Role of a membrane glycoprotein in friend virus-induced erythroleukemia: Studies of mutant and revertant viruses

VIROLOGY

144,158-172

(1985)

Role of a Membrane Glycoprotein in Friend Virus-Induced Erythroleukemia: Studies of Mutant and Revertant Viruses CURTIS A. MACHIDA,’

RICHARD K. BESTWICK, BRUCE A. BOSWELL, AND DAVID KABAT

Department qf Biochemistry, School of Medicine, Oregon Health Sciences University, Portland,

Oregon 97.1

Received January 7, 1985;accepted March 15, 1985 We previously reported the isolation and characterization of spontaneous, transmissible mutants of Friend spleen focus-forming virus (SFFV) that are nonpathogenic in adult NIH/Swiss mice and that contain abnormalities in nonoverlapping regions of their envelope glycoprotein (env) genes (M. Ruta, R. Bestwick, C. Machida, and D. Kabat, 1983, Proc. N&l. Acud Sci USA 80, 4’704-4’708).In newborn NIH/Swiss mice, these mutant SFFVs form revertants that are pathogenic in mice of all ages. At least two of three studied revertants contain second site env mutations which affect the sizes and proteolytic fragmentation patterns of their encoded glycoproteins. A variety of structural and genetic evidence suggests that the xenotropic- and ecotropic-related regions of the SFFV glycoprotein fold into separate globular domains that are connected by a flexible proline-rich joint. A glutamyl peptide bond within this joint is exceptionally susceptible to cleavage with Staphylococcus aureus V8 protease. Moreover, disulfide bonds occur within the xenotropic-related domain, but not between the globular domains. These results provide strong additional evidence that the env gene is required for SFFV pathogenesis, and they provide a new system for identifying the features of glycoprotein 0 1985 structure and localization which are essential for its leukemogenic activity. Academic

Press. Inc.

has a recombinant envelope gene (env) Friend erythroleukemia virus is a com- (Bosselman et aC, 1982; Chattopadhay et plex of two components. One component al, 1982; Koch et al, 1984) and that binds is a replication-defective highly patho- to a cell surface receptor different than genic spleen focus-forming virus (F-SFFV) that used by F-MuLV (Rein, 1982; Kozak, 1983). that causes rapid formation of erythroid Interestingly, the Friend SFFV (Fcolonies in the spleens of susceptible mice SFFV) and the similar Rauscher SFFV and in bone marrow cultures (Axelrad (R-SFFV) contain env genes that have a and Steeves, 1964; Anand and Steeves, recombinant-type structure closely related 1980; Hankins and Troxler, 1980). The to those of dual tropic MuLVs (Troxler et second component is a replication-comaZ., 1978; Bosselman et al, 1980, 1982; petent murine leukemia virus (F-MuLV) that serves as a helper for SFFV and by Amanuma et aL, 1983; Bestwick et al, itself causes relatively long latency lym- 1983: Clark and Mak, 1983; Wolff et aZ., 1983; Bestwick et al, 1984; Wolff et al, phatic leukemia in adult mice or eryth1984). Consequently, it has been proposed roleukemia in newborns (Dawson et al, that the wide variety of progressive he1966; Oliff et ah, 1980). The disease in matopoietic leukemias and lymphomas newborns seems to require the intermediate formation of a dual tropic MuLV caused by different dual tropic MuLVs (called F-MCF) (Ruscetti et a& 1981) that (Cloyd et aC, 1980; Haas and Patch, 1980; Famulari, 1983) and by SFFVs might be i Author to whom requests for reprints should be initiated by a common mechanism that addressed. involves their structurally related env INTRODUCTION

0042~6822/85 $3.00 Copyright All rights

0 1985 by Academic Press, Inc. of reproduction in any form reserved.

158

MUTANT

AND

REVERTANT

genes (Famulari, 1983; Bestwick et al, 1983). Recent evidence supports the conclusion that leukemogenesis by SFFVs requires their ewu genes (Linemeyer et cd, 1981, 1982; Ruta et aL, 1983; Machida et aL, 1984). For example, we have isolated three spontaneous env gene mutants of Friend SFFV and two of Rauscher SFFV and all five mutants are either nonleukemogenic or weakly leukemogenic in adult NIH/Swiss mice (Ruta et aL, 1983; Machida et al, 1984). We have also molecularly cloned and sequenced the env genes of three of these mutants (Machida et al, 1985; J.-P. Li, R. Bestwick, and D. Kabat, unpublished observations). Despite their negligible pathogenicities in adults, we have now observed that the F-SFFV mutants reproducibly cause erythroleukemias in newborn NIH/Swiss mice. As described below, this pathogenesis occurs after a lag and involves the formation of revertant SFFVs that contain second-site or intragenic suppressor mutations in their env genes. These revertants are pathogenic in adult mice and they provide a new system for identifying structural features of the glycoprotein that are necessary for its leukemogenic activity. MATERIALS

AND

METHODS

Cells and viruses. All virus-infected NIH-3T3 cell lines were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum and antibiotics. Clonally isolated wild-type F-SFFV and R-MuLV and mutant clone 4,62, and 63 F-SFFVs have been described previously (Dresler et uL, 1979; Machida and Kabat, 1982; Ruta et uL, 1983). MuLVs were routinely titered by using the S+Lmethod (Bassin et uL, 1971). Isolation of revertant viruses. SFFV pathogenesis was detected by previously described methods (Metcalf et uk, 1959; Rowe and Brodsky, 1959; Axelrad and Steeves, 1964; Dresler et ah, 1979). Newborn (l- to 2-day-old) NIH/Swiss mice were injected intraperitoneally with 0.1 ml virus complexes containing R-MuLV and wild-type or mutant F-SFFVs. Control mice were uninfected or infected with R-MuLV alone. Animals were sacrificed

