Antigens of murine leukemia viruses

Biochimica et Biophysica Acta, 355 (1974) 105-118 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - Printed in T h e N e t h e r l a n d s BBA 87004

ANTIGENS FRANK

OF

MURINE

LEUKEMIA

LILLY and RICHARD

VIRUSES

STEEVES

Departments of Genetics and of Developmental Biology and Cancer, AIbert Einstein College of Medicine, Bronx, N. Y. 10461 (U.S.A.) (Received N o v e m b e r 8th, 1973)

CONTENTS I.

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

II.

Biochemically defined antigens . . . . . . . . . . . . . . . . . . . . . . . . . .

105 106

A. Protein 30 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

107

B. Glycoprotein 69-71 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Protein 15 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

108 109

D. Protein 12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

109

E. Protein 10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

109

F. Reverse transcriptase . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

109

Ill. Other antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

110

A. Virus envelope antigens . . . . . . . . . . . . . . . . . . . . . . . 1. M u L V laboratory isolates . . . . . . . . . . . . . . . . . . . . . 2. Naturally occurring, e n d o g e n o u s M u L V s . . . . . . . . . . . . . B. Cell surface antigens . . . . . . . . . . . . . . . . . . . . . . . . . i. G r o s s leukemia antigens . . . . . . . . . . . . . . . . . . . . . . 2. F M R leukemia antigens . . . . . . . . . . . . . . . . . . . . . . IV. C o n c l u d i n g remarks

. . . . . .

. . . . . .

. . . . . .

. . . . . . .

110 II1 1I I 112 113 113

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

114

A. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

114

B. Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

115

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I 16

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I 16

I. I N T R O D U C T I O N Antigens immunologists concentrated

associated [I]

and

with murine by

on antibodies

virologists

leukemia [2,3].

viruses have been studied The

(and also cell-mediated

immunologists immune

have

responses)

by tumor generally

obtained

in

Abbreviations: M u L V , murine leukemia virus; s u b g r o u p s G a n d F M R , s u b g r o u p s G r o s s a n d F r i e n d - M o l o n e y - R a u s c h e r of M u L V ; CSA, cell surface antigen; VEA, virus envelope antigen.

106

I

co E

VIRION [

f O 0 0 - 1200

750 - 800 ~-

I

L \ NUCLEOID

Fig. 1. Anatomy of a mature MuLV particle. the natural host of the virus, ill order to study their role in controlling the disease. On the other hand, virologists have used antibodies, generally obtained from a different species, as probes for dissecting the molecular anatomy of the virus particles. This review concerns the virologic approach and includes antigens discovered by both approaches. Morphologically the various strains of murine leukemia virus (MuLV) are indistinguishable from one another (Fig. 1). They are spherical particles, 1200 A in diameter, consisting of a core surrounded by an envelope acquired during maturation of the virus at the surface of infected cells [4]. The core consists of an outer shell, perhaps constructed of capsomers, and an inner nucleoid containing the genetic material and associated proteins. The envelope has surface projections which have been visualized only recently with special preparative techniques [5]. Although biochemical analysis has revealed a vast array of electrophoretically distinguishable molecular entities in these virus particles, it is now apparent that there are only a small number of major molecular components. Our aim in this review is to relate each of the numerous antigens associated with MuLVs with one of these molecular components of the virus, to the extent that this is now possible.

11. BIOCHEMICALLY DEFINED ANTIGENS In prelude to the biochemical definition of MuLV antigens, it was found that sera from rats bearing MuLV-induced tumors had complement-fixing and precipitating activity when tested against viral extracts [6,7]. The antigens detected in these reactions were species- or group-specific (gs), since the sera reacted with all MuLVs but not with leukemia viruses of other species. The first major antigen of this type to be isolated was called gs-I [8]. Another antigen detected, called gs-3 [9,10], was shown to be an interspecies antigen, for the antibodies which defined it cross-reacted with leukemia viruses of cats, rats and hamsters. Although clearly defined as distinct antigenic specificities, gs-I and gs-3 were later shown to reside on the same major protein [11,12].

