some proviral information in each tumor is found integrated at one of a limited number of common sites (Neel et al., 1981; Payne et al., 1981; Fung et al., 1981). As proviral integration is known to occur at a larger number of sites on the host chromosome, possibly at random (Hughes et d.,1978; Steffen and Weinberg, 1978; Ringold et d . , 1979; Quintrell et al., 1980; Shimotohno et al., 1980a,b),this apparent specificity of integration in lymphomas suggests that neoplastic transformation requires integration adjacent to specific genes. In addition, it would appear that integration leading to transformation does not occur frequently, since most primary and metastatic tumors are clonal (Neel et al., 1981; Payne et uZ., 1981; Fung et al., 1981; Neiman et al., 1980); that is, neoplastic cells isolated from various organs of the same neoplastic animal contain similar or identical patterns of proviral integration as reflected by similar restriction maps of the integrated viral information throughout all organs. Recent evidence suggests that these viruses cause neoplasia by activating a normal cellular gene(s). It has, for example, been found that many ALV-induced lymphomas do not contain viral 35 S and/or 21 S

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mRNAs (Neel et al., 1981; Payne et al., 1981) and that many of the integrated proviruses in these tumors are defective (Neel et al., 1981; Payne et al., 1981; Fung et aZ., 1981).Therefore, it is unlikely that the replicating virus is itself able to sustain the transformed state. Furthermore, in at Ieast some cases, such as ALV, the oncogenic potential seems to reside within the 3' portion of the viral genome-within approximately 500 nucleotides from the poly(A) tract (Robinson et al., 1980; Crittenden et al., 1980, Tsichlis and Coffin, 1980).This segment of the viral genome does not seem to code for a viral protein (Czernilovsky et al., 1980). Finally, ALV-induced lymphomas contain new tumor-specific RNAs consisting of viral 5' terminal sequences covalently linked to cellular sequences (Neel et al., 1981; Payne et al., 1981).These mRNAs fall into a limited number of size classes (Neel et al., 1981) and these tumor-specific mRNAs are expressed at moderately high levels [loo-300 copies per cell (Neel et aZ., 1981)l. Based on the above observations, several investigators (Neel et al., 1981; Hayward et al., 1981; Payne et al., 1981; Robinson et al., 1980; Tsichlis and Coffin, 1980; Quintrell et al., 1980) have suggested a model for oncogenesis termed oncogenesis by promoter insertion (Neel et al., 1981; Hayward et al., 1981). We described earlier (see Section II1,B) that the integrated provirus consists of the viral structural genes flanked by sequences of approximately 600-350 nucleotides termed long terminal repeats (LTRs) (Hsu et al., 1978; Shank et al., 1978; Sabran et al., 1979; Hughes et aZ., 1979). These LTRs contain a putative promoter sequence (Czernilovsky et al., 1980; Shimotohno et al., 1980a,b; Yamamoto et al., 1980). Initiation of viral RNA synthesis normally occurs within the left LTR. However, initiation could also occur within the right LTR because the promoter sequence is repeated at the right end. If the provirus integrated adjacent to a potentially oncogenic cellular gene, transcription initiated from the viral promoter could generate an RNA molecule such as those found in ALV-induced lymphomas, containing both viral and cellular sequences. The resultant enhanced expression of this cellular gene might lead to neoplastic transformation. The model has found support recently from the identification of such a mechanism in at least one leukemia system. Using cDNA probes specific for five o-onc genes of avian acute transforming viruses, Hayward et al. (1981) have managed to identify one such gene. These authors have shown that most of the ALV-induced lymphomas they studied resulted from activation of the c-myc gene, the cellular counterpart of the transforming gene of MC29 virus, by a promoter insertion mechanism. More recently, however, Payne et al. (1982) have shown that enhanced expression of c-myc can

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occur when flanking proviruses are found in any of three configurations: (1)on the 5‘ side (“upstream”) of the c-myc in the same transcriptional orientation, (2) on the 3‘ side (“downstream”) of c-myc in the same orientation, or ( 3 )upstream, in the transcriptional orientation opposite to that of c-myc. These authors have, therefore, postulated that activation of adjacent cellular genes by retroviral DNA can involve mechanisms other than provision of a transcriptional promoter. An hypothesis concerning murine leukemia which is consistent with this protooncogene activation schema has been proposed by Dec k v e et al. (1977b) as a mechanism by which fractionated irradiation induces leukemia. As stated previously, most radiation-induced C57BLJKa thymic lymphomas do not express viral antigens detectable by immunofluorescence or radioimmune competitive assay (Ihle et al., 1976a) despite the fact that RadLV, a virus with thymotropicleukemogenic properties (Kaplan, 1967), can usually be recovered from such tumors by passage of cell-free extracts in uiuo. Decleve et al. (197713) have suggested that this paradox can be resolved by postulating that RadLV is initially activated by X-irradiation in a replication-defective form (RadLV-0) in which only the oncogenic segment of the RadLV genome is expressed (this would go undetected by immunofluorescence or radioimmune competition assays which use antibodies directed at non-oncogene-derived antigens) and that RadLV acquires infectivity in uiuo secondarily, possibly by a recombination mechanism. Such an infectious recombinant virus would be expected to carry the oncogenic element encoded by RadLV-0. Kaplan and collaborators have shown that the leukemogenic recombinant virus (but not the nonleukemogenic viruses) encapsulates two RNA species of molecular weights 8 and 5.6 kb. Nonleukemogenic viruses lack the 5.6-kb RNA species. This 5.6 RNA codes for 100,000- and 36,000-molecular weight species which have certain similarities to the putative transforming “fusion” proteins of certain acute defective viruses (e.g., Abelson MuLV). That is, they contain viral gag-derived determinants linked to possibly cell-derived sequences, and may be important for oncogenicity (Manteuil-Brutlag et al., 1980). It is possible that these findings will apply to leukemogenic (as opposed to nonleukemogenic) viruses of the MCF or recombinant type, although presently no such similarities have been uncovered. We hope the above thoughts have served to illustrate our ignorance regarding the transformation phase of leukemogenesis while reviewing current thoughts, and raising awareness to the fact that genetic elements are required on the part of both host and virus for transfor-