FRIEND

VIRUSES

159

at weekly intervals from 2 to 7 weeks postinfection and were examined for splenic erythroblastosis and for hematocrit. Revertant viruses were recovered from homogenates of enlarged spleens and were subsequently used for infection of secondary recipients and of NIH-3T3 fibroblasts. Analysis of leukemic progression. Adult (6- to 8-week-old) DBA/Z mice were infected with wild-type, mutant, and revertant virus complexes; spleens from these infected mice were then used 7 weeks later in an in vitro methylcellulose colony assay (Mager et ah, 1981) for detection of leukemic cells. Spleen cell suspensions (5 X lo6 cells) were plated in Iscove’s modified Dulbecco’s medium supplemented with 15% heat-inactivated fetal calf serum, 2 X lop5 M 2-mercaptoethanol, and 2% methylcellulose and incubated at 37” for 14 days. Large colonies (containing 3 lo4 cells) were then scored by eye. Individual colonies were transferred into microtiter wells containing 2 ml of Iscove’s modified Dulbecco’s medium plus 15% fetal calf serum and were subsequently labeled with L-[35S]methionine to analyze expression of env gene products. Other methods. All other methods have been described. These include methods for metabolic labeling of cells with L-[~~S]methionine (Dresler et uZ., 1979; Ruta and Kabat, 1980), immunoprecipitation of proteins from labeled cell extracts with antiserum (Dresler et ah, 1979; Ruta and Kabat, 1980), polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate (Laemmli, 1970; Ruta and Kabat, 1980), fluorographic detection of radioactive protein components on dried gels (Bonner and Laskey, 1974; Ruta and Kabat, 1980), and protein fragmentation with Stuphylococcus aweus V8 enzyme (Cleveland et uL, 1977; Machida et uL, 1984). Previously described materials include goat antiserum to Rauscher MuLV gp70 (Dresler et uL, 1979; Machida and Kabat, 1982) and a rat antiserum that reacts with F-SFFV gp55 and with the gp7Os of dual tropic MuLVs but not with the gp7Os of ecotropic MuLVs (Ruscetti et uL, 1979; Kabat et cd., 1980).

MACHIDA

160 RESULTS

Injection of Nonleukemogenic F-SFFV Mutants into Newborn Mice Results in Formation of Pathogenically Active Revertants Figure 1A shows an electrophoretic analysis of viral-encoded membrane glycoproteins synthesized in cell lines infected with mutant or wild-type Friend virus complexes. Cells infected with the wild-type virus complex synthesize the MuLV env gene product gPr90 and its processed derivatives gp70 and p15E in

I2

34

ET AL.

addition to the F-SFFV env gene product gp55 (lane 2). Cells infected with complexes containing clone 4, 62, or 63 SFFV env gene mutants synthesize normal MuLV components and SFFV-related env gene products with apparent M,s of 40,000 (gp40), 54,000 (gp54), and 58,000 (gp58), respectively (lanes 3, 4, and 1, respectively). These SFFV env gene mutants are either nonleukemogenic (clones 4 and 63) or weakly leukemogenic (clone 62) in adult NIH/Swiss mice (Ruta et aZ., 1983). However, since pathogenesis caused by ery-

123456

78

FIG. 1. Electrophoretic analysis of L-[35S]methionine-labeled proteins encoded by wild-type, mutant, or re-.ertant Friend virus complexes. (A) NIH-3T3 cells infected with virus complexes containing replication competent R-MuLV and wild-type (lane 2) or mutant clone 4 (lane 3), 62 (lane 4), or 63 (lane 1) F-SFFVs were pulse-labeled for 2 hr with L-[?S]methionine as described previously (Dresler et CLL,1979; Ruta and Kabat, 1980). Viral proteins were immunoprecipitated from cell extracts with monospecific antiserum to MuLV gp7Oenv. Immunoprecipitated proteins were electrophoresed in 10 to 20% polyacrylamide gels containing 0.1% sodium dodecyl sulfate. Both gp40 and the Mr 45,000~51,000 component seen in lane 3 cosegregate during virus cloning and become rapidly labeled during brief pulses with L-[35S]methionine. Consequently, the larger component is apparently not a more processed derivative of the major smaller J4, 40,000 component. (B) NIH-3T3 cells were infected with virus complexes containing wild-type or mutant Friend virus complexes or with viruses recovered in cell-free extracts of diseased spleens from newborn mice (see Table 1). Viral proteins were immunoprecipitated from @S]methioninelabeled cell extracts with monospecific antiserum to MuLV gp’i’oenv (lanes l-3, 7, and 8), or with a rat antiserum (lanes 4-6) that recognizes antigenic determinants that occur specifically on gp55 and on gp7Os of dual tropic MuLVs. Lanes: 1 and 4, cells infected with R-MuLV and wild-type F-SFFV; 2 and 5, cells infected with R-MuLV and F-SFFV mutant clone 4; 3 and 6, cells infected with virus complex recovered in cell-free extract of diseased spleen of a mouse initially infected with clone 4 (contains revertant F-SFFV clone 4asv); 7, cells infected with R-MuLV and F-SFFV mutant clone 63, 8, cells infected with virus complex recovered in cell-free extract of diseased spleen of a mouse initially infected with clone 63 (contains revertant F-SFFV clone 63asv).

MUTANT

AND

REVERTANT

throleukemogenic retroviruses may depend upon host age (Oliff et d, 1980), we analyzed disease in newborn mice. For this purpose, we used as the helper a cloned Rauscher MuLV (R-MuLV) that causes only a long latency B-cell leukemia in adult or newborn mice (Fieldsteel et al, 1969; Reddy et ok, 1980; Teich et cd, 1982). Moreover, because SFFV pathogen-

FRIEND

161

VIRUSES

esis depends on virus titer and on relative quantities of helper and defective components, aliquots of the viruses were adjusted to contain equivalent titers of the virus components before intraperitoneal injection into newborn NIH/Swiss mice (see Table 1 for details). Mice were sacrificed between 2 and 7 weeks postinfection and were examined for splenic erythro-

TABLE

1

ANALYSIS OF SPLEEN WEIGHTS AND HEMATOCRITS OF MICE INFECTED AS NEWBORNS WITH WILD-TYPE OR MUTANT MURINE RETROVIRUSES Spleen weights Virus inoculated”

[g] and hematocrits

(in parentheses)

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Uninfected

0.06

0.10 0.09 0.10

0.14 0.13

0.13 (45)

0.12 (46)’

0.11 (46)

R-MuLV

0.06

0.11 0.08

0.16 0.17

0.17 (45) 0.13

0.19 (44)C

0.19 (44)

F-SFFV + R-MuLV

0.39 0.25

0.85 0.60

-*

Clone 4 SFFV + R-MuLV

0.07

0.13 0.14

0.12 0.86

Clone 62 SFFV + R-MuLV

0.06

0.13 0.15

Clone 63 SFFV + R-MuLV

0.06

0.11 0.11

-

-

1.50 (65)

1.39 (68)’ 1.46 (65)

2.79 (55)

0.30 0.29

0.39 (46) 0.38

0.46 (35)” 0.43 (50)

-d

0.15 0.14

0.16 (48) 0.20

1.61 (56)’ 0.97 (53)

2.33 (66)