107 It seems now to be a fair assumption that all leukemia viruses contain a homolog of this major protein. That portion of this polypeptide which, in MuLVs, bears gs-1 activity thus appears to vary considerably in other leukemia viruses, according to their species of origin. In contrast, the portion of this polypeptide bearing gs-3 (interspecies) activity is relatively but not totally invariant from one species to another. Furthermore, a third class of antigenic differentiation has been observed among different strains of leukemia viruses obtained from the same species, and such typespecific differences also exist on this major MuLV protein [13]. It is probably more accurate to think of a continuous spectrum of antigenic specificities associated with this major protein, rather than three distinct classes (interspecies, group and type), and this same principle undoubtedly applies to other protein constituents of MuLV [I 3]. Therefore, rather than discuss the antigenic specificities individually, we shall consider now the antigenic properties of those proteins that have been isolated, purified and biochemically characterized. Separation of the proteins from disrupted MuLV particles has been carried out by chromatography on agarose columns saturated with g u a n i d i n e HCI and by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. It has been agreed* to designate these proteins according to their molecular weights, using the former method for low molecular weight (10000-30000) proteins and the latter method for the higher molecular weight (30000-100000) proteins. IIA. Protein 30 (p30) (Synonyms: gs-1 [8], component Ill [14], protein 1 (pl) [15], polypeptide 4 (P4) [3], interspec 1 [16].) Around 30 ~ of the total virion protein of MuLVs consists of a major structural molecule of about 30000 daltons [11]. Bolognesi [3] has suggested that this molecule constitutes the core shell of MuLVs, by analogy with tile homologous p30 molecule of avian myeloblastosis virus. In keeping with this role as an internal virion component, p30 does not elicit the formation of virus-neutralizing antibodies [17], and p30 antibodies do not react by immunodiffusion with intact MuLV particles [7]. The amino acid sequences of p30 from several strains of MuLV and from leukemia viruses of other species are currently being determined [18], and this information will be of value in inferring evolutionary relationships among these viruses. The MuLV p30 molecule is highly immunogenic in non-murine hosts, but mice do not appear to respond immunologically to this substance. Whether antibodies to gs-I or gs-3 are obtained depends on the species immunized and on the method of immunization. Guinea pigs immunized with purified p30, for example, respond principally to the gs-I determinant, whereas rats bearing MuLV-induced tumors respond to the gs-3 as well as to the gs-1 determinants. These antibodies can be used to detect p30 not only by complement fixation [6], but also by immunodiffusion * In this review we identify the protein components of MuLV according to the nomenclature accepted at the "Oncornavirus Discussion Group" on June 4, 1973, at the Sloan-Kettering Institute for Cancer Research, New York City.

108 [7] and by the very sensitive radioimmune assay [19,20]. That both gs-1 and gs-3 are on the same p30 molecule was demonstrated by Gilden et al. [11 ] with reciprocal blocking in immunodiffusion gels and subsequently confirmed by Parks and Sco!nick [19] using competitive inhibition with heterologous and homologous combinations of antigens and antibodies in the radioimmune assay. The latter analysis was confirmed by Strand and August [13], who demonstrated type-specific determinants on p30 as well. Gs-I antigen activity, and hence presumably p30, is widely distributed in the reticular tissues of normal mice. Its presence does not, however, correlate consistently with the presence of infectious MuLV. For example, mice with a high incidence of spontaneous leukemia (AKR) express both gs-I antigen and infectious MuLV throughout postnatal life. Mice with a low incidence of spontaneous leukemia fall into three categories: (1) mice which are gs-l-positive but virus-negative throughout life (e.g., N IH Swiss), (2) mice which are gs-l-positive as early embryos, which become gs-l-negative prior to birth and become positive both for gs-1 antigen and frequently for infectious virus late in life (e.g. BALB/c, C3H), and (3) mice which are negative for both gs-I antigen and infectious virus throughoutlife (e.g. C57L) [21 ]. In studies of crosses of A K R mice with low-leukemic strains, there appears to be considerable correlation in the expression of gs-I antigen and infectious virus [22-24]. However, the occasional finding of the antigen in the absence of infectious virus, noted above in certain mouse strains, suggests that other regulatory genes may be found to affect these factors individually.