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mation to occur. The latter point is of special importance to this article and is, therefore, worth rephrasing. Clearly in the absence of genes favorable to recombination (e.g., those coding for ecotropic and xenotropic virus sequences, or facilitating recombination), resistance to the disease may ensue. Furthermore, if cellular genes are involved, the presence of the “transforming alleles” for the potential locus is required for susceptibility to the disease, unless of course the virus carries in its genome the required information. Much work will still be required to elucidate the mechanism(s) of transformation and genetic resistance at this level.

F. IMMUNESURVEILLANCE AGAINST VIRAL INFECTION AND TRANSFORMATION 1. The Murine Major Histocompatibility Complex, H-2 Once transformation of normal cells has occurred, the only mechanisms known which can interfere with the maintenance of transformation (short, of course, of the hypothetical loss of the gene for transformation itself) are immunological. While the host immune response may be capable of eliminating the transformed cells, the capacity of the host to mount an effective response is markedly influenced by its genetic constitution at a variety of loci. Perhaps the most important of these loci are clustered together and known as the major histocompatibility complex, H-2. This complex mapping on the mouse chromosome 17 affects a considerable number of immunological phenomena (Meruelo and McDevitt, 1978). It is a complex of closely linked genes responsible for the rapid rejection of skin grafts, graft-to-host reactions, and other immunologic as well as nonimmunologic phenomena. The present knowledge of the genetic map of the H-2 region (Fig. 6) is based on analysis of an extensive series of congenic resistant strains and their recombinants produced by numerous workers (Amos et al., 1955; David and Shreffler, 1972; Gorer and Mikulska, 1959; Klein et al., 1970; Shreffler and David, 1975; Snell and Cherry, 1974; Stimpfling and Reichert, 1970). The gene products of the H-2K and H-2D regions are detected serologically and are found on most cell types including lymphocytes, although their concentration may vary in different cell types. The normal physiologic functions of histocompatibility genes in the K and D regions of the mouse are still poorly understood, although tests employing mice with recombinant H-2 chromosomal segments have shown that incompatibility at either the K or D end of the H-2 complex causes rapid rejection of tumor and skin

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grafts (Shreffler and David, 1975). Biochemical analysis has shown that the D region product(s) has an apparent molecular weight of 43,000, while that of the K region product is 47,000 (Nathenson and Cullen, 1974). The complete amino acid sequence and molecular structure of some K and D region gene products has been obtained (Coligan et al., 1978; Vitetta and Capra, 1978; Hood et al., 1983). H-2K and H-2D gene products are often obligatorily involved in the induction and effector phases of T cell “killer” function. For example, in several studies cytotoxic T cells specific for viruses and for minor transplantation antigens appeared to recognize not only the antigen to which they had been sensitized, but also the H-2K and/or D gene product on the immunizing cells. In addition, products of the major histocompatibility complex (MHC) play an important role in cellular interaction in the immune response (Benacerraf and Katz, 1975) as well as during embryogenesis (Snell, 1968). The Z region was recognized as an important segment of the MHC when genetic control of immune responses to several synthetic polypeptides and low doses of natural antigens were shown to map in the chromosomal region lying between the K and S regions of the H - 2 system (McDevitt et al., 1972). It was subsequently shown that this region also codes for determinants on cell surfaces eliciting a proliferative cellular response in the mixed lymphocyte culture reaction and the graft-versus-host reaction (Bach et aZ., 1972; Meo et al., 1973). Efforts by several investigators (David et al., 1973; Gotze et al., 1973; Hammerling et al., 1974; Hauptfeld et al., 1973; Sachs and Cone, 1973), designed to raise antisera against Z region gene products, resulted in the identification of a new class of cell surface alloantigens, the I immune response region-associated (Ia) antigens. Biochemical analysis of these alloantigens showed them to be cell surface glycoproteins of two classes with molecular weights of approximately 25,000 and 33,000 (Cullen et al., 1974; Vitetta et al., 1974). Recent studies have identified a third “invariant” chain of MW 31,000 (Jones et ul., 1978; &cloosicet al., 1980). These antigens may be found on the surface of lymphocytes, niacrophages, and epidermal cells (Hammerling et al., 19’75). Within the last few years, systematic study of the Z region in a number of distinct inbred mouse strains, and in recombinant H-2 strains derived by crossing over between the H-2K and H-2D loci of distinct H - 2 haplotypes, led to the subdivision of the Z region into the Z-A, Z-B, Z-C, Z-E, and Z-J subregions (Lieberman and Humphrey, 1972; Murphy et al., 1976; Shreffler et al., 1977; Delovitch et al., 1977).In addition, this genetic analysis resulted in mapping of genes