“The SFFV and MuLV components were present in similar ratios and quantities in all preparations tested. The MuLV titers were measured by the S+L- method. The SFFV/MuLV ratios were estimated by measuring the relative amounts of SFFV- and MuLV-encoded glycoproteins formed in NIH-3T3 fibroblasts newly infected with these virus preparations. Ratio estimates determined by this method were consistent with actual SFFV and MuLV concentrations titered by limiting dilution cloning into NIH-3T3 fibroblasts. All injections contained approximately 1 X 10’ MuLV and 4 X lo3 SFFV per dose. Mice inoculated with RMuLV alone were injected with 1 X 10’ MuLV. *All remaining mice infected with virus complex containing wild-type F-SFFV and R-MuLV died between the 3rd and 4th weeks postinfection. Autopsies performed on these mice showed the presence of grossly enlarged spleens. ’ Cell-free extracts of these spleens were prepared and subsequently injected into the tail veins of several adult (4- to g-week-old) female NIH/Swiss mice (see Table 2). The viruses contained in extracts of these spleens from mice initially infected with clone 4,62, or 63 mutants (these recovered viruses are called clone were also used to infect NIH-3T3 cells (see Fig. 1B). The 4ssv, clone 62asv, and clone 63 ssv, respectively) enlarged spleens of mice initially infected with the F-SFFV env gene mutants were also examined histologically. d Four mice infected with virus complex containing clone 62 and R-MuLV died between the 3rd and 4th weeks. Spleens obtained from dead mice were all enlarged and each weighed in excess of 0.6 g. In addition, two mice from both the clone 4 and clone 63 litters died of splenomegaly at the end of 6 weeks postinfection.

MACHIDA

162

blastosis and for hematocrit. As expected, newborn mice infected with our wild-type Lilly-Steeves polycythemia strain of FSFFV developed a rapidly fatal splenomegaly within 2 weeks postinfection (see Table 1). Mice infected with R-MuLV alone did not develop substantial splenomegaly and had normal hematocrits even after the 7th week. Unexpectedly, newborn mice infected with the mutant SFFVs developed splenomegaly, although not as fatal and only after a longer lag phase compared with mice infected with wild-type F-SFFV. In addition, like mice infected with our wild-type F-SFFV, mice infected with two of these mutants reproducibly developed polycythemia (see Table 1). Histological examinations of these enlarged spleens revealed extensive erythroblastosis, with scattered abnormal mitotic figures including tripolar mitoses, and with substantial effacement of normal splenic architecture (data not shown). Surprisingly, all of the viruses recovered from the grossly enlarged spleens of newborns differed from the original mutants in causing pathogenesis, including poly-

ET AL.

cythemia, in adult NIH/Swiss mice (Table 2). Figure 1B shows an electrophoretic analysis of L-[YS]methionine-labeled glycoproteins encoded by viruses recovered from these diseased spleens. In two of the three recovered viruses which were analyzed, the env gene products were electrophoretically different than those encoded by either the original mutants or by wildtype F-SFFV. Thus, the clone 4 mutant (lanes 2 and 5) yielded a recovered virus (clone 4anv) (lanes 3 and 6) which encodes an ilf, 56,000 glycoprotein (gp56). Similarly, the clone 63 mutant (lane 7) formed a recovered virus (clone 63asv) (lane 8) which encodes a glycoprotein with an apparent M, of 62,000 (gp62). However, the clone 62 mutant and its recovered virus (clone 62asv) encode gp54 glycoproteins which coelectrophorese in the one-dimensional gel system (data not shown). Figure 1B (lanes 4-6) also shows that gp55, gp40, and gp56 can all be precipitated by a rat antiserum (Ruscetti et aZ., 1979; Kabat et aZ., 1980) that recognizes an antigenic determinant found in the aminoterminal regions of gp55 and of the gp7Os

TABLE

2

ANALYSIS OF SPLEEN WEIGHTS AND HEMATOCRITS OF ADULT NIH/SWISS INFECTED WITH RECOVERED VIRUSES Spleen weights Virus inoculated” Virus-free extract R-MuLV F-SFFV + F-MuLV Clone 4axv + R-MuLV Clone 62ssv + R-MuLV Clone 63ssv + R-MuLV

[g] and hematocrits

MICE

(in parenthesis)

Week 2

Week 3

Week 4

0.12 0.13 1.53 1.21 2.29 2.49

0.13 (45) 0.14 (44) -b

0.13 (46) 0.13 (44) -

2.52 (66) 1.50 (55) 3.06 (73)

3.36 (76) 1.35 (50) -c

(45) (45) (55) (77) (56) (66)

a Cell-free extracts of the spleens designated in Table 1 (footnote c) were injected into three different 4to g-week-old NIH/Swiss mice. An extract of an enlarged spleen (1.5 g) obtained from a mouse infected with wild-type F-SFFV and R-MuLV was also prepared and injected into adult mice. Spleen weights and hematocrits of injected mice were analyzed at various weeks postinfection. a All remaining mice infected with the wild-type Friend complex died from splenomegaly by the end of the 3rd week postinfection. “One mouse infected with the clone 63ssv complex died from splenomegaly before the end of the 4th week.

MUTANT

AND

REVERTANT

encoded by dual tropic MuLVs but not ecotropic MuLVs. This antigen is also present in the glycoproteins encoded by the clone 62 mutant and its revertant, but not in the glycoproteins encoded by the clone 63 mutant or its revertant (Ruta et aZ., 1983; also unpublished observations). These results are consistent with the regions of the glycoproteins altered by the mutations (see below). Proteolytic Fragmentatim Analyses of the Glycoproteins Enuw!~d by Mutant, Rem&ant, and Wild-Type SFFVs The gp55 encoded by wild-type F-SFFV is cleaved by S. aureus V8 protease to form two primary fragments, V-l (2M, 32,500) and V-2 (J!& 20,500) (see Fig. 2; panel A, lane 2, panel B, lane 1, and panel C, lane 1). The larger primary fragment V-l derives from the amino-terminal region of the glycoprotein and it (but not V-2) contains the antigenic site(s) speeifically related to the gp7Os of dual tropic MuLVs (Ruta et al, 1983; Machida et aL, 1984; Wolff et al, 1984). By increasing the amount of V8 enzyme used for gp55 digestion, V-l is further cleaved to produce secondary fragments V-3 (M,. 21,000) and V-4 (ikf, 12,400) (Fig. 2; panel A, lane 3), and tertiary fragments are observed when the protease concentration is further increased (panel A, lane 4). Although V-2 and V-3 have similar sizes, they can be separated by extensive electrophoresis (Machida et al, 1984; also unpublished observations), and can be distinguished by the fact that under appropriate conditions only V-2 forms during incubation with the lowest concentration of V8 enzyme. Figure 2 also shows comparative cleavage analyses of L-[35S]methionine-labeled glycoproteins encoded by the mutant and revertant F-SFFVs. The V-l fragments of clone 4 gp40 and clone 4zEv gp56 are electrophoretically identical to V-l obtained from the wild-type gp55 molecule (panels A and B). However, gp56 contains a carboxyl-terminal V-2 fragment (M, 21,500; panel B, lane 3) that is larger in