HB. Glycoprotein 69-71 (gp69-71) (Synonyms: antigen 11 [25], M2 [15], interspec 11 [16], OSA [26].) A high molecular weight glycoprotein was identified by chromatography [15] and by immunodiffusion [25] in extracts of MuLV. Electrophoretic analysis of the substance, recently purified by Strand and August, has revealed that it consists of two separable, but apparently antigenically related molecules, with approximate weights of 69000 and 71000 daltons [16]. They comprise 5-10% of the total viral protein, and, because they contain small amounts of sialic acid, glucosamine and galactose, they are thought to be part of the viral envelope. This suggestion is supported by the observations that these molecules are iodinated when intact virions are treated by the lactoperoxidase procedure [27], and that antibodies to purified gp69-71 neutralize the infectivity of MuLV [17]. An apparently analogous antigen has been detected on the surfaces of cells productively infected with MuLV and also in small amounts on the surfaces of uninfected cells [27]. The interspecies-specific determinant (interspec 11) of MuLV gp69-71 was demonstrated initially by gel diffusion with antiserum against feline leukemia virus, against which a precipitin pattern of non-identity was observed with p30. In the radioimmune assay, a complete lack of antigenic cross-reactivity between gp69-71 and p30, as competing antigens, was confirmed [16]. In further studies with the radioimmune assay [13], extracts of a variety of leukemia viruses were used as

109 competing antigens in combinations of labeled MuLV gp69-71 with either feline leukemia virus antiserum or homologous antiserum, and the results of these studies demonstrated both group- and type-specific determinants, as well as the interspecies determinant, on gp69-71. These group- and type-specific determinants may represent the major virus envelope antigens revealed in virus neutralization studies, to be discussed below. IIC. Protein 15 (pl5) (Synonyms: protein 2 (p2) [15], polypeptide 3 (P3) [3].) Following the recent identification of the three major low molecular weight components of MuLVs, these proteins have now been isolated and purified, and studies of their biochemical and antigenic characterization have begun to yield information. Purified MuLV p15 has a molecular weight of 15000 [75]. Studies with detergents and sucrose gradients show that this product is present in the viral cores [76], but it is not associated with ribonucleoprotein isolated from the cores [77]. Green et al. [75] found little or no precipitating antibody activity specific for this protein in any of several antisera obtained by immunizing with whole or disrupted virus preparations. IID. Protein 12 (p12) (Synonyms: protein 3 (p3) [15], polypeptide 2 (P2) [3].) Another of the three small MuLV proteins demonstrated by column chromatography in guanidine' HCI [15] has been further analyzed by polyacrylamide gel electrophoresis [75] and by the radioimmune assay [28]. It is a polypeptide of molecular weight 12000 as determined by chromatography and 16000 by electrophoresis. Green et al. [75] concluded that this protein contains about 5 ~o carbohydrate and shows staining properties with Coomasie blue dye similar to those of a smaller polypeptide (pl0) obtained from avian myeloblastosis virus and thought to be located at or near the surface of the virus particle. MuLV p12 has been purified, labeled and tested by Tronick et al. [28] in the radioimmune assay with antisera to several purified MuLVs. Competition experiments revealed both group- and type-specific determinants on the p12 molecule, but the presence of interspecies determinants has not yet been investigated. Also untested is the possibility that antigenic determinants of this molecule might be involved in virus neutralization. llE. Protein 10 (plO) (Synonyms: protein 4 (p4) [15], polypeptide 1 (PI) [3].) The smallest of the major MuLV proteins has a molecular weight of 10000 and is associated with ribonucleoprotein in the viral core [77]. This protein, which is relatively rich in arginine, has shown group-specific antigenic activity [75,77]. HF. Reverse tranvcriptase A protein of molecular weight 70000, possibly a subunit of a larger molecular