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controlling the immune response to particular foreign antigens in the I-A, I-B, or I-C regions. These were designated Zr-lA, Zr-lB, and Ir-1C, respectively. In several instances, it also became apparent that two distinct I region genes could complement each other to develop a high response to a particular antigen (Benacerraf and Dorf, 1976). This complementation phenomena raised questions regarding the role of Ia antigens in immune responses. Recent evidence suggests that the two polypeptide chains that make up the I-E antigen are separately encoded within the I region. Jones et al. (1978), using two dimensional gel electrophoresis, discovered that the location of the I-E p chains on two-dimensional gels differed between BIO.A (5R) (AbEk)and B1O.A (AkEk).This suggested that expression of the I-E molecule is under two gene control with one gene mapping in the I-E subregion and another gene mapping to the left of the I-J subregion. This was confirmed by the peptide mapping studies of Cook et al. (1979, 1980). These authors showed that among recombinant mice B1O.A (5R) (AbEk),BIO.A (AkEk), and BIO.S (9R) (AsEk)heterogeneity exists in the peptide maps of the p chain of the I-E molecule, while no heterogeneity was found in the fy chain. Together these studies suggest that the I-E subregion moIecule is formed by complementation of a gene in the I-A subregion coding for the p chain (Ae) and a gene in the I-E subregion coding for the a chain ( E a ) .Tryptic peptide mapping studies from several other laboratories have confirmed these results (McMillan et al., 1981; Silver and Russel, 1979; Wakeland and Klein, 1979). The regulation of expression of cell surface antigens coded for by the I-A subregion by a locus mapping between I-J and H-2D suggests one mechanism by which two complementary genes might control immune responsiveness (Jones et al., 1978; McNicholas et al., 1982; Matis et al., 1982).As is true for Ir (immune response) and Is (immune suppression) genes, complementation allowing I-A antigen expression can occur in either the cis or trans position. Combining the b and k or d haplotypes allows the expression of the Aeb :E complex on the cell surface; this molecular structure is not found on cells of either parental haplotype. Functional capabilities unique to this complex of I-A and I-E polypeptide chains also would not be shared by either parental heplotype. In this context, it is interesting to note that in a number of complementary l r and I s gene systems, there is a good correlation between expression of Ia antigens coded for by the I-E or I-C and the presence of a complementary Ir or Is gene on the right side of the I region (Jones et al., 1978; McNicholas et al., 1982; Matis et al., 1982). The Ira gene thus might regulate the expression of Irp

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gene product (the Ae protein) on the cell surface, perhaps by coding for the I-E antigen. Further implications regarding the importance of immunologic mechanisms in MHC-associated phenomena come from the recent finding that the S gene of the H - 2 system, which was known to affect serum levels of a p-globulin, is in fact the structural gene for one of the polypeptide chains of the fourth component of complement, C4 (Shreffler et al., 1976). 2. H - 2 Linked Genes and Resistance to Virus-Induced Leukemogenesis

It was apparent from the initial studies of Corer (1956) and Gross (1970) that some relation existed between susceptibility to leukemogenesis and the H-Zk haplotype. The high leukemia mouse strains (AKR, C58, C3H/Figge, and RF), as well as two strains utilized by Gross to obtain a filterable agent from AKR mice, C3H/Bi and C57BR, were all of the H-Zk haplotype. In a formal genetic study of mice of various H - 2 types and their hybrids, Lilly and co-workers (Lilly, 1966; Lilly and Pincus, 1973)inoculated neonatal mice with Gross virus and recorded the ensuing development of the disease. The most extensiveIy studied hybrids were those of the C3H/Bi ( H-2k) and C57BL/6 (H-Zh) cross; in Fz and backcross hybrids, mice of the homozygous H-2klk type showed greater than 90% incidence of leukemia, whereas H-Zhb and H-2hlkmice showed leukemia incidence of approximately 30-50% which was also delayed in onset. Confirmation of the importance of H - 2 in affecting susceptibility to Gross virus-induced leukemia came from studies with congenic strains of mice, differing from each other only in the H - 2 chromosomal segment. Thus mice of the congenic C3H ( H - 2 k ) and C3H.SW (H-Zb) strain pair differed radically in their response to Gross virus, being susceptible and resistant, respectively. Similarly, C57BWlOSn (B10) mice, which are of the H-2b haplotype, were resistant to Gross virusinduced leukemia, but mice of the congenic B1O.BR strain ( H - 2 k ) were susceptible, although with latent periods significantly longer than those of C3H mice (probably because of their differences at Fu-1) (Lilly et al., 1964). Other lines of evidence indicated the importance of H - 2 in susceptibility to viral leukemia in mice. For example, Tennant and Snell (1968),studying leukemogenesis by the BT/L virus, observed a considerably greater level of resistance in C57BL/10 (H-Zb) than in congenic B1O.BR ( H - 2 k )mice.