FRIEND

VIRUSES

163

apparent jl& than that obtained from gp55 (panel B, lane 1). The V-2 from gp40 apparently lacks methionine. In addition, the V-l fragment from clone 63 gp58 is electrophoretically larger (M, 33,500;panel C, lane 2) than that obtained from gp55 (panel C, lane 1). The corresponding revertant, clone 63sEv, encodes a gp62 glycoprotein which is not cleaved by V8 protease at concentrations of enzyme (0.1 pg/ ~1) normally employed for primary fragmentation of the wild-type molecule (panel C, lane 3). Furthermore, gp62 is resistant to V8 proteolysis even when lo-fold higher concentrations (1 pg/pl) of enzyme are employed (data not shown). These results indicate that S. aureus V8 protease cleaves gp55 into nonoverlapping amino-terminal V-l and carboxyl-terminal V-2 primary fragments, and that the glycoproteins of mutant and revertant SFFVs have alterations which affect different V8 fragments of the glycoprotein. Although S. aureus V8 protease cleaves both aspartyl and glutamyl peptide bonds in sodium or potassium phosphate buffers, cleavage is limited to glutamyl peptide bonds in an ammonium bicarbonate (50 m&f, pH 7.8) buffer (Houmard and Drapeau, 1972). V8 protease digestion of FSFFV gp55 in the latter conditions caused rapid cleavage to form the V-l and V-2 primary fragments (data not shown). These results suggest that a glutamyl peptide bond links the V-l and V-2 regions of gp55. Additional important information concerning V8 protease susceptibility was obtained by comparing the fragmentation of nonreduced and reduced preparations of the SFFV-encoded glycoproteins. Similar results were obtained using F-SFFV and R-SFFV. We present the R-SFFV gp54 data because the V-2 (apparent M, 19,300) and V-3 (apparent M, 20,000) fragments are more completely separated by electrophoresis (see Fig. 3). In the absence of a reducing agent such as 2-mercaptoethanol, gp54 has a faster electrophoretic. mobility (apparent M, 51,000) (lanes 1 and 2). Moreover, a similar decrease in apparent M, affects the nonreduced V-l frag-

MACHIDA

164

I2

B

34

ET AL.

5 6 7 8 9 IO II 12

123

FIG. 2. Proteolytic fragmentation analyses of L-[%]methionine-labeled glycoproteins encoded by wild-type, mutant, and revertant F-SFFVs. (A) L-[%]Methionine-labeled F-SFFV gp55, clone 4 gp40, and clone 4asv gp56 were eluted from polyacrylamide gels and were subjected to proteolysis for 30 min at 37” with increasing amounts (0, 0.1, 1.0, and 10.0 pg) of S. aure~~ V8 protease as described previously (Ruta et al, 1983, Machida et &, 1984). The digests were then reelectrophoresed, and the radioactive components were visualized by fluorography. Lanes: 1-4, gp55 digested with increasing amounts of protease; 5-8 and 9-12, same experiments but with gp40 or gp56, respectively. Apparent A&s of V8 fragments: V-l gp55 (&f, 32,500), V-2 gp55 (&f, 20,500), V-3 gp55 (A& 21,000), V-4 gp55 (Af, 12,400), V-l gp40 (A& 32,500), V-l gp56 (A& 32,500), V-2 gp56 (M, 21,500). (B) L-[85S]Methionine-labeled gp55, gp40, and gp56 (lanes 1, 2, and 3, respectively) were subjected to proteolysis with 0.1 fig S uureu.s V8 protease under conditions specified in panel A. (C) L[asSJMethionine-labeled F-SFFV gp55, clone 63 gp58 and clone 63aav gp62 (lanes 1, 2, and 3, respectively) were subjected to proteolysis with 0.1 pg S. aureuS V8 protease under conditions specified in panel A. Apparent M,s of V8 fragments: V-l gp 58 (33,500), V-2 gp58 (20,500).

ment but not the V-2 fragment (compare lane 2 with lanes 5 and 8). These results suggest that intrachain disulfide bond(s) occur within V-l, but not between the V-

1 and V-2 regions disulfide bonds in relatively compact. age site remains

of gp54, and that the V-l cause it to become The primary V8 cleavhighly susceptible re-

MUTANT

AND

REVERTANT

123456789

Nonreduced 9P 54 Nonreduced Vl

3

v2 -c

FIG. 3. Proteolytic fragmentation analyses of nonreduced (lanes l-3) and reduced (lanes 4-9) preparations of L-pS]methionine-labeled SFFV envelope glycoprotein. R-SFFV-infected NIH-3T3 cells were pulse-labeled with 509 pCi L-[86S]methionine for 2 hr at 3’7” and subsequently extracted with immunoprecipitation lysis buffer (IPB) (20 m&f Tris-HCl [pH 7.51, 1 II4 NaCl, 1 mAf EDTA, 0.1% SDS, 0.5% deoxycholate, 1% Triton X-100,0.02% sodium azide (lanes ‘7-9) as described previously (Dresler et oL, 1979; Ruta and Kabat, 1930) or extracted with a modified lysis buffer containing 20 mM Tris-HCl (pH 7.5), 0.15M NaCl, 1 mM EDTA, 1% Triton X-100, and 0.02% sodium aside (lanes l-6). The radioactive proteins in cell lysates prepared with the modified extraction buffer, were immunoprecipitated as described previously (Dresler et al, 1979; Ruta and Kabat, 1930), with the exception that immunoprecipitates were not routinely treated with 2-mercaptoethanol prior to electrophoresis. Both the reduced (lanes 7-9) and nonreduced forms of gp54 (lanes l-6) were then isolated as described in the legend to Fig. 2 and subjected to proteolysis with 0 (lanes 1, 4, and 7), 0.1 (lanes 2, 5, and 8) or 1.0 (lanes 3, 6, and 9) pg V8 protease for 30 min at 37”. The digests were then reelectrophoresed, and the radioactive components were visualized by fluorography. Preparations of nonreduced gp54 and its fragmentation products were also reduced with 2-mercaptoethanol (lanes 4-6) prior to reelectrophoresis in polyacrylamide gels.

gardless of disulfide bond reduction, but secondary and tertiary fragment yields from V-l seem to be influenced by the presence of disulfide bonds (see Fig. 3).