110 complex but present in the core in quantities too small to be detected as a major virion component, bears the enzymatic activities of reverse transcriptase. We shall discuss here the antigenic properties of the molecule, and those interested in its enzymatic properties are referred to a review by Temin and Baltimore [29]. Initial studies showed that antibodies from the serum of rats bearing MuLVinduced tumors could inhibit the enzymatic activity of MuLV reverse transcriptase [30]. Subsequently, rabbit antisera to partially purified MuLV reverse transcriptase were used to demonstrate that reverse transcriptases carry both interspecies- and group-specific determinants [31]. The interspecies determinants of this MuLV molecule are closely related to those of leukemia viruses from other small mammals (rat, cat and hamster), but are not related to those from primate or chicken leukemia viruses; in contrast, the interspecies determinants of MuLV p30 are related to those of leukemia viruses from both small mammals and primates, although again not to those from chicken leukemia viruses [32,33].

1II. OTHER ANTIGENS The major antigens associated with MuLV infection which were defined in biologic studies have received at most only preliminary biochemical characterization. These antigens have been defined in two completely different systems: the cytotoxic test, which detects cell surface antigens (CSAs) on infected cells, and the virusneutralizing test, which detects virus envelope antigens (VEAs) on the surface of virus particles. Because VEAs are also expressed on the cell surface at sites of budding virus particles, there has been some uncertainty in the past as to whether CSAs and VEAs are the same or different from each other. However, immunoelectron microscopic studies have clearly shown that the two types of antigens are located in topographically separate regions of the cell surface.

HIA. Virus envelope antigens Neutralizing antisera may be produced by immunization with virus-infected cells or with crude or purified preparations of infectious or inactivated virus. With the recent availability of highly purified VEA molecules, the choice of host for immunization is of little consequence. With crude or even gradient-purified MuLV preparations, on the other hand, the immunized host should ideally be syngeneic with the donor of the virus-infected tissue. This precaution avoids the induction of antibodies to host-specified antigens which may occur on the MuLV particle. However, mice of strains from which the virus was harvested rapidly succumb to the disease induced by some MuLVs, and in these cases immunization can be initiated with a sub-threshold dose of infectious virus and the dose increased in succeeding immunizations. The alternative approach of inactivating MuLVs (e.g. formalin treatment) may reduce their immunogenicity. Methods for detecting VEAs include: electron microscopy with visually labeled