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a. Role of Zmmune Response Genes in Leukemia Resistance. Studies with H - 2 recombinant mice were performed to localize the gene coding for resistant to Gross virus, Rgu-1 ,within the known fine structure of the H - 2 complex. The data obtained indicated that Rgu-1 maps toward the K-end of the H - 2 complex, comprising the K and Z regions (see Fig. 6). Evidence suggestive of the nature and mechanism of the Rgu-1 influence was obtained in studies with Friend virus (FV). Crosses between susceptible (DBN2 and BALB/c) and resistant (C57BL) mice showed the expected responses according to their Fu-1 and F v - 2 types, but in addition, they showed an H-2 specific component in their response. Because C57BL mice are rendered completely resistant by their Fv-2' genotype (which blocks the spleen focus-forming component of the virus from infecting such mice), Lilly et al. (1964) bred a strain of BALB/c mice (termed BALB.B) that was congenic at the H-2 locus, carrying the C57BL-derived H-2b haplotype instead of H-2d, and the permissive allele at Fu-2. BALB/c (H-2d)mice showed a 10fold lower virus dose threshold for splenomegaly induction and were much less prone to recovery from splenomegaly than BALB.B (H-2b) mice, indicating that the H - 2 difference of the host appeared to significantly alter the cause rather than the onset of the disease. The close or identical mapping of Rgu-l with the Z region, to which the great majority of Zr genes have been mapped, and the indication from FV disease studies that the H - 2 effect may influence a late event in the disease, namely recovery from splenomegaly (Lilly, 1968), led to the suggestion (Lilly et al., 1964; McDevitt and Bodmer, 1972) that the strength of the immune response to virus-specific or tumor-specific transplantation antigens (TSTA) might be regulated by Zr genes. Thus H-2 linked resistance to virus-induced leukemogenesis might result from a stronger immunologic response to a given virus induced antigen. One indication that this hypothesis could be correct was the finding of Aoki et al. (1966) that among progeny of the cross AKR ( H - 2 k and positive for the G antigen induced by Gross virus) x C57BL ( H-2b and G antigen negative), a significant number of mice homozygous or heterozygous for the H-2b haplotype showed detectable levels of anti-G antibodies but no H-2khomozygotes showed these antibodies. In addition, subsequent experiments by Sat0 et aZ. (1973) found that certain leukemias derived from BALB/c mice were rejected by hybrids of BALB/c with other inbred strains, contrary to the usual rules of transplantation. Similar studies with a series of recombinant hybrids estab-

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lished that the responsiveness to these tumors was linked with the K end of H-2, the location of Rgc-l and H-2-linked Ir genes. Furthermore, animals resistant to the tumor had high titers of antibody to Gross-specific antigens, whereas susceptible animals did not have similar titers. More recent studies by Lonai and Haran-Ghera (1977) showed a gene Rrtj-I located in the H-2 region, that influences resistance to the A-RadLV strain of radiation leukemia virus. This strain of RadLV is distinct from the Kaplan strain of RadLV (Lieberman and Kaplan, 1959). Chesebro and Wehrly (1978) also suggested an Z region influence on Friend virus (FV)-induced leukemogenesis Rfu-2, located in the K or Z regions, appears to affect recovery from FV leukemia. All of these results imply, but do not prove, that H-2-linked immune response genes are directly involved in conferring resistance to the development of malignancy. Recent studies from our laboratories have provided more direct evidence for the existence of H-2 linked immune response gene(s) to a virus- or tumor-specific transplantation antigens (Meruelo et aE., 1977b, 1980b). In these studies, AKR mice were crossed with animals of various H-2 congenic strains on the C57BL/10 or C3H genetic background and the hybrid mice injected intraperitoneally with AKR thymoma cell BW5147. (AKR X C3H.Q)Fl (H-2") but not (AKR X C3H)FI (H-2kk)mice were shown to generate a strong humoral response against BW5147 cells. A direct correlation could be demonstrated between survival to the injected BW5147 cells and humoral responsiveness. Cellular immunity appears to play no role in resistance to the proliferation of tumor cells. Humoral immunity and survival to BW5147 cells can be shown to be due to genes in either the B , J , or E subregions of H-2 (Meruelo et al., 1980b). The development of effective humoral immunity depends on B cells and Ly-1+,2-,3- helper T cells bearing the Z-Jk phenotype. These studies appear directly applicable to the spontaneous disease, and results of studies using transformed cells from an overtly leukemic AKR mouse parallel those obtained using BW5147 cells (Meruelo et al., 1980b). Further analysis of the humoral response has shown it to be directed against a protein, which appears to be distinct from virally coded gp70, p30, p12, and p10 by a variety of criteria (Zalman and Meruelo, 1982). Current investigations revolve around several issues: (1) Is this protein a true TSTA molecule; (2) Is it involved in transformation and, if so, does it bear any resemblance to other transforming proteins (e.g., like STC); and (3)What is the nature of the Zr defect in low responder animals (i.e., H-2kk). b. H-2D Effects on Susceptibility to Virus-Induced Leukemogene*