FRIEND

VIRUSES

165

Eflect.s of SFFV env Mutations on Leukemic Progression Erythroleukemia induced by Friend virus is a multistage process which is poorly understood. In particular, it is uncertain whether the advanced leukemia cells which occur late in the disease form within the primary neoplasm or whether they arise elsewhere perhaps by a different pathogenic mechanism. To study this issue, we prepared single-cell suspensions of spleens infected with wild-type, mutant clone 4, or revertant clone 4aEVF-SFFVs for use in an in vitro methylcellulose colony assay as described by Mager et al. (1981). This assay detects cells which develop late during leukemic progression. Spleen cells recovered from mice infected with either wild-type F-SFFV or clone 4aEV, but not with clone 4 or with RMuLV alone, grew as large colonies in methylcellulose when analyzed by this assay (see Table 3). In all cases, only those SFFVs which initiate splenic erythroblastosis were capable of causing leukemic progression as measured by this assay. DISCUSSION

Rewrtant SFFVs Form in Newborn NIH/ Swiss Mice This paper describes the reproducible recovery of pathogenic SFFVs from NIH/ Swiss mice infected as newborns with nonleukemogenic env gene mutants. These recovered SFFVs are pathogenic not only in newborn mice (Table 1) but also in adult mice of the same strain (Table 2). Moreover, in two of three studied cases, the recovered viruses encode env gene products clearly distinct in size from those of the original mutant or wild-type viruses (Figs. 1 and 2). Therefore, formation of revertant SFFVs is often associated with secondary env gene mutations which frequently but perhaps not always affect the sizes of the encoded glycoproteins. Based on these results, we propose that the secondary env mutations are intragenic suppressors which are responsible for the

166

MACHIDA

ET AL.

TABLE

3

ANALYSIS OF LEUKEMOGENICITY OF WILD-TYPE, MUTANT, AND REVERTANT SFFVs IN ADULT DBA/2

Week 2

Week 3

Week 4

Week 7

Colonies detected in Mager et al assaye

0.10, 0.11 0.12, 0.11

0.12, 0.12 0.14, 0.12

0.13, 0.12 0.14, 0.14

0.14, 0.13 0.14, 0.12

0 0

0.90, 1.35

1.6Ob

1.96b

2.25, 1.55

47,73

1.10, 1.41

1.50, 2.15

2.03, 3.10c

3.01, 2.65

32, 68

-d

-d

-d

0.14, 0.13

0

Spleen weights Virus inoculated” No virus R-MuLV F-SFFV + R-MuLV clone 4REV + R-MuLV Clone 4 + R-MuLV

MICE

(g)

“Several adult (6- to 8-week-old) DBA/2 mice were injected intravenously with the wild-type, mutant clone 4, or revertant clone 4azv virus complexes analyzed in Table 2, or with R-MuLV alone. Uninfected mice were also maintained as a negative control. Spleen weights of infected mice were analyzed at various weeks postinfection. b Four mice infected with F-SFFV plus R-MuLV died during Weeks 3-5 postinfection. ‘Two mice infected with clone 4asv plus R-MuLV died during Weeks 4-5 postinfection. d Not analyzed. e Animals that survived long-term infection (7 weeks) were sacrificed; spleens were removed and weighed and single cell suspensions were prepared for use in an in vitro methylcellulose assay as described by Mager et al (1981). This assay detects cells which develop late during leukemic progression. Large colonies (alO” cells) were observed (F-SFFV, 4’7 and 73 colonies in two plates containing cells derived from two different animals, 120 total; clone 4REv, 32 and 68 colonies in two plates containing cells derived from two different animals, 100 total), picked, grown in microtiter wells, and subsequently labeled with Lf%$nethionine to analyze enu gene expression. These cell cultures synthesized env gene components that were equivalent in apparent M, to the envelope glycoproteins encoded by the viruses inoculated into the mice (unpublished observations).

recovery of SFFV pathogenic activity. The recovered SFFVs described here are now being molecularly cloned and their env genes sequenced. Analysis of a battery of such SFFVs should provide an excellent system for identifying the features of glycoprotein structure and intracellular localization which are essential for its leukemogenic activity. Retroviruses which cause cell proliferation have an enormous replicative advantage compared with nonpathogenic mutants. This advantage derives not only from growth of the neoplastic cells, but also from the fact that retroviruses can only infect and bud from the surfaces of proliferating cells (Temin, 1967; HobemSchnegg et aL, 1970; Fischinger et al, 1975; Sherton et d, 1976). Consequently, patho-

genic revertants or new oncogenic retroviruses undergo rapid amplification and selection in target tissues and they can be readily isolated from the neoplastic cells. Previous workers have exploited this characteristic of retroviruses to isolate pathogenic revertants of transformationdefective Rous sarcoma virus mutants (Enrjetto et a& 1983; Wang et al, 1934). Similarly, F-MuLV causes erythroleukemia only in newborn mice (Oliff et al, 1980), and this pathogenesis apparently depends upon env gene recombination events which generate dual tropic MuLVs (Ruscetti et al, 1981). Our results show that pathogenic virus recovery occurs with different SFFV mutants and that this recovery also occurs in newborn but not in adult mice. Because of the dependency

MUTANT

AND REVERTANT

of retroviral infection and budding on cell proliferation (Temin, 1967; HobemSchnegg et aC, 1970; Fischinger et a& 1975; Sherton et aL, 1976), retroviruses which do not cause neoplasia would be expected to have a better opportunity to mutate or to recombine in the growing and mitotitally active tissues of newborns. These observations and approaches may be applicable to studies of other retroviruses. A Structural Model for gp55 With previous evidence, these results indicate that gp55 of F-SFFV and gp54 of R-SFFV contain a glutamyl peptide bond which is exceptionally susceptible to S. aureus V8 protease and that cleavage at this site produces an amino-terminal V-l fragment (apparent M, 32,500) and a carboxyl-terminal V-2 fragment (apparent M, 20,500). Figure 4 shows a partial amino acid sequence of gp55 containing the relevant V8 protease primary cleavage region compared with the related sequences of F-MCF and F-MuLV env glycoproteins. Of the five glutamic acid residues shown, only cleavage at Glu 238 would yield primary V8 fragments consistent with those observed. Specifically, cleavage at Glu 238 would yield an amino-terminal fragment with an apparent M, 31,100-33,500 with the size range resulting from uncertainty in the placement of the four Asn-linked high-mannose oligosaccharides (Polonoff et ah, 1982; Amanuma et al, 1983; Clark and Mak, 1983; Wolff et ak, 1983). The primary V8 cleavage site could not be at glutamic acid positions 154, 273, or 286 because cleavage at these sites would yield amino-terminal fragments with apparent M,s of 21,000~23,500, 37,400-39,900, and 39,200-42,700, respectively. The primary cleavage site also could not be at position 273 which is Asp rather than Glu in the case of the R-SFFV gp54 glycoprotein (Bestwick et aZ., 1984). Moreover, the primary cleavage site is intact in the gp40 glycoprotein encoded by the clone 4 mutant (see Fig. 2). Molecular cloning and nucleic acid sequence analyses indicate that gp40 terminates prematurely and