111

antibody, hemagglutination-inhibition and neutralization. With the first method, antibodies specific for VEAs may be coupled directly with a visual marker or may be hybridized with antibodies directed against a visual marker such as ferritin [34] or southern bean mosaic virus [35]. On MuLV-infected cells, such antibodies label only virions and those sites of the ceil surface involved in virus maturation [36]. The acquisition of VEA molecules at sites of virus budding either replaces or masks most cell surface alloantigens on most, but not all, virus particles. The presence of a residuum of alloantigens, plus the finding that sera from rats and rabbits immunized with normal murinetissues rapidly inactivate MuLVs [38,39], reinforces the importance of immunizing mice with tissue from syngeneic donors. Hemagglutination of sheep erythrocytes can be induced by MuLVs, but only with virus treated with neuraminidase and phospholipase C. This treatment damages the virion envelope and exposes its subunits so that they may interact with the erythrocyte membrane in the hemagglutination reaction [40]. These subunits are associated with the surface projections and glycoproteins of the virion, with viral infectivity and with group- and type-specific VEAs. Antibodies to MuLV will inhibit the hemagglutination reaction specifically after elimination of nonspecific inhibitory factors from the antisera [41 ]. Virus neutralization is the most sensitive and discriminating of these three tests for VEAs. The test is most easily carried out with an enumerative response assay method, such as the XC test in vitro [42] or the spleen focus assay in vivo [43 ]. Antiserum activity may be expressed in terms of dilution end-point or, better, rate of virus inactivation [44]. 1. MuLV laboratory isolates. Established MuLV isolates have recently been classified on the basis of their type-specific antigens by virus neutralization with murine antisera. These antisera were prepared against strains of MuLV which are nonpathogenic but usually immunogenic in adult mice. Because these MuLVs can serve as helpers for two defective, easily titrated viruses (spleen focus-forming virus, SFFV, and murine sarcoma virus, MSV), the MuLVs were used to produce pseudotypes of the defective viruses, which bear the VEAs of their helper MuLVs. The results of cross-neutralization experiments with pseudotypes of SFFV [45] are summarized in Table 1, in which the viruses are grouped into five categories. A similar study, carried out with pseudotypes of MSV [46] did not discriminate between Types 2a, b, c and d of Table I, but separated the Graffi and Tennant MuLVs into distinct categories. The use of neutralizing antisera rendered more specific by selective absorption may help to resolve these minor differences. 2. Naturally occurring, endogenous MuL Vs. Strains of mice with a high incidence of spontaneous leukemia (AKR, C58 and C3H/Fg) produce throughout their postnatal lives MuLVs which appear to be serologically related or identical to Gross virus [47]. Morphologically identical viruses also appear spontaneously in clonal lines of BALB/3T3 cells, and these viruses, some of which bear a distinct antigen, xVEA, do not carry the Gross MuLV antigen, GVEA [48]. It is thus clear that there are type-specific differences among VEAs of naturally occurring viruses, as well as among VEAs of laboratory isolates.

112 TABLE 1 A PRELIMINARY CLASSIFICATION OF MuLVs ACCORDING TO THEIR VEAs This is modified slightly from ref. 45. Antibodies to subgroup 2 MuLVs do not neutralize subgroup 1 virus (Gross pseudotype). Antibodies to Types 2a and 2b MuLVs do not cross-react with each other, but these types are included within the same subgroup because antibodies to Type 2c MuLVs neutralize both Types 2a and 2b MuLVs. Buffett MuLV has been placed in a separate category (2d) because it was neutralized by antisera to some but not all Type 2a and 2c MuLVs. The MuLV strains are named according to the investigators who first isolated them. LLV-Friend and LLV-Rauscher are the lymphatic leukemia-inducing helper viruses in the Friend and Rauscher virus complexes, respectively. SimLV is a MuLV isolated from the spontaneously enlarged spleen of a Swiss inbred (SIM) mouse. Subgroup

Type

1

2

Strains G ross LLV-Friend, Graft], Tennant (B/T-L), SimLV, Rowson-Parr Moloney, Abelson LLV-Rauscher, Rich, Breyere-Moloney Buffett (334C)