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sis. Resistance or susceptibility to FV (Chesebro et al., 1974) and the Kaplan strain of RadLV (Meruelo et al., 1977a) is associated with the D region of the H - 2 complex. It is important to note that while previous findings with FV were used to help explain the mechanisms of Rgu-I, the mapping location of the major component of resistance to FV is at the opposite ( H - 2 D ) extreme of the H - 2 complex from Rgu-l, which maps to the H-2K end (see Fig. 6). Such mapping argues that a second gene, distinct from Rgu-1, is responsible for the observed resistance. There are some other data indicating a second location for Ir genes within the H - 2 complex. For example, Young et al. (1976)have indicated the existence of an Ir-type mechanism to ferritin which maps to the TL region. However, most Ir genes mapped are located distant from H - 2 D . Therefore, the implied functional similarity between Rgu-l and Ir genes based on near if not identical mapping cannot be as readily applied in associating H - 2 D linked resistance to FV or RadLV with Ir gene effects. Experiments designed to test the role of H - 2 linked genes in resistance to FV and RadLV-induced neoplasia have revealed two general observations. First, virus infection alters quantitative expression of H-2 molecules. Second, cell-mediated immunity plays an important role in recovery from the disease. We shall detail these observations below. H-2 genes appear to affect the expression of surface antigens on FVinfected spleen cells (Lilly et al., 1964). One such antigen whose expression is affected by H-2 gene(s) is Friend-Moloney-Rauscher (FMR), an antigen probably encoded in the FV genome. FMR is abundantly present with the FV although not detectable on the surface of intact viruses. It appears, within 3-5 days after virus inoculation, on the surface of spleen cells of both BALB/c (H-2d, susceptible) and BALB.B (H-2", resistant) attaining its maximum level of expression in both strains 7-14 days after virus injection. Thereafter the level of expression of this antigen (as determined both by direct cytotoxicity of anti-FMR on the cells and by the quantitative ability of cells to absorb cytotoxic antibodies) declines in both strains of mice. However, in BALB/c mice (susceptible) the decline is rapid and much more complete, such that FMR is often difficult to detect at all during the terminal stages of the disease (about 21-28 days after virus administration). Associated with the apparent loss of FMR antigen in BALBlc mice is a concurrent and equally severe decrease in the level of expression of H-2 antigens that is not seen in BALB.B (resistant) mice. These changes in antigenic expression may significantly affect host defenses as discussed below. In addition, Bubbers and Lilly (1977) have obtained evidence that H-2" antigenic determinants might be incorpo-

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rated into virus particles in the process of budding from the membranes of resistant H-2b hosts, whereas H-2k antigenic determinants are never incorporated into virus budding off from H-2k (susceptible) hosts. Different levels of expression of FMR on infected cells could elicit different levels of immune reactivities in the two kinds of hosts and be the basis of H-2 linked resistance to FV-induced leukemia. Similarly, the appearance of H-2 antigenic determinants on budding virus may allow its rapid elimination by the host's immunologic defenses, while the lack of such H-2 determinants on virus may preclude such immune reactivity. Alternatively, quantitative differences in expression of cell surface antigens may, in and of themselves, constitute a host of defense mechanism independent of the immune surveillance system. Such perturbations on the cell surface may have profound effects on virus penetration in and out of cells. Studies from our laboratories during the past several years have shown that H-2 has a marked effect on RadLV-induced neoplasia. H-2associated resistance or susceptibility to RadLV maps to the D end of the complex (Meruelo et al., 1977a). The H-2Dd allele confers resistance to the disease, whereas the H-2Dq and H-2Ds haplotypes are associated with susceptibility. Thus, for example, BIO.S (7R) (H-2Dd) mice are resistant, while B1O.S (H-2Ds)animals are susceptible. H-2linked resistance to RadLV appears to be expressed as a dominant trait in hybrid offspring of crosses between susceptible and resistant mice. Further studies on virus replication have indicated no effect on actual infection (Meruelo et al., 1978). This is in accord with early studies with Friend virus by Lilly (1968) that indicates that H-2 effects on virus-induced leukemogenesis are noted at a late stage in the disease, namely recovery from splenomegaly. If, after intrathymic inoculation the course of virus replication in the thymus is followed over a 12-week period with the aid of an immunofluorescent anti-MuLV serum, a major difference is discernible 5 weeks after virus inoculation between susceptible BIO.S and resistant B1O.S (7R) mice. The number of immunofluorescence-positive thymus cells increases markedly during the 3- to 9-week interval after virus inoculation in susceptible mice, while little increase is seen in virus-positive cells among thymocytes of resistant mice. A remarkable effect on quantitative expression of H-2 antigens occurs on the cell surface following intrathymic RadLV inoculation (Meruelo et al., 1978). We have described some aspects of these changes in expression earlier in Section XII,H,2. Here we shall concentrate on the role of these changes in H-2D-linked resistance. When thymocytes