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contains only 271 amino acids (J.-P. Li, R. K. Bestwick, and D. Kabat, unpublished results). These results suggest that the primary V8 protease cleavage site occurs at Glu 238. Interestingly, Glu 238 is contained within a proline-rich sequence (see boxed region in Fig. 4). Such a sequence would be expected to lack helical secondary structure and to be relatively flexible and a similar sequence occurs at a flexible hinge which links independently folding domains in immunoglobulins (Tucker et ah, 1981; Honjo, 1983). The high degree of sequence hypervariability in this region (Koch et aL, 1984) is consistent with the hypothesis that its overall length and high proline content may be functionally more important than its precise sequence. Since protein unfolding enhances susceptibility to proteases, the probable lack of helical secondary structure in this region of gp55 is consistent with an exceptional proteolytic cleavage rate for Glu 238. Figure 4 also indicates that the junction site which links the xenotropic-related and ecotropic-related amino acid sequences of gp55 occurs in this region of the glycoprotein. We identify this as the position at which the amino acid sequence of gp55 becomes highly homologous to the F-MuLV env sequence, and it is noteworthy that it occurs precisely at the end of the proline-rich sequence. The sites of recombination are based on studies of nucleotide sequence homology (Amanuma et al, 1983; Clark and Mak, 1983; Wolff et a.& 1983; Bestwick et al., 1984) and these occur slightly downstream from the junction site described above (see Fig. 4). This displacement results from nucleotide sequence differences which do not affect the amino acid sequence. This positioning is consistent with the observation that only V-l contains the antigens specific for the dual tropic MuLV glycoproteins (Ruta et al, 1983; Machida et al, 1984; Wolff et aC, 1984). There are several additional lines of evidence consistent with the hypothesis that the V-l and V-2 portions of gp55 are independently folded domains which are

168

MACHIDA l)F-SFFVp 2)F-MCF 3)F-MuLV

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ET AL.

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Ser Trp Asp Ala Pro Lys Val ___ ___ --- Gly --- -_- -__-- --- Thr Thr Gly His Tyr

Gly Arg LyS Ala (162) --- LYS --- ----- LYS Gl" x.7=1

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Val Thr Arq Phe Ser Leu Thr Arg Gin Val Leu Asp Ile Gly r0 Arg Val --- --- --- --- --- --- --- --- Arq --- _-- Asn __- ---I"-1 Gly Leu Thr --- Gly Ile Arg Leu Arq Tyr Gln Am Leu --- --- --- ---

r0 Ile ---

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(202)

---

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Glu Lys Cys Cys Phe (307) ___ Gl" --- -_- --___ Gl" --- --- --FIG. 4. Comparison of a partial amino acid sequence of SFFV gp55 with related sequences of F-MCF and F-MuLV en2r glycoproteins. The sources of these sequences are as follows: (1) FSFFVp (polycythemia strain), Wolff et al (1983); (2) F-MCF, Koch et al. (1984); (3) F-MuLV, Koch et al. (1983). Only those F-MCF and F-MuLV sequences that have homology with F-SFFV sequences are shown. Hyphens are those amino acids identical to the F-SFFV-encoded gp55. Proline residues are in bold type. Asterisks have been placed above glutamic acid (Glu) residues to denote potential V8 protease cleavage sites. The boxed region denotes the proline-rich sequence found in gp55 and in related env glycoproteins. Amino acid sequences downstream from the point marked Eco-related (amino acid 247) are homologous to the sequences in the gp70 of ecotropic FMuLV. The alanine at position 248 in F-SFFV is unique to this particular F-SFFV sequence (Wolff et al, 1983); the other strains of SFFV have a threonine at this position, the same as FMuLV. Although the ecotropic F-MuLV amino acid homology starts at position 247, the potential ecotropic-xenotropic recombinational sites as determined by nucleotide sequence analysis (Clark and Mak, 1984) are marked with hatched boxes. The site for R-SFFV was determined using the same approach as Clark and Mak (1984) and is reported here for the first time. The numbers refer to different strains of SFFV as follows: 1, Axelrad and Steeves strain of F-SFFVp (Clark and Mak, 1983); 2, Rauscher SFFV (Bestwick et al, 1984); 3, Ikawa F-SFFVp strain (Amanuma et aL, 1983); 4, Lilly-Steeves F-SFFVp strain (Wolff et &, 1983). The discrepancy between the recombination site and the site of amino acid sequence homology occurs because of nucleotide sequence changes that do not influence the amino acid code words.

connected by a flexible proline-rich joint. First, mutations affecting V-l do not alter proteolytic susceptibility of V-2 and vice-

versa (Fig. 2) (Ruta et al, 1983; Machida that disulfide bonds occur within V-l but not et aL, 1984). Second, the observation

MUTANT

AND REVERTANT

between V-l and V-2 (Fig. 3) supports the idea that these regions have separate tertiary structures. V-l contains eight Cys and V-2 contains four Cys residues (Wolff et al, 1983). We cannot preclude the possibility that disulfide bonds occur in V-2 but do not affect electrophoretic mobility because its four Cys residues occur in closely clustered pairs. Third, the clone 4 mutation affects V-2 and is suppressed by a secondary mutation that also affects the same domain (Fig. 2). Similarly, the clone 63 mutation affects V-l and our data is consistent with the possibility that the suppressor in clone 63asv may repair V-l and span incidentally into the adjacent linker portion of the glycoprotein. Other evidence (Machida et aL, 1985) supports the hypothesis that V-l may contain an active site for causing erythroblast proliferation and that V-2 may perform an ancillary function such as glycoprotein stabilization or facilitation of proper intracellular placement. Progressive Erythroleukemia Our results support previous evidence (Linemeyer et aZ., 1981, 1982; Ruta et al, 1983; Machida et al, 1984) that the SFFV env genes are oncogenes which initiate progressive leukemogenesis. Consistent with this idea, a revertant SFFV which causes erythroblast hyperplasia also causes leukemic progression whereas the original SFFV mutant causes neither (see Table 3). This suggests that the formation of the advanced leukemia cells requires the env gene and that these cells develop directly from the premalignant neoplasm. This progression clearly distinguishes these viral diseases from nonleukemogenic erythroblast proliferative disorders such as chronic anemias. Although tumor progression is not well understood, it clearly involves hereditable changes which are rare at the level of the individual cell but are relatively common or even certain at the level of the whole neoplasm (Foulds, 1969; Fidler and Hart, 1982). Accordingly, progressive neoplasms are characterized by subclonal diversity