llIB. Cell surface antigens Surface antigens on M u L V - i n f e c t e d cells were first d e m o n s t r a t e d in transp l a n t a t i o n studies with virus-induced tumors. Mice rejecting such t u m o r grafts could often be shown t o possess a n t i b o d i e s specifically cytotoxic in the presence o f c o m p l e m e n t for cells o f the same t u m o r line and of other t u m o r s induced by the same strain o f virus. In m o s t cases these cytotoxic sera were found to contain virusneutralizing activity as well as cytotoxic activity. The possibility t h a t these two activities were due to the same p o p u l a t i o n o f a n t i b o d i e s was disproven by the d e m o n stration t h a t washed, intact virus particles a b s o r b e d the virus-neutralizing activity b u t left the c y t o t o x i c activity o f the serum undiminished [49,50]. These experiments also indicated t h a t the C S A c o r r e s p o n d i n g to the cytotoxic a n t i b o d i e s was not present on the surface of virions in a n t i b o d y - a c c e s s i b l e form. Nevertheless, similar cytotoxic antisera can also be p r e p a r e d in some cases by i m m u n i z i n g directly with M u L V p r e p a r a t i o n s , rather t h a n cells: in this case it a p p e a r s t h a t the cytotoxic a n t i b o d i e s are f o r m e d in response either to C S A material c o n t a m i n a t i n g the virus p r e p a r a t i o n (usually an extract o f infected tissue), or to C S A induced on the cells o f the i m m u n i z e d host by infection with the i m m u n i z i n g virus. The pattern o f cross-reactivity o f c y t o t o x i c a n t i b o d i e s p r e p a r e d against leukemias induced by various strains o f M u L V resulted in the designation of t w o m a j o r s u b g r o u p s of m u r i n e teukemias: G (Gross) and F M R ( F r i e n d - M o l o n e y - R a u s c h e r ) [l]. Since the cross-reactions observed a m o n g antisera to Friend, M o l o n e y and R a u s c h e r l e u k e m i a s are incomplete, this classification scheme is u n d o u b t e d l y an oversimplification, and true type-specific C S A d e t e r m i n a n t s have yet to be analyzed. The existence o f an interspecies C S A d e t e r m i n a n t has been suggested by the b r o a d i m m u n o f l u o r e s c e n c e reactivity o f a r a b b i t a n t i - d i s r u p t e d feline leukemia virus antiserum with cells infected by M u L V s o f both the G and F M R s u b g r o u p s [51]: the r e l a t i o n s h i p o f this antigen with other C S A s is u n k n o w n .

113

1. Gross leukemia antigens. Antibodies specific for G antigen were made by immunization of C57BL mice with Gross virus-induced tumors [52,53] and were also found in some normal sera from mice of this strain. The antibodies are cytotoxic for leukemias induced by both Gross virus and radiation leukemia virus [54], both of which are laboratory-passaged, endogenous MuLVs. Normal, pre-leukemic spleen cells of high-leukemic mouse strains (e.g. A K R ) will absorb G antibodies, and this trait segregates in a mendelian fashion in crosses with negative mouse strains. Immunoelectron microscopy has confirmed that the G CSA is widely distributed on the membranes of virus-infected cells, but it is not found on the surfaces of budding or mature virions [36]. The antigen has been solubilized from leukemic cell membranes, and chromatography revealed molecules of two different sizes bearing the antigenic determinant [55]. Serum from Gross virus-infected mice specifically inhibits the cytotoxicity of anti-G antibodies, and sedimentation of virus particles from the infectious serum does not significantly diminish this inhibitory activity [56]. This G soluble antigen is also found in the sera of pre-leukemic A K R mice [57]. Large amounts of G soluble antigen can adsorb onto the surfaces of cells in vitro and in vivo [58]. Another antigenic determinant associated with endogenous MuLV is G~x, identified by cytotoxic antibodies from rats immunized with Gross virus-induced, syngeneic tumors [59]. This antigen is present not only on murine Gross virusinduced leukemias and on normal and leukemic lymphocytes of A K R mice, but also on thymocytes of mice of Strain 129 and of a few other low-leukemia, apparently MuLV-free strains. Genetic control of the G:x antigenic determinant in normal mice is complex. A close relationship between the G and Gix antigens is indicated by the fact that in crosses o f A K R (G- and G~x-positive) and C57BL (G- and G~x-negative) mice, the genes governing these two traits are completely linked [24]. This might be interpreted as indicating that the two antigenic determinants are located on the same molecule, although other explanations are possible. 2. FMR leukemia antigens. 1.eukemias induced by several strains of MuLV laboratory isolates have been shown to induce antibodies cytotoxic to their homologous t u m o r cells (e.g. Friend [60,61], Moloney [62], Rauscher [63] and Graffi [64] leukemias). These antibodies and tumor cells cross-react to a greater or lesser extent among themselves, and this fact led to the concept of the F M R group of virusinduced leukemias [63]. In contrast with the G antigen specificity, the F M R determinant is not expressed in normal cells of any known mouse strain. Like the G determinant, F M R is found both on the surfaces of virus-infected cells and in soluble form in viremic serum [65]. Washed, intact virions from the serum of Friend virusinfected mice are capable of only a slight inhibition of the cytoxicity of anti-FM R antibodies [66], but disruption of the virions by freezing and thawing (Fig. 2), ether extraction or detergent treatment releases large amounts of antigenic activity from an intravirion location apparently external to the cores [67]. The release of F M R antigen from disrupted virions was also suggested by the finding that ether extraction o,f purified Friend virus rendered the particles capable of inducing immunity to