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from RadLV-inoculated mice and uninfected mice are incubated with alloantisera to H-2D and H-2K and then reacted with a fluoresceinlabeled rabbit anti-mouse IgG reagent, their cell surfaces can readily be seen stained with the aid of a fluorescent microscope or the fluorescent-activated cell sorter (Loken and Herzenberg, 1975). After intrathymic inoculation of RadLV, there is an early increase in cell surface expression of H-2K and H-2D molecules on thymocytes of BIO.S (7R) and B1O.S mice. The subsequent patterns of expression of H-2K molecules on thymocytes appear similar when these two strains of mice are compared. However, the subsequent effect of virus infection on levels of H-2D molecules differs in the two strains. Expression of H-2D molecules appears to be more markedly increased, for a more prolonged period in thymocytes of RadLV-infected BIO.S (7R) (resistant) mice than in thymocytes of RadLV-infected BIO.S (susceptible) animals. Furthermore, the increased expression of H-2D on B1O.S (7R) cells is greater and persists longer than the changes observed in expression of H-2K molecules in these same cells. It is remarkable that the differences observed with regard to H-2D expression in comparing BIO.S (7R) and BIO.S thymus cells correlate reciprocally with the changes in virus-positive cells seen in these same animals (Meruelo et al., 1978).BIO.S (7R) mice, which are resistant to leukemogenesis, do not appear to show a marked increase in the number of virus-positive cells in the thymus during the first 9 weeks after infection and show dramatically increased cell surface expression of H-2D molecules. Exactly the opposite phenomenon is seen when thymus cells of B1O.S mice (susceptible to RadLV) are examined. A number of additional factors suggest that changes in H-2 antigen expression may be important in the host’s response to infection by RadLV: (1)changes in expression occur very rapidly; (2) genes in the D region confer resistance to RadLV-induced neoplasia, and antigens coded for by the D region show the most marked and prolonged changes in expression and differential regulation between susceptible and resistant animals; (3) there is an inverse correlation between expression of H-2D and viral antigens (uide infra); and (4)finally, our studies (Meruelo et al., 1978) have shown that H-2 antigens disappear from the surface of RadLV-transformed cells. Thus, while resistance to the disease is associated with increased H-2 antigen expression, the neoplastic state is associated with disappearance of these antigens. Although the effects of increased H-2 expression on other steps required for oncogenesis remain to be evaluated, several observations suggest that elevated H-2 antigen expression enhances the effective-

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ness of the host’s cellular immune response to virus infected cells (Meruelo, 1979). First, cell-mediated immunity (CMI) against RadLVtransformed or infected cells can be detected with ease when H-2positive target cells are used in assaying ceil-mediated lympholysis ( C M L ) . Second, resistant mice develop greater numbers of effectors when injected with RadLV than do susceptible mice. Third, injection of normal (uninfected) thymocytes into syngeneic recipients of resistant or susceptible H - 2 type does not stimulate a CML response. However, injection of RadLV infected thymocytes from resistant mice produces a vigorous CML response and such thymocytes elicit the strongest response at a time when both H-2 and viral antigen expression is elevated. By contrast, injection of infected thymocytes from susceptible mice, which express viral antigens but low levels of H-2 antigens, does not stimulate a CML reaction. Thus, H-2D resistance to RadLV induced leukemogenesis has two important characteristics associated with it: (1)it is accompanied with specific changes in H-2 antigen expression; and (2) it appears to be mediated by a celi-mediated response. The effects of H-2D on FVinduced splenomegaly discussed previously strongly suggested similar ef’tects of H - 2 and viral antigen expression. However, the role of CMI has not yet been described. In a series of experiments designed to test the role of Zr genes on FV disease, Chesebro and Wehriy (1976a,b) found that while the antibody response showed little correlation with recovery from splenomegaly, there was a clearcut correlation between recovery from the disease and cell-mediated immunity. It was not clear, however, that CMI played a crucial role in the recovery or ensued after recovery had begun. In addition, no correlation existed between H - 2 type and ability to mount either a cell-mediated or humoral response. Both susceptible (H-2df and resistant ( H-2b) animals were capable of vigorous humoral and cell-mediated cytotoxic responses, despite the clear association between H - 2 type and resistance to the disease. An important consideration, however, is that Chesebro and Wehrly (1976a,b) tested for cell-mediated Zr gene(s) after inoculating virus concentrations so low that normally susceptible animals showed a high percentage of recovery from the disease-i.e., H - 2 control was not detectable. Under such circumstances, equal cell-mediated responsiveness in resistant and susceptible animals does not argue for the lack of an Zr gene. In fact, more recent experiments by Chesebro and collaborators (B. Chesebro, personal communication) has revealed that the kinetics of cell-mediated responses may be different with effectors from resistant animals proliferating earlier than those of

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Susceptible mice. Timing of the host response would, of course, be critical in containing the rapid spread of FV and the replicating erythroleukemic cells. Further evidence for the role of CMI in H-2D control of FV-induced erythroleukemia has come from the studies of Blank et al. (1976a). Using an experimental system different from that of Chesebro et al. (1976a), these authors have shown that resistant mice (H-2b) can mount a CMI response to FV-induced tissue culture-adapted tumor cells, whereas susceptible animals (H-2k)cannot. In the case of mice of the H-2b genotype immunized with FV, where the major factor governing the relative resistance of the mice to the virus-induced disease is the H-2Db-associated Rfu-l locus, resistance to the disease is associated with the generation of CTL specific for an unidentified viral antigen and the H-2Db gene product but not for the H-2Kb gene product (Blank and Lilly, 1977; Bubbers and Lilly, 1977). Present evidence indicates that FV-encoded molecules become associated with H-2Db molecules on the surface of infected cells, and that this molecular complex possess an antigenicity which induces the CTL response and which is recognized by the effector CTL population. In FV-infected cells of other H-2 genotypes, such H-2hiral protein complexes may involve H-2K or H-2D molecules, or both, or neither, and these complexes may or may not possess the capacity to induce a strong CTL response. By this mechanism, the H-2 genotype of the host plays a major role in determining whether or not virus-infected cells bear an antigen appropriate for eliciting a CTL response capable of destroying tumor cells.