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and by competitive evolution, and they are correspondingly pleiomorphic compared with benign neoplasms (Foulds, 1969; Nowell, 1976; Fidler and Hart, 1982; Hill et al, 1984). Our results and related earlier studies of Friend virus (Linemeyer et al., 1981,1982; Ruta et al, 1983; Machida et ah, 1984) support the hypothesis that cancer can be caused (i.e., rendered inevitable) by a seed oncogene that acts during the premalignant phase to initiate progression. The fact that natural cancers develop preferentially from premalignant progressive neoplasms also implies that these seed or establishment stages can play a critical causal role. Other oncogenes are believed to cause the static characteristics (e.g., immortalization or ability to grow in soft agar or to form tumors in nude mice) that have often been associated with relatively advanced cancers or with transformed fibroblasts (Bishop, 1983; Land et al., 1983). We suggest that both types of oncogenes must be considered if we are to understand the pleiomorphism and change which typify cancer. ACKNOWLEDGMENTS We are indebted to our colleague Jing-Po Li for allowing us to cite his unpublished nucleotide sequence results and for stimulating discussions. In addition, we thankfully acknowledge David Linder for continued helpful advice concerning the characteristics of cancer and the pathologic analysis of tissue sections and Jennifer Au for expert technical assistance. R.K.B. is a Special Fellow of the Leukemia Society of America. This research was supported by U. S. P.H.S. Grant CA 35810and in part by American Cancer Society Grant MV-169. REFERENCES AMANUMA, H., KATORI, A., OBATA, M., SAGATA, N., and IKAWA, Y. (1933). Complete nucleotide sequence of the gene for the specific glycoprotein (gp55) of Friend spleen focus-forming virus. Proc N&l Ad Sci. USA 80,3913-391’7.

ANAND, R., and STEEVES,R. (1980). How many types of erythroleukemia are induced by retroviruses in mice? Nature &m&m) 286, 615-61’7. AXELRAD, A. A., and STEEVES,R. A. (1964). Assay for Friend leukemia virus: Rapid quantitative

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DRESLER,S., RUTA, M., MURRAY, M. J., and KABAT, D. (1979). Glycoprotein encoded by the Friend spleen focus-forming virus. J. Vi& 30, 564-575. ENRIE?TO, P. J., PAYNE, L. N., and WYKE, J. A. (1983). Analysis of the pathogenicity of transformation-defective partial deletion mutants of avian sarcoma virus: Characterization of recovered viruses which encode novel src specific proteins. Virology 127,397-411. FAMULARI, N. G. (1983). Murine leukemia viruses with recombinant f?nugenes: A discussion of their roles in leukemogenesis. Cuw. Top. MicrobioL ImmunoL 103, l-108. FIDLER, I. J., and HART, I. R. (1982). Biological diversity in metastatic neoplasms: Origins and implications. Science (Washington, D. Cl) 217,9981003. FIELDSTEEL,A. H., DAWSON,P. J., and KURAHARA, C. (1969). Induction of lymphatic leukemia in Balb/c mice from the original isolate of Rauscher virus. Brit. J. Cancer 23, 806-812. Biochem 46,83-88. BOSSELMAN,R. A., VAN GRINESVEN,J. L. D., VOGT, FISCHINGER,P. J., TUTIZE-FULLER, N., HUPER, G., and BOLOGNESI,D. P. (1975). Mitosis is required M., and VERMA, I. M. (1980). Genome organization for production of murine leukemia virus and of retroviruses. IX. Analysis of the genomes of structural proteins during de nova infection. J. Friend spleen focus-forming (F-SFFV) and helper ViroL 16, 267-274. murine leukemia viruses by heteroduplex mapping. FOULDS,L. (1969). “Neoplastic Development,” Vol. 1, virologg 102, 234-239. pp. 41-86. Academic Press, New York/London. BOSSELMAN,R. A., VAN STRAATEN,F., VAN BEVEREN, C., VERMA, I., and VOGT,A. (1982). Analysis of the HAAS, M., and PATCH,V. (1980). Cell-surface antigens associated with dual tropic and thymotropic murine enw gene of a molecularly cloned and biologically leukemia viruses inducing thymic and nonthymic active Moloney mink cell focus-forming proviral lymphoms. J. Exp. Afed 151,1321-1333. DNA. J. Vi& 44, 19-31. CHA~OPADHAY, S. K., CLOYD, M. W., LINEMEYER, HANKINS, D., and TROXLER,D. (1980). Polycythemia D. L., LANDER, M. R., RANDS, F., and LOWY, D. R. and anemia inducing erythroleukemia viruses ex(1982). Origin and role of mink cell focus-forming hibit differential erythroid transforming effects viruses in murine thymic lymphomas. Nature in vitro. Cell 22, 693-699. (London) 295, 25-31. HILL, R. P., CHAMBERS,A. F., LING, V., and HARRIS, CLARK, S. P., and MAK, T. W. (1983). Complete J. F. (1984). Dynamic heterogeneity: Rapid genernucleotide sequence of an infectious clone of Friend ation of metastatic variants in mouse B/6 melaspleen focus-forming provirus: gp55 is an envelope noma cells. Science (Washington, D. C.) 224, 998fusion glucoprotein. Proc. NatL Ad Sci USA 80, 1001. 5037-5041. HOBEM-SCHNEGG, B., ROBINSON,H. L., and ROBINSON, CLARK, S. P., and MAK, T. W. (1984). Fluidity of a W. S. (1970). Replication of Rous sarcoma virus in retrovirus genome. J. Viral 50, 759-765. synchronized cells. J. Gen ViroL 7, 85-93. CLEVELAND, D., FISCHER, S., KIRSCHNER, M., and HONJO, T. (1983). Immunoglobulin genes. Ann Rev. LAEMMLI, U. (1977). Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol Chem 252, 11021106. CLOYD, M. W., HARTLEY, J. W., and ROWE, W. P. (1980). Lymphoma-genicity of recombinant mink cell focus-inducing murine leukemia viruses. J. Exp. &fed 151,542-552. DAWSON,P. J., ROSE,W. M., and FIELDSTEEL,A. H. (1966). Lymphatic leukemia in rats and mice inoculated with Friend virus. Brit. J. Cancer 20,114121.

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HOUMARD,J., and DRAPEAU, G. R. (1972). Staphylococcal protease: A proteolytic enzyme specific for glutamoyl bonds. Proc NatL Ad Sk USA 69, 35063509. KABAT, D., RUTA, M., MURRAY,M. J., and POLONOFF, E. (1980). Immunoselection of mutants deficient in cell surface glycoproteins encoded by murine erythroleukemia viruses. Proc NatL Ad Sci USA 77, 57-61. KOCH, W., HUNSMANN,G., and FRIEDRICH,R. (1983).