i14 I00

~r~

o

4

5¢

2

7

3

5

10

20

1- 2 5

50

100

ANTIGEN DILUT$ON

Fig. 2. Release of FMR antigen activity from Friend MuLV particles by freezing and thawing. Aliquots of Fractions 1-7 were tested for their ability as antigens to inhibit the cytotoxicity of FMR antiserum; the stronger the inhibitory activity, the further the curve was displaced to the right in the figure. Whole serum (1) was collected from infected mice; it was centrifuged (100000 ~, g for 1 h), the supernatant (2) removed and the pellet resuspended to the original volume in saline; the resuspended pellet was recentrifuged, yielding another resuspended pellet (3) and supernatant (4); this second resuspended pellet was subjected to five cycles of freezing (I-70 °C) and thawing (5); the frozen and thawed material was centrifuged as before, yielding another supernatant (6) and resuspended pellet (7). The effect of disruption of the virions by freezing and thawing was a 9.6-fold increase in inhibitory activity (Fractions 3 vs 5).

Friend t u m o r c o l o n y - f o r m i n g cells [68]. The presence o f the F M R C S A within Friend virions suggests t h a t other M u L V C S A s might also be f o u n d in the virus particles, and t h a t these antigenic d e t e r m i n a n t s m i g h t be located on a m a j o r virion protein or glycoprotein.

IV. CONCLUDING REMARKS IVA. Discussion R N A t u m o r viruses are a class of biologic entities which have u n d o u b t e d l y h a d as much o p p o r t u n i t y to evolve as their hosts. Given the s t r o n g species-specificity o f these viruses, t h o s e o f each host species have b e h a v e d in general as isolates free to evolve s e p a r a t e l y from o t h e r isolates. F u r t h e r , s e p a r a t i o n o f different varieties o f the p r o t o t y p e virus within a single species is by no m e a n s an unlikely event. Each o f these genetic divergences is p o t e n t i a l l y the source o f v a r i a t i o n in the antigenic p r o p erties o f the m o l e c u l a r c o m p o n e n t s o f the virus. The functional integrity o f these molecules requires t h a t viable m u t a t i o n s be o f limited scope, affecting only those p o r t i o n s o f the molecules where v a r i a t i o n w o u l d n o t interfere with their function. Thus, the relatively invariable p o r t i o n s o f the molecules m i g h t be the sites o f interspecies antigenic d e t e r m i n a n t s , whereas the m o r e variable p o r t i o n s w o u l d b e a r g r o u p - a n d type-specificities. There is now c o n s i d e r a b l e evidence t h a t the g e n o m e s of e n d o g e n o u s M u L V s are vertically t r a n s m i t t e d in mice, in keeping with the t h e o r y o f H u e b n e r and T o d a r o [69,70]. The existing evidence is inadequate, however, t o indicate whether the viral