3. Other Immune Reactivities Many indications are available that natural immunity against endogenous type C viruses in many mouse strains may be important in regulation of virus protein expression and virus replication. As discussed in Section IV, seroIogists had noted reactivity with type C associated antigens in a variety of reagents and normal mouse sera. More direct evidence for a role of humoral immunity in regulating virus protein expression came from studies of group specific antigen (gs) expression in R F mice. Gs expression was found to increase in these mice until approximately 50 days of age, at which time a sharp decrease in expression could be correlated with germinal center and gs antigen localization by immunofluorescence (Hanna et al., 1972). While R F autogenous mice seem to be protected by such type of immunity against lymphoid neoplasia, this response seemed detri-

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mental in the sense that it promoted glomerular disease (Hanna et al., 1972).By contrast, AKR mice also generate a natural humoral antiviral response, and while the antibodies can be eluted from kidneys (Oldstone et al., 1976), glomerular disease does not become fatal because death from leukemia occurs more rapidly. Alternatively, in AKR mice, production of immunoglobulin and immune complexes does not lead to glomerular damage. It should be noted that AKR mice have a complement deficiency (C5) which might explain why humoral immunity is not effective in protection against viremia and leukemia. With the introduction of potent radioimmunoassay techniques (Ihle et al., 1973), it was soon determined that occurrence of this type of immunological reactivity was found in many strains, including I, 129, NZB, C3H, A, and DBM2 (Nowinski et al., 1974). In most cases, the antiviral response was directed at gp70 and p15E (Ihle et al., 1976b; Oldstone et al., 1976). H-2-linked genes may affect the generation of these immune reactivities (Nowinski, 1975). However, the in vivo significance of these reactions remains unclear. Although selected normal mouse sera react with isolated viral gp70 (Ihle et al., 197613; Stephenson et al., 1976), many normal sera reactive with intact virus show no reactivity with isolated viral proteins. This result may be explained on the basis of weak-affinity interactions with determinants on gp70 and/or p15E which may be altered. However, the biological role of such antibodies is unclear, e.g., there exists high reactivity in AKR mice and low reactivity in C58 mice (Nowinski et al., 1974) although these strains have similar virology and leukemia incidence. It is of further interest that antibodies reactive with ecotropic viruses are seen only in certain strains, and antibodies to xenotropic viruses are seen only in the C57BLJ6 strain, but all strains appear to have antibodies to the recombinant MCF virus (Stockert et al., 1979). The relationship of these natural antibodies to prevention or enhancement of disease is under active investigation. Chesebro et al. (1979) and Doig and Chesebro (1979) have shown that H-2 and non-Hi-&linkedgenes are required for recovery from FVinduced leukemia. The non-H-2-linked gene, Rfu-3,appears to influence the production of anti-FV antibody independent of the H-2 genotype. Although mice of the Rfu-3‘l” genotype produce high levels of anti-FV antibody, they fail to recover from FV-induced leukemia unless the appropriate H-2 associated genes are present (Chesebro et al., 1979; Doig and Chesebro, 1979). Thus, FV leukemia cells continue to proliferate even in the presence of anti-FV antibody, possibly because of their resistance to antibody-complement-mediated lysis (Doig and Chesebro, 1979). Furthermore, FV leukemic spleen cells late in the

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disease express decreased amounts of FV cell surface antigens and release less infectious virus than leukemia spleen cells early in the course of the disease (Doig and Chesebro, 1979).The low levels of FV cell surface antigens and infectious virus observed in mice with antiFV antibody were found to be reversible after transfer of leukemic spleen cells into nonimmune animals. Therefore, anti-FV antibody appeared to play a role in altering the expression of viral antigens and infectious virus release in these cells. The presence of anti-FV antibody late in the disease is associated with decreased expression of Friend helper virus (F-MuLV)-encoded intracellular and cell surface proteins, whereas the expression of the replication-defective spleen focus forming virus (SFFV)-encoded proteins appeared to be minimally altered. Other investigators have suggested that cell surface antigen loss induced by antiviral antibody might protect infected cells from virus-specific cell-mediated lysis. This did not appear to be the case in the FV system. Anti-FV CMI effectors could recognize late leukemic spleen cells even though these cells expressed greatly decreased amounts of F-MuLV cell surface antigen. It appears likely that these CTL are able to recognize residual low amounts of F-MuLV gp70 on late leukemic cells. VI. Prospects for Control of Human Leukemia