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Nucleotide sequence of the envelope gene of Friend murine leukemia virus. J. Viral 45, l-9. KOCH, W., ZIMMERMANN,W., OLIFF, A., and FRIEDRICH,R. (1984). Molecular analysis of the envelope gene and long terminal repeat of Friend mink cell focus-forming virus: Implications for the functions of these sequences. J vird 49, 828-840. KOZAK, A. (1983). Genetic mapping of a mouse chromosomal locus required for mink cell focusforming virus replication. J. Vird 48, 300-303. LAEMMLI,U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227. 680-685. LAND, H., PARADA, L. F., and WEINBERG, R. A. (1983). Cellular oncogenes and multistep carcinogenesis. Science (Washington, D. C!) 222, 771-777. LINEMEYER, D. L., MENKE, J. G., RUSCEWI, S. K., EVANS,L. H., and SCOLNICK,E. M. (1982). Envelope gene sequences which encode the gp52 protein of spleen focus-forming virus are required for the induction of erythroid cell proliferation. J. Viral. 43,223-233. LINEMEYER,D. L., RUSCETTI,S. K., SCOLNICK,E. M., EVANS, and DUESBERG,D. H. (1981). Biological activity of the spleen focus-forming virus is encoded by a molecularly cloned subgenomic fragment of spleen focus-forming virus DNA. Proc Natl. Acad. Sci USA 78, 1401-1405. MACHIDA, C. A., BESTWICK,R. K., and KABAT, D. (1984). Reduced leukemogenicity caused by mutations in the membrane glycoprotein gene of Rauscher spleen focus-forming virus. J. viral. 49, 394-402. MACHIDA, C. A., BESTWICK,R. K., and KABAT, D. (1985). A weakly pathogenic mutant of Rauscher spleen focus-forming virus has lost the carboxylterminal membrane anchor of its envelope glycoprotein. J. ViroL 53, 990-993. MACHIDA, C., and KABAT, D. (1982). Role of partial proteolysis in processing murine leukemia virus membrane envelope glycoproteins to the cell surface. A viral mutant with uncleaved glycoprotein. J. Biol. Chem 257, 14018-14022. MAGER, D., MAK, T. W., and BERNSTEIN,A. (1980). Friend leukemia virus-transformed cells, unlike normal stem cells, form spleen colonies in Sl/sld mice. Nature (Lo&m) 288, 592-594. MAGER,D. L., MAK, T. W., and BERNSTEIN,A. (1981). Quantitative colony method for tumorigenic cells transformed by two distinct strains of Friend leukemia virus. Proc. Nat1 Ad Sci. USA 78, 1703-1707. METCALF, D., FURTH, J., and BUFFETS, R. (1959). Pathogenesis of mouse leukemia caused by Friend virus. Cancer Res. 19, 52-58. NOWELL,P. C. (1976). The clonal evolution of tumor

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cell populations. Science (Washington, D. C.) 194, 23-28. OLIFF, A. I., HAGER, G. L., CHANG,E. H., SCOLNICK, E. M., CHAN, H. W., and LOWY, D. R. (1980). Transfection of molecularly cloned Friend murine leukemia virus DNA yields a highly leukemogenic helper-independent type C virus. J. ViroL 33,475486. POLONOFF,E., MACHIDA, C. A., and KABAT, D. (1982). Glycosylation and intracellular transport of membrane glycoproteins encoded by murine leukemia viruses. Inhibition by amino acid analogues and by tunicamycin. J. BioL Chem 257,14023-14028. REDDY, E. P., DUNN, C. Y., and AARONSON,S. A. (1980). Different lymphoid cell targets for transformation by replication-competent Moloney and Rauscher mouse leukemia viruses. Cell 19, 663669. REIN, A. (1982). Interference grouping of murine leukemia viruses: A distinct receptor for the MCFrecombinant viruses in mouse cells. V+ology 120, 251-257. ROWE, W. P., and BRODSKY,I. (1959). A graded response assay for the Friend leukemia virus. J. Natl. Cancer Inst. 23,1239-X248.

RUSCETTI, S., DAVIS, L., FEILD, J., and OLIFF, A. (1981). Friend murine leukemia virus-induced leukemia is associated with the formation of mink cell focus-inducing viruses and is blocked in mice expressing endogenous mink cell focus-inducing xenotropic viral envelope genes. J. Eq. Med. 154, 907-920. RUSCETTI,S. K., LINEMEYER,D., FEILD, J., TROXLER, D., and SCOLNICK,E. M. (1979). Characterization of a protein found in cells infected with the spleen focus-forming virus that shares immunological cross-reactivity with the gp70 found in mink cells focus-inducing virus particles. J. viral 30, 787798. RUTA, M., BESTWICK,R., MACHIDA, C., and KABAT, D. (1983). Loss of leukemogenicity caused by mutations in the membrane glycoprotein structural gene of Friend spleen focus-forming virus. Proc Natl

Acad Sci. USA 80,4704-4708.

RUTA, M., and KABAT, D. (1980). Plasma membrane glycoproteins encoded by cloned Rauscher and Friend spleen focus-forming viruses. J. Irirol. 35, 844-853. SHERTON,C. C., EVANS, L. H., POLONOFF,E., and KABAT, D. (1976). Relationship of Friend murine leukemia virus production to growth and hemoglobin synthesis in cultured erythroleukemia cells. J. Viral. 19, 118-125. TEICH, N., WYKE, J., MAK, T., BERNSTEIN,A., and HARDY, W. (1982). Pathogenesis of retrovirusinduced disease. In “RNA Tumor Viruses, Molecular Biology of Tumor Viruses” (R. Weiss, N.

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Teich, H. Varmus, and J. C&in, Eds.). Cold Spring sequence required for the generation of recovered Harbor Laboratory, Cold Spring Harbor, N. Y. avian sarcoma viruses and characterization of a TEMIN, H. M. (1967). Studies on carcinogenesis by series of replication-defective recovered avian saravian sarcoma virus. V. Requirement for new coma viruses. J. Viral 49, 881-891. DNA synthesis for cell division. J. Cell PhysioL WENDLING, F., MOREAN-GACHELIN,F., and TAM69, 53-54. BOURIN,P. (1981). Emergence of tumorigenic cells TROXLER,D. H., YUAN, E., LINEMEYER,D., RUSCETTI, during the course of Friend disease. Proc NatL S., and SCOLNICK,E. M. (1978). Helper-independent Acad Sci USA 76,3683-3687. mink cell focus-inducing strains of Friend murine WOLFF, L., HUBBERT, N., and RUSCEITI, S. (1984). type C virus: Potential relationship to the origin Structural analysis of the spleen focus-forming of replication defective spleen focus-forming virus. virus envelope gene product. I&-d 133,376-385. J. Exp. Med 148,639-653. TUCKER, P. W., SLIGHTOM, J. L., and BLA~NER, WOLFF, L., SCOLNICK,E., and RUSCETTI,S. (1983). F. R. (1981). Mouse IgA heavy chain gene sequence: Envelope gene of the Friend spleen focus-forming Implications for evolution of immunoglobulin hinge virus: Deletion and insertions in 3’ gp70/p15Eexons. Proc. NatL Ad Sci USA 73,7684-7688. encoding region have resulted in unique features WANG, L.-H., BECKSON,M., ANDERSON,S. M., and in the primary structure of its protein product. HANAFUSA, H. (1984). Identification of the viral Proc. NatL Acaa! Sci USA 80, 4718-4722.