115 genome is transmitted in a mendelian manner (i.e. chromosomally integrated) or if it is located elsewhere (i.e. in an episome) in the host zygotes. In either case it is certain that the endogenous MuLV genome is subject to regulation by host genes which may act by several different mechanisms. Expression of the viral genome in the form of mature, infectious particles could be prevented by a host gene suppressing a single, indispensible viral gene product. Second, a host gene might influence the virus genome as a whole, e.g. by interfering with the production of RNA copies suitable for packaging in virions or by immunologic destruction of completed virions. A further control mechanism is immunologic surveillance, whereby host cells which spontaneously express all or part of the endogenous MuLV genome might become susceptible to immunologic elimination unless the individual is tolerant to MuLV antigens (e.g. all mice appear to be tolerant to the p30 gs-l antigenic determinant, and mice of the H-2 k genotype do not generally respond immunologically to the G CSA). The concept of host gene regulation of individual virus genes is particularly relevant to the observation of the presence of MuLV antigens in mice in the absence of infectious MuLV itself [69]. All mice which spontaneously express high levels of MuLV also express the p30 antigen, gs-l, and the G and Gtx CSAs in their tissues, but some MuLV-free mice also do so. It seems likely that, in this latter case, the virus is blocked from maturing because some other essential portion of its genome is either suppressed (host regulatory gene) or not present (defective virus genome). An alternative which has been proposed, i.e. that the so-called virus genes expressed in virus-free mice are in fact host genes, is untenable if M u l V s routinely include these genes within the infectious particle. IVB. Hypothesis

Starting with the evidence that F M R CSA also exists within the Friend virion, we propose an hypothesis that relates this fact to a number of other observations currently available. The hypothesis is presented in five separate but related ideas: (1) Molecules with F M R antigenic specificity exist at the proximal ends of the surface projections of the virion envelope; in this inaccessible configuration the FMR antigenic specificities are not immunogenic, nor do they absorb FM R antibodies. (2) Molecules bearing the VEA specificities exist at the distal ends of the same surface projections that bear F M R specificities at their proximal ends; there may be other types of surface projections as well. (3) MuLV envelope surface projections are released into the extracellular space either directly from cells or from virus particles during or after their synthesis, and in this state the projections are independent of virus particles. (4) MuLV receptor sites exist on the surfaces of mouse cells (by analogy with the chicken leukemia virus system [71 ]), and these sites recognize and bind the VEA ends of the envelope surface projections, whether they are virion-attached or free. (5) F M R antigenic activity is transferred to the cell surface when the virion surface projections attach by their VEA ends to virus receptor sites (or perhaps to

116 lr-I gene-related antigen receptors [72]) on the cell surface, leaving the F M R ends of the projections exposed. The hypothesis takes into a c c o u n t the fact that significant a m o u n t s of soluble F M R antigen activity exist in the serum of virus-infected mice, and preliminary experiments suggest that this serum also contains antigen which inhibits virusneutralizing antibodies [73]. The hypothesis explains the adsorption, observed by Stack et al. [65] of soluble F M R antigen o n t o spleen cells from mice of certain strains, by a t t a c h m e n t of the VEA a n d of the antigen to virus receptors. Extensive blocking of virus receptor sites on the cell surface by virus surface projections could a c c o u n t for the broad interference patterns observed a m o n g M u L V s [74]. This might also explain why A K R mice, which release MuLV s p o n t a n e o u s l y , are more resistant to Friend M u L V than mice of other strains with similar sets of M u L V susceptibility genes. We do not yet know if G and other CSAs are on a molecule h o m o l o g o u s to the F M R - b e a r i n g molecule, but if so, then our hypothesis should apply to all MuLVs.

ACKNOWLEDGEMENTS We are grateful to J. T h o m a s August, David Baltimore, Erwin Fleissner, M a u r e e n F r i e d m a n , Alice S. H u a n g and Mette Strand for discussions helpful in the p r e p a r a t i o n of this manuscript. O u r work is supported by contract N O I CP NCI 33249 a n d grant N | H 5 RO1 CA 14529 from the N a t i o n a l Cancer Institute, Bethesda, Md.

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