We began this articIe with two questions: (1)Are viruses involved in human neoplasias? and (2) How can the knowledge on hand be applied to arrest or control the malignant process? In the face of the voluminous literature described, it is clear that biologists have done much to understand viral-induced leukemogenesis. The point is rapidly approaching when we can answer the first of these two questions and begin to provide an answer to the second question. Let us recapitulate the central core of knowledge that should permit such answers. First, with the demonstration that transforming genes of retroviruses are derived from cellular genes, designated protooncogenes, a door has been opened in search of the desired answers. Thus if certain cellular genes are oncogenic when placed under the influence of viral control elements, perhaps other genes, or indeed the same ones, mediate carcinogenesis by various agents when they are expressed at high levels as a result of exposure to harmful environmental agents. Second, identification and cloning of endogenous retroviral sequences present in human DNA appear to suggest an affirmative answer to the first of our two questions. For example, Martin et al. (1981) have shown that under nonstringent annealing conditions, a

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2.75 kb segment of cloned African green monkey DNA that specifically hybridizes to the provirus of AKR ecotropic murine leukemia

virus (MuLV) and baboon endogenous virus (BaEV), detects related sequences in three different preparations of human brain DNA fragments. Furthermore, the evidence for human viral-like DNA is strengthened by the fact that restriction analysis of the annealing human sequences yields similar results to those obtained for mouse DNA annealing with the MuLV cDNA probe. In both cases, multiple bands suggested the presence of numerous copies of retrovirus-related sequences integrated in the genomes (mouse and human). Even more recent and convincing data come from the laboratory of R. C . Gallo (Poiesz et al., 1981a,b), which has directly isolated a newly discovered retrovirus in association with certain types of adult T cell lymphonidleukemia. The novel retrovirus has been isolated from a Tlymphoma cell line established in culture from a patient with mycosis fungoides. Subsequent results by Poiesz et al. (1981b) demonstrated the isolation of a similar virus from a patient with Sezary syndrome, and Kalyanaraman et al. (1981)showed that sera from two patients and one of the patient’s spouses contained antibodies which specifically reacted with the major core protein of the virus. Mycosis fungoides and Sezary syndrome are clinical variants of cutaneous T cell lymphoma and leukemia, respectively, a rare disease in human adults (Lutzner et al., 1975). Probably several technical difficulties account for the failure to detect such viruses earlier. One is the fact that culturing of leukemia cells is often required to detect virus expression, and second, culturing of human T cells was impossible until recently. With the advent of T cell grow factor (TCGF), in witro T cell culturing and detection of human retroviruses became possible. The virus called HTLV (for human T-lymphoma virus) seems to be quite distinct from the numerous types of animal retroviruses previously described. HTLV is morphologically and biochemically more closely related to bovine leukemia virus, which causes lymphoma in cattle, although the human and bovine viruses seem unrelated antigenically or by nucleic acid homology. The epidemiological most convincing data come from Japanese workers. Miyoshi et al. (1982a-c), by cocultivated T leukemic cells with umbilical cord leukocytes, have made the cord leukocytes TCGF-independent cells. These cultures have subsequently been shown to be producing virus similar to HTLV particles. These workers have thereby demonstrated for the first time with human cells experimental transmission of and transformation by a human virus.

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Even more striking, however, is the association between the virus and leukemia in uiuo. In major Japanese cities, there is a relatively high incidence of an aggressive form of adult T cell leukemia. An HTLV-like virus seems to be associated with such an aggressive, particularly malignant, fast growing type of T cell leukemia. Association of this type of leukemia with HTLV is strongest in Kyushu, Japan’s extreme southwest island. For example, a recent study (Uchiyamo et al., 1981) of 272 T cell leukemia cases shows a remarkable clustering of places of birth, in the Kagoshima and Nagasaki prefectures of Kyushu, and indicates that sera from all the leukemia patients tested and from 25% of healthy adults sampled react positively with the Miyoshi’s virus isolate (Miyoshi et al., 1982a) (similar or identical with HTLV). While not all T cell leukemia patients tested were positive (Kalyanaraman et al., 1981; Miyoshi et al., 1982a), the evidence for the involvement of viruses in human neoplasms becomes strengthened by such findings. Furthermore, the above evidence is, of course, not the only one supporting a role for viruses in human neoplasia. Previously, other viruses had been implicated as etiological agents for several human neoplasms. For example, Burkitt’s lymphoma and nasopharyngeal carcinoma have been associated with Epstein-Barr virus. Hepatitis B virus infection has been associated with liver cancer. Herpes simplex and papilloma virus have been suspect in the etiology of cancer of the uterine cervix. What is perhaps most clear now is that our ability to identify transforming genes, whether cellular or virus encoded, and our ability to isolate, clone, sequence, and study the transforming function of these genes and their regulatory mechanisms are increasing. At the same time, our understanding of the immune system, our ability to produce monoclonal antibodies of high specificity, and to identify other host mechanisms of defense against neoplasia are improving. Thus it is not unreasonable to foresee the day when this knowledge will be brought to bear successfully in the control of human cancer.

ACKNOWLEDGMENT Dr. Hugh 0. McDevitt introduced one of us (Daniel Meruelo) to the fundamental notions embodied in this article. His contributions in many of the areas covered, particularly those dealing with involvement of immune response and other H-2 and H L A genes in disease susceptibility, have been helpful in formulating our approach to the subject. His careful reading of this manuscript and constructive comments and advice are much appreciated.